Effect of astaxanthin nanoparticles in protecting the post-thawing quality of rooster sperm challenged by cadmium administration Davood Najafi,* Ramezan Ali Taheri,*,1 Abouzar Najafi,y Mohammad Shamsollahi,y and Manuel Alvarez-Rodriguezz,x *Nanobiotechnology Research Center, Baqiyatallah University of Medical Sciences, 17166 Tehran, Iran; yDepartment of Animal Science, College of Agriculture, University of Tabriz, 51666 Tabriz, Iran; zDepartment of Clinical and Experimental Medicine (IKE), Division of Clinical Sciences, Obstetrics and Gynecology, Link€oping University, oping, Sweden; and xDepartment of Animal Health and Anatomy, Veterinary Faculty, Universitat 58113 Link€ Autonoma de Barcelona, 08193 Bellaterra, Barcelona, Spain ABSTRACT The protective role of astaxanthin this change compared to the Cd group. Cd-treated group nanoparticles (Ast NPs, 25 mg/kg p.o) against cadmium resulted in significantly (P , 0.05) lowest total (37.29 6 (Cd, 1 mg/100 g b.w. SC), a known inductor of lipid 2.46) and progressive (5.84 6 0.47) motility and peroxidation and changes in the antioxidant defense decreased antioxidant enzyme activity (CAT, TAC, and system in the Ross 308 breeder roosters sperm, was GPx), as well as producing a significant (P , 0.05) examined. Sperm motility (computer-assisted sperm decrease in testis weight (mg) compared to the control motility analysis), membrane integrity (hypoosmotic group. Treatment with Ast NPs (Ast NPs 1 Cd) had swelling test), viability, total abnormality, and enzy- reversed Cd-induced changes in the antioxidant defense matic parameters were assessed after thawing. The system and significantly prevented Cd-induced testis testis/body weight (mg/kg) ratio and HE staining re- damage. In conclusion, the results of our work suggest sults of testis were also performed. The obtained results that Ast NPs at 25 mg/kg act as a potent antioxidant in showed that Cd induced detrimental effects on testis and protecting rooster testes against oxidative stress induced sperm, while Cd treated by Ast NPs (Cd Ast) diminished by Cd. Key words: astaxanthin, nanoparticle, cadmium, rooster, toxicity 2020 Poultry Science 99:1678–1686 https://doi.org/10.1016/j.psj.2019.12.006 INTRODUCTION Sood et al., 2013; Rasaie et al., 2014). Liquid storage of avian semen is a technique utilized in poultry to The nanotechnology industry has matured and optimize the management of genetically superior males expanded at a rapid pace in the last decade, leading to (Rad et al., 2016). The cell membrane of avian sperm the research and development of nanomaterials with contains high amounts of polyunsaturated fatty acids enormous potential (Bagdeli et al., 2017; Fasihi- that can easily undergo lipid peroxidation (LPO) in Ramandi et al., 2018; Hajipour et al., 2018). the presence of reactive oxygen species (ROS), thus mak- Nanoparticles (NPs), due to their nanoscale and huge ing avian spermatozoa highly susceptible to LPO surface area, may interact with biological systems by (Cerolini et al., 1997, 2006; Fattah et al., 2017). The more efficient approaches (Azin et al., 2017; Khansary effects of LPO include irreversible loss in motility, and et al., 2017). The improved drug bioavailability of NPs damage to spermatozoa DNA and performance (Rad is attributed to the fact that particles in the nanosize et al., 2016; Mehdipour et al., 2017; Najafi et al., range are efficient in crossing permeability barriers. It 2019a). Antioxidant supplementation of semen has been demonstrated that absorption and extenders, for the purposes of LPO inhibition and bioavailability of some drugs were significantly maintenance of membrane integrity during semen enhanced when administered as NPs (Dian et al., 2013; storage, has previously been described in many mammalian and bird species (Partyka et al., 2013; Najafi et al., 2014; Swami et al., 2017). Ó 2019 The Authors. Published by Elsevier Inc. on behalf of Poultry Science Association Inc. This is an open access article under the CC BY- Cadmium (Cd), one of the most toxic heavy metals, NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). has no known beneficial biological function, and it poses 