Research Article

 

Supplement Effects of Vitamin C, Vitamin E and the Combinations in Semen Extenders of Kub Chicken Quality

 

Muhammad Farid1, Makruf Arif1, Kurniawan Dwi Prihantoko1, Asmarani Kusumawati2*, Agustina Dwi Wijayanti3, Erif Maha Nugraha Setyawan2

1Department of Food Hygiene and Control, Faculty of Veterinary Medicine, Cairo University, Giza square, Giza 12211, Egypt; 2Department of Veterinary Hygiene and Management, Faculty of Veterinary Medicine, Cairo University, Giza square, Giza 12211, Egypt; 3Faculty of Biotechnology, MSA University, 6th October City, Egypt.

 

Received | February 06, 2021; Accepted | March 17, 2021; Published | June 15, 2021

*Correspondence | Amarani Kusumawati, Department of Veterinary Hygiene and Management, Faculty of Veterinary Medicine, Cairo University, Giza square, Giza 12211, Egypt; Email: [email protected]

Citation | Farid M, Arif M, Prihantoko KD, Kusumawati A, Wijayanti AD, Setyawan EMN (2021). Supplement effects of vitamin c, vitamin e and the combinations in semen extenders of kub chicken quality. Adv. Anim. Vet. Sci. 9(7): 1034-1039.

DOI | http://dx.doi.org/10.17582/journal.aavs/2021/9.7.1034.1039

ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331

Copyright © 2021 Kusumawati et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 

INTRODUCTION

 

Native chicken brings benefits to most households in developing countries and participates in economic growth (Padhi, 2016). The KUB Chicken is a moderate local chicken breed by Indonesian Research Institute for Animal Production (IRIAP) with high-productivity eggs and better weight gain ability compared to ordinary native chickens (Erwan, 2020). Artificial Insemination (AI) can be administered in the process of mating to increase the KUB Chicken population, but spermatozoa of KUB chicken contains high polyunsaturated fatty acids (PUFA) that are easily oxidized by reactive oxygen species (ROS) and caused lipid peroxidation (Khan, 2011). Lipid peroxidation can produce toxic biological substances that cause permanent damage to the sperm (Wen et al., 2020). Lipid peroxidation can lead to decreased motility, damaged the DNA spermatozoa, and reduced fertility (Peruma et al., 2013).

 

In the storage, spermatozoa usually expose to a cold shock and oxidative damage which results in decreased motility and fertility. The production of ROS is a normal physiological process in spermatozoa. However, the over-produced ROS may cause structural damage to the sperm membrane (De Lamirande et al., 1997). Oxidative-stressed is a factor that increases cell damage due to reactive oxygen species (ROS). The excessive ROS production in sperms is dangerous due to its negatives effect on counting the active spermatozoa (Prihantoko et al., 2020a). To prevent peroxidative damage as a result of ROS, the membrane must be layered by a highly effective antioxidant system. Catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) are examples of the enzyme with roles as natural inhibitors which are similar to the antioxidant mechanism in sperm: vitamin A, C, E, uric acid, glutathione, and carotenoids (Bréque et al., 2003; Cerolini et al., 2006; Partyka et al., 2012). ROS production and the antioxidant system have to be balanced to maintain the viability and fertility of spermatozoa. Several studies have examined the effect of antioxidants on variable chicken sperm after cryopreservation (Amini et al., 2015a; Amini et al., 2015b; Moghbeli et al., 2016). Other several antioxidant studies applied to different species, for example, the additions of genistein antioxidant to Ongole grade bull semen (Prihantoko et al., 2020a).

 

Vitamin C is naturally a free radical exposure with its impact on optimizing sperm performance by reducing cell damage include the lipid peroxidase (Hu et al., 2010). The ascorbic acid will bring the best effect on the spermatozoan quality performance through its radical-scavenging by reducing cell injury (Amini et al., 2015a). Vitamin E is a fat-soluble compound with a membrane-stabilized inhibitor that performs effectively on cell membranes (Tabatabaei et al., 2011). According to Jeong et al. (2009), vitamin E can break the covalent bond of ROS that is formed within the fatty acids, besides, it is the major protectant against ROS and lipid peroxidation. Vitamin C and vitamin E functioned as antioxidants may decrease lipid peroxidation and increase the quality of semen. The combination of both vitamins in dietary has been observed to provide a marked increase in better protection against spermatozoa damage (Uzochukwu et al., 2020), therefore this study aimed to evaluate the supplementation effects of vitamin C, vitamin E, and the combinations in semen extenders to the variable of KUB chicken semen quality for 48 hours stored at 4°C.

 

MATERIALS AND METHODS

 

Research Animals

The study used a total of 12 male KUB chickens aged 1.5-2 years. The KUB chickens were housed in individual cages at 32°C in the Chicken Breeding Cage, Faculty of Veterinary Medicine, Gadjah Mada University in Yogyakarta, Indonesia. The chickens were fed by a commercial feed containing 19% crude protein without antioxidant supplementation and ad libitum water. The chickens were adapted for two weeks and at the same time trained to collect the semen.

 

Semen Collection

Semen samples were collected by dorsal-abdominal massage method every twice a week for four weeks. The sampling with semen volume of 0.2-0.6 mL, a concentration of ≥ 3×109 sperm/mL, motility ≥ of 80%, and abnormal morphology ≤ 10% were used in this study. Then the sampling was thawed in a water bath (37°C) and analyzed macroscopically and microscopically.

 

Extender preparation

The base extender used in this study contained ringer lactate, egg yolk, penicillin, and streptomycin. Then the base was added with vitamin E 5µg/mL (Sigma, St. Louis, MO, USA), vitamin C 200µg/mL (Merck KGaA, Darmstadt, Germany), and the combinations (vitamin E 5µg+vitamin C 200µg/mL). Control diluent was used the base extenders composition only.

 

Semen storage

The semen mixed with the extenders fluids, the solution was diluted to a final concentration of 5×106 sperm/mL and placed in a 0.25 ml straw (Minitube, Slovakia) according to the control label (P0), vitamin C (P1), vitamin E (P2). The combinations (P3) were placed on a cooling shelf at 4°C. Spermatozoa quality was evaluated at 2 (T1), 4 