1 Corresponding author: taheri@bmsu.ac.ir a significant public health hazard, including 1678 ASTAXANTHIN NANOPARTICLES IN PROTECTING ROOSTER SPERM 1679 Table 1. Detailed composition of the basic freezing medium activity of Ast is approximately 10 times higher than extender. other carotenoids such as zeaxanthin, lutein, canthaxan- Ingredient thin, and b-carotene and about 100 times than that of a-tocopherol (Miki, 1991). Potassium citrate tribasic monohydrate (g) 0.64 Sodium-L-glutamate (g) 8.67 Thus, the main aim of the present study was to inves- Magnesium chloride anhydrous (g) 0.34 tigate the protective effect of Ast NPs in 1) roster sperm D-(2)-Fructose (g) 5 cryopreservation and 2) against LPO, ROS production, Potassium phosphate dibasic trihydrate (g) 7.59 Potassium phosphate monobasic (g) 0.7 changes in the enzymatic (MDA, CAT, SOD, TAC, and N-[Tris (hydroxymethyl) methyl]-2 2.71 GPX) antioxidant activity of fresh and post-thawed Sodium acetate trihydrate (g) 3.1 sperm, and testicular damage Cd-induced model in Soybean lecithin (%) 1 Glycerol (%) 8 rooster. Purified water (mL) 1,000 pH 7.1 Osmolarity (mOsm/kg) 310 MATERIALS AND METHODS Chemicals reproductive toxicity (Benoff et al., 2008). Cd poisoning All materials and chemicals were obtained from Merck has been reported in individuals engaged in the battery (Darmstadt, Germany) and Sigma-Aldrich Corporation and paint processing industries. Testicular injury is a (St Louis, MO). major symptom of Cd poisoning, and it also results in blood–testes barrier destruction, germ cell apoptosis, Animals and Treatment testicular edema, hemorrhaging, necrosis, increased LPO, and infertility (Siu et al., 2009; de Souza Predes A total of 20 adult male Ross 308 breeder roosters (25 et al., 2010; Alkhedaide et al., 2016). The Cd wk old) were selected from a commercial flock and were reproductive-injury effect has been considered by housed individually in cages (70 ! 70 ! 85 cm) at 18 to numerous studies in recent decades in species including 22 C in a 15 L: 09 D photoperiod program. The animals fish (Acosta et al., 2016), mouse, and rat (Sayed et al., were fed on a diet composed of 10% crude protein, 3,170 2014; Wang et al., 2017; Zhang et al., 2017), as well as kcal ME/kg, 0.9% calcium, and 0.45% accessible phos- in humans (Li et al., 2016; Riaz et al., 2016; Zhou phate without any additional antioxidant in feed. All et al., 2016), avian (Massanyi et al., 2008), and bovine birds were handled in accordance with methods (Tvrda et al., 2012, 2013), which most of them have approved by the Animal Care and Use Committee of focused on strategies to modify testicular damages. Baqiyatallah University of Medical Sciences. Using antioxidants such as sulforaphane (Yang et al., The roosters were divided into 4 experimental groups 2016), quercetin, and catechin (Jamalan et al., 2016), co- each consisting of 5 animals and were treated as follows: enzyme Q10 and vitamin E (Ognjanovic et al., 2010) are group 1– control roosters (treated subcutaneously (SC) among those strategies to decrease Cd-induced testicular with isotonic saline); group 2– Cd (received i.p. a single damage and improve sperm recovery post- dose of 1 mg Cd/100 g body weight in 0.1 mL isotonic sa- cryopreservation. Moreover, it has been shown that line once a week) subcutaneously (SC); group 3—Ast various antioxidants protect cells from Cd-induced NPs 25 mg/kg peroral (p.o) for 5 consecutive days a toxicity, by reducing GSH, SOD, and MDA presence week for 5 wk; group 4—a single dose of Cd (1 mg/100 (Tandon et al., 2003). g b.w. SC) 1 Ast NPs (25 mg/kg p.o) for 5 consecutive Recently, different types of nanotechnology-based days a week for 5 wk. methods, including nanoemulsion, have been used for Roosters received, twice a day in the morning and the delivering drugs to the targeted site without any adverse evening, approximately 90 g of diet within the experi- effects. The antioxidant activity of astaxanthin (Ast) ment period. Water was provided ad libitum. has been shown to be 10 times that of lutein, canthaxan- thin, and b-carotene. Astaxanthin like other carotenoids Semen Collection is very lipophilic compound which recorded low bioavail- ability. Therefore, a nanopreparation of Ast has been Semen samples were collected weekly from the developed that is more stable and well bioavailable. roosters, after 2-wk habituation, by the dorso- However, the current study investigates the protective abdominal massage method (Burrows and Quinn, effects of Ast, as a lipid-soluble carotenoid against Cd- 1937). Briefly, ejaculates were collected using the induced toxicity in rooster testis and reduced sperm dorso-abdominal massage method. The semen was quality. Recently, the red carotenoid pigment Ast, a caught in a 15-mL graduated centrifuge tubes, which is potent antioxidant without provitamin-A activity pre- transferred immediately to the laboratory. To maintain sent in some marine organisms (Lai et al., 2004), has the temperature of the ejaculates, the tubes was placed been used in numerous studies to improve sperm quality in a water bath (37 C), for primary evaluation in the lab- (Mansour et al., 2006; Fang et al., 2015; Tizkar et al., oratory. Normal quality of sperm was approved in the 2015b; Vahidinia et al., 2017). Furthermore, it has experiment if the subsequent parameters were met: vol- been reported in the literature that the antioxidant ume between 0.2 and 0.6 mL; sperm concentration of  4 1680 NAJAFI ET AL. ! 109 spermatozoa/mL; sperm motility  80%; and  Japan), including total motile sperm (TM, %), progres- 10% abnormal morphology. Instantly after the sive motile sperm (PM, %), average path velocity (VAP, initial assessment, semen samples were pooled (Najafi mm/s), straight linear velocity (VSL, mm/s), curvilinear et al., 2019c). velocity (VCL, mm/s), amplitude of lateral head displacement (ALH, mm), straightness (STR, %), and Extender Preparation and Semen linearity (LIN, %). Cryopreservation Procedure Sperm Viability The basic extender composition used in this study is shown in Table 1 (Moghbeli et al., 2016). Sperm samples Sperm viability was evaluated using the eosin-nigrosin from treated roosters were diluted with modified Belts- stain method (Safari Asl et al., 2018). To prepare the ville in their groups in order to aspirate into 0.25 mL sperm suspension smears, a drop of sperm sample (10 French straws (IMV, L’Aigle, France) to get a definitive mL) was mixed with 2 drops (20 mL) of stain on a pre- concentration of 400 ! 106 spermatozoa/mL (100 ! 106 warmed slide and immediately expanded with another spermatozoa per straw). After that, the straws were slide. After drying, the viable and non-viable sperm on sealed with polyvinyl alcohol powder and stored at 4C the slide were assessed by counting 200 cells at !400 for 3 h .The straws were placed in 4 cm over the liquid magnification under phase-contrast microscopy nitrogen for approximately 7 min in a 40 ! 20 ! 20 (CKX41; Olympus). Sperm with partial or complete cm cryobox with 8,000 cm3 liquid nitrogen. Afterwards, purple staining were considered non-viable, and only liquid nitrogen was used to store the straws at –196 C cells that showed complete exclusion of stain were (Najafi et al., 2014). After a month, the frozen straws considered viable. were thawed separately at 37 C for 30 s in a water bath and then evaluated individually. Sperm Morphology Astaxanthin Nanoemulsion Preparation Thirty microliters of semen was placed in 1 mL Han- cock’s solution to evaluate total sperm abnormalities In order to prevent the light sensitivity effect of Ast, (Mehdipour et al., 2018). This solution included 62.5 the nanoemulsion was prepared with minimal exposure mL formalin (with 37% formaldehyde), PBS buffer to light. The oil-in-water red color Ast emulsion was pre- (150 mL), sodium saline solution (150 mL), and pared using ultrapure water as the continuous phase and double-distilled water (500 mL). To specify acrosome, palm olein containing Ast as the disperse phase. Ast head, and tail abnormalities, 10 mL of processed sperm extract was first mixed with pure palm olein (15% was handled on a slide. The abnormalities percentage w/w). The solution was then mixed with purified water was determined by counting a total number of 300 sperm (82.5% w/w) containing Tween 80 and lecithin as the with a phase-contrast microscope (!1,000 magnifica- emulsifier (2.5% w/w). The premix was homogenized us- tion; oil immersion). ing high shear homogenizer (Silverson Homogenizer, UK) at 3,000 rpm for 5 minu followed by high pressure Plasma Membrane Integrity Evaluation homogenizer (model APV 1000, APV Systems, Alberts- land, Denmark). The hypoosmotic swelling test was used to evaluate the functionality of sperm plasma membrane after Assessment of Fresh and Post-Thawed freeze-thawing (Bazyar et al., 2019). This test was per- Sperm formed by placing 5 mL of extended semen in a 50 mL hypoosmotic solution with 100 mOsm/l (19.2 mM so- Fresh and post-thawed motion characteristics of dium citrate and 57.6 mM fructose). After incubation sperm were analyzed by computer-assisted sperm for 20 min, the sperm was verified under a phase- motility analysis (CASA; IVOS version 12; Hamilton- contrast microscope (!1,000 magnification), and at Thorne Biosciences, MA) with settings programmed to least 300 spermatozoa with swollen and non-swollen tails detect rooster sperm (30 frames acquired, minimum were recorded. contrast 5 50, minimum average path velocity (VAP) 5 5 mm/s, minimum straight line velocity (VSL) 5 6 Lipid Peroxidation mm/s, non-motile head size 5 7 pix and nonmotile head intensity 5 95) (Moce et al., 2010). A pre- The malondialdehyde (MDA) level, as indicative of warmed (37 C) Leja chamber (Leja 4, Leja Products LPO in the sperm membrane phospholipids, was Luzernestraat B.V., Holland) was inserted under the measured using the thiobarbituric acid (TBA) reaction thermal plate of the microscope, and then the semen (Feyzi et al., 2018). Briefly, 1 mL of diluted sperm (250 from the 3 straws in the chamber was assessed individu- ! 106 mL21) was added to 1 mL of cold 20% (w/v) ally. Five fields, randomly selected by the computer and TBA to precipitate protein. The precipitate was centri- estimated for each semen sample (Najafi et al., 2019b), fuged (960 ! g 15 min), and 1 mL of 0.67% (w/v) and 9 motility parameters were recorded using a TBA was incubated with the supernatant (1 mL) in a phase-contrast microscope (CKX41; Olympus, Tokyo, boiling water (100 C) bath for 10 min. After cooling, a ASTAXANTHIN NANOPARTICLES IN PROTECTING ROOSTER SPERM 1681 Figure 1. HE staining results of testis. The testes of the control group showed different stages of spermatogenesis (A). The Cd group showed pyk- nosis undergoing necrosis of cells. There were also fewer Leydig cells (B). Ast-treated group increased sperm cell proliferation and improved seminif- erous tubule structure (C). The Cd1Ast-treated group increased sperm cell proliferation and maturation as well as the number of Leydig cells in the seminiferous tubule and also the structure of the seminiferous tubule was more complete, and closely connected with other tubules (D). 400X. spectrophotometer (UV-1200, Shimadzu, Japan) was min. GPx activity was expressed as international units used to read the absorbance at 532 nm. All MDA values (IU) per g protein. were reported as nmol/mL. Superoxide Dismutase and Total Histology of the Testes Antioxidant Capacity Levels The roosters were killed at the end of the experi- ment, and the testes were isolated and fixed in Bouin’s The supernatant absorbances were recorded by a spec- fixator. The samples were removed from fixative and trophotometer (UV-1200, Shimadzu, Japan) at 532 nm. dehydrated using a graded series of ethanol (50, 70, The SOD value was recorded according to the adapted 80, 90, 96, and 100%), cleared in xylene, and procedure explained elsewhere. In this reaction, superox- embedded in paraffin. The paraffin sections were sliced ide anions cause pyrogallol oxidation. Pyrogallol oxida- using a microtome. The 5 mm microscopic sections tion inhibition by SOD was followed by using a were prepared, and at least 2 slides from each testis spectrophotometer at 420 nm. TAC was measured by were stained with hematoxylin and eosin for histolog- monitoring hydrogen peroxide decomposition. ical assessment. GPx Activity Assessment Statistical Analysis The GPx value was determined according to Najafi et al. (2018b). The reaction substance consisted of potas- The experiment was performed in 5 replicates. Data sium phosphate buffer (50 mM, pH 7.0), EDTA (1 mM), were checked for normal distribution using a univari- 1 mM sodium azide (NaN3), 0.2 mM decreased nicotin- ate procedure and the Shapiro–Wilk test. Arc sine amide adenine dinucleotide phosphate (NADPH), gluta- (for percentage data) transformation of data was thione reductase (1 EU mL–1) and 1 mM GSH. The used when appropriate. Single and repeated measure- sperm sample (0.1 mL) was blended with 0.8 mL of the ments data were analyzed by PROC GLM and PROC reaction substance, incubated at 25 C for 5 min before Mixed SAS 9.1 (version 9.1, 2002; SAS institute Cary, the beginning of the reaction, and induced by the addi- NC), respectively. The results are presented as the tion of 0.1 mL of a H2O2 solution (final H2O2 dose: Lsmean 6 SEM. The Tukey test was applied to 0.25 mM). A spectrophotometer (UV-1200, Shimadzu, compare Lsmeans, and the level of significance was Japan) was used to read absorbance at 412 nm in 5 adjusted to P , 0.05. 1682 NAJAFI ET AL. Table 2. Effect of different antioxidants on motion parameters of fresh and post-thawed rooster sperm assessed by CASA (mean 6 SEM). Treatment Fresh Post-thawed Parameter Control Cd Ast Cd 1 Ast SEM Control Cd Ast Cd 1 Ast SEM a b a a b c a b MOT (%) 91.32 80.61 92.27 88.36 1.23 57.70 37.29 68.67 54.67 2.46 PROG (%) 51.22b 39.61c 57.74a 50.45b 1.34 13.97b 5.84c 17.67a 13.25b 0.47 VAP (mm/s) 38.21b 30.11c 42.95a 36.41b 0.88 28.58b 19.49d 33.30a 26.10c 0.47 VSL (mm/s) 24.74b 19.42c 28.92a 23.14b 0.74 15.91b 12.68c 19.26a 16.36b 0.64 VCL (mm/s) 63.09 60.21 64.39 62.24 2.14 56.51 51.13 58.19 54.53 1.8 LIN (%) 38.13a,b 30.1b 43.8a 36.7a,b 1.94 28.28a,b 24.93b 33.35a 30.20a,b 1.75 STR (%) 65.42 61.04 67.89 64.24 2.32 55.72 65.22 57.85 62.71 2.48 ALH (mm) 4.13 3.94 4.20 4.01 0.43 2.96 2.65 3.44 3.13 0.51 BCF (Hz) 19.07 17.05 19.8 18.9 1.32 17.55 16.9 18.41 17.75 0.93 Abbreviations: MOT, total motility; PROG, progressive motility; VCL, curvilinear velocity; VAP, average path ve- locity; VSL, straight-line velocity; LIN, linearity; STR, straightness; ALH, amplitude of lateral head displacement; BCF, beat/cross frequency. Different letters exhibit differences among treatment groups (P , 0.05). RESULTS Viability, Membrane Integrity, and Sperm Abnormalities Histological Result The influence of Cd and Ast NPs on sperm viability, The testes of the control group showed different stages membrane integrity, and abnormal forms is presented of spermatogenesis (Figure 1A). Compared with the con- in Table 3. Post-thawed, higher sperm viability and trol group (Figure 1A), the Cd group showed pyknosis membrane integrity (75.76 6 1.86, 50.43 6 1.54; respec- undergoing necrosis of cells (Figure 1B). There were tively) were found in the Ast NPs group compared to the also fewer Leydig cells (Figure 1B). No significant differ- others, but not in fresh sperm, where only membrane ences were seen between the control and Ast groups integrity was higher (87.48 6 1.39).The percentage of (Figure 1C). Compared with the Cd group, the abnormal forms decreased both in fresh (4.90 6 0.51) Cd1Ast-treated group increased sperm cell proliferation and post-thawed (17.01 6 1.25) sperm in Ast group and maturation as well the number of Leydig cells in the compared with the rest of the groups, including control. seminiferous tubule and also the structure of the seminif- Moreover, Cd induced lower (P , 0.05) viability (fresh: erous tubule was more complete, and closely connected 82.4 6 1.57 and post-thawed: 43.56 6 1.86) and integ- with other tubules (Figure 1D). rity (fresh: 72.31 6 1.39 and post-thawed: 31.87 6 1.54) and higher abnormal forms (fresh: 9.81 6 0.51 and post-thawed: 37.12 6 1.25) among the control and Motility and Kinetic Parameters treatment groups. Table 2 shows data for the effect of Cd and Ast NPs on sperm motility variables under the following conditions: when the rooster sperm was fresh and then after Oxidative Variables freezing-thawing. Cd (1 mg/100 g/body SC) resulted in The effects of Cd and Ast NPs on the oxidative vari- significantly (P , 0.05) lower total (80.61 6 1.23) and ables status of rooster sperm after freeze-thawing are progressive (39.61 6 1.34) motility, and it is restored to summarized in Table 4. There were no differences in similar control values after treatment with Ast (group the MDA value in the control group as compared with Cd 1 Ast; 88.36 6 1.23 and 50.45 6 1.34, respectively). the other groups in post-thawed values. However, fresh Post-thawed total (37.29 6 2.46) and progressive (5.84 values of MDA were significantly higher in Cd group 6 0.47) motility showed the same patterns, also restoring compared with the control and treatment groups. to similar values relative with control in Ast 1 Cd group Furthermore, the lowest values for CAT, TAC, and (54.67 6 2.46 and 13.25 6 0.47, respectively). In addition, GPX activity (fresh: 29.17 6 3.51, 0.66 6 0.12, post-thawing values using Ast NPs (25 mg/kg p.o) 42.80 6 1.63 and post-thawed: 32.31 6 3.27, 0.79 6 resulted in significantly (P , 0.05) higher total (68.76 0.21, 48.20 6 1.50; respectively) were obtained in the 6 2.46) and progressive (17.67 6 0.47) motility compared Cd group. In general, addition of Ast NPs to the to other groups. Moreover, the VAP (fresh: 42.95 6 0.88 roosters’ diet in the Cd group (Cd 1 Ast) improved and post-thawed: 33.30 6 0.47) and VSL (fresh: 28.92 6 CAT (P , 0.05), TAC, and GPX activity (fresh: 45.23 0.74 and post-thawed: 19.26 6 0.64) values in Ast NPs 6 3.51, 1.66 6 0.12, 51.47 6 1.63 and post-thawed: group were significantly (P , 0.05) higher than the other 48.80 6 3.27, 1.71 6 0.21, 55.34 6 1.50; respectively) groups. No significant differences were observed in LIN, compared to the Cd group. However, there were no dif- STR, ALH, and BCF in the treatment groups compared ferences in SOD activity among the control and treat- to the control. ment groups. ASTAXANTHIN NANOPARTICLES IN PROTECTING ROOSTER SPERM 1683 Table 3. Percentage of viability, membrane integrity, and abnormal forms of fresh and post-thawed rooster sperm (mean 6 SEM). Treatment Fresh Post-thawed Parameter Control Cd Ast Cd 1 Ast SEM Control Cd Ast Cd 1 Ast SEM a b a a b c a b Viability (%) 92.01 82.4 94.30 90.36 1.57 64.69 43.56 75.76 57.24 1.86 Membrane integrity (%) 82.23b 72.31c 87.48a 81.34b 1.39 40.04b 31.87c 50.43a 41.91b 1.54 Abnormal forms (%) 6.59b 9.81a 4.90c 7.21b 0.51 25.67b 37.12a 17.01c 30.15b 1.25 Different letters exhibit differences among treatment groups (P , 0.05). Testis/Body Weight Ratio quality that has decreased during Cd exposure (adminis- trated subcutaneously). Results for the testis/body weight (g/kg) ratio from The antioxidant defense system plays a key role in treated roosters and controls are shown in Table 5. protecting the male reproductive system against oxida- Cd-treated roosters showed a significant (P , 0.05) tive damage (Yang et al., 2016). It has been reported decrease in testis weight (g) compared to the control that food supplementation with Ast in 30 infertile men group (38.45 6 0.08, 41.30 6 0.08, respectively). Using resulted in a significant reduction in seminal ROS and Ast NPs reversed (P , 0.05) Cd induced testicular at- improved sperm motility and morphology (Comhaire rophy (Figure 1B) in Cd 1 Ast NPs group. However, et al., 2005). Another study documented that Ast treat- there were no differences in body weight and testis/ ment significantly restored sperm DNA damage induced body weight ratio among the control and treatment by cyclophosphamide and showed a protective effect groups. against cyclophosphamide-induced testicular toxicity confirmed by testes histology in mice (Tripathi and DISCUSSION Jena, 2008). Our findings in the present study showed that diet The main aim of this experiment was to study the effect supplementation with Ast NPs enhanced sperm quality of Ast NPs antioxidant activity and also the combined ef- in the Ast group compared to the control. Furthermore, fect on an induced model by Cd exposure on testis weight, we proved that Ast NPs in the diet could decrease the ROS generation, quality parameters, and the oxidative detrimental effect of Cd in sperm motility, viability, status of fresh and cryopreserved rooster sperm. In this and oxidative status. In another study, it was noted study, we also determined Ast NPs antioxidant activity that treatment of rats with CoQ10 and Vit E alone against Cd-induced oxidative stress by assessing motility and/or in combination with Cd decreased the toxic effect parameters and oxidative variables in fresh and post- of Cd on antioxidant enzyme activity (Ognjanovic et al., thawed rooster sperm. The improvement of semen char- 2010). In the present study, Ast NPs reduced cellular acteristics in fresh semen seemed to be a result of the toxicity caused by Cd-induced ROS and protected the dietary supplementation of antioxidants. The findings post-thawing sperm samples in the anti-oxidative of the present study show that the results of fresh and system. cryopreserved sperm are in consistency. One of the reported roles of carotenoids is their antiox- Nanotechnology is a rapidly progressing field. Nano- idant role in animals. Oxidative stress is of great concern particles hold tremendous potential as an effective in poultry industries with breeder roosters. Normally, drug delivery system. Nanosystems with different com- various antioxidants and enzymes in seminal plasma positions and biological properties have been extensively protect sperm from oxidative attack (Min et al., 2016). investigated for drug delivery applications (Koo et al., The efficiency of dietary carotenoid supplementation 2005; Najafi et al., 2018a). In this study, we on antioxidant status has been investigated and shown demonstrated that dietary Ast NPs have a protective to rescue various cells and tissues from oxidative damage effect against Cd-induced LPO and can improve sperm (Pulido et al., 2000; Wang et al., 2006). Ast is a naturally Table 4. Malondialdehyde (MDA) concentration and antioxidant enzyme activity status of rooster sperm after fresh and freeze-thawing (mean 6 SEM). Treatment Fresh Post-thawed Parameter Control Cd Ast Cd 1 Ast SEM Control Cd Ast Cd 1 Ast SEM b a c b a,b a b a,b MDA (nmol/mL) 1.72 2.69 1.13 1.84 0.26 2.02 3.14 1.78 2.54 0.31 CAT (U/mL) 53.14b 29.17c 59.66a 45.23b 3.51 61.00a,b 32.31c 69.51a 48.80b 3.27 SOD (U/mL) 88.42 74.34 98.45 87.90 9.88 96.02 85.13 117.42 98.37 10.3 TAC (mmol/l) 1.73b 0.66c 2.10a 1.66b 0.12 1.86a 0.79b 2.31a 1.71a 0.21 GPX (IU/g protein) 54.11b 42.80c 59.73a 51.47b 1.63 58.47a,b 48.20c 63.46a 55.34b 1.5 Different letters exhibit differences among treatment groups (P , 0.05). 1684 NAJAFI ET AL. Table 5. Testis/body weight (g/kg) ratio of treated roosters and controls at 5 wk following treatment (birds at 25 wk old). Treatment Testis weight (g) Live body weight (kg) Testis/body weight (g/kg) a Control 41.30 4.07 10.16 Cd 38.45b 3.85 10.03 Ast 45.95c 4.12 11.17 Cd 1 Ast 39.93d 3.92 10.19 SEM 0.08 0.10 0.28 Different letters exhibit differences among treatment groups (P , 0.05). occurring carotenoid with strong antioxidant properties, damage to rooster sperm quality parameters. Combined which shows low oral bioavailability due to its treatment with Ast NPs and Cd showed protective ef- lipophilicity. The use of nano emulsion-based formula- fects against LPO and changes in enzymatic (MDA, tions is one of several approaches found to be efficient CAT, SOD, TAC, and GPX) antioxidant systems in in improving the bioavailability of lipophilic entities rooster sperm. The results of our work suggest that such as Ast (Affandi et al., 2011). Ast NPs at 25 mg/kg may act as a potent antioxidant Ast NPs (25 mg/kg) seemed to be an effective dose for in protecting rooster testes against oxidative stress enhancing sperm total and progressive motility, vitality induced by Cd. The present study suggests that Cd im- and membrane integrity as outcomes improved relative pairs the reproductive function in male Ross 308 breeder to the controls. Furthermore, our results confirmed roosters through toxic effects on the testis. that oral treatment with Ast NPs in rooster could coun- teract the detrimental effect of Cd in sperm motility, morphological appearance, and oxidative status indexes. ACKNOWLEDGMENTS These results are in agreement with Ognjanovic et al. 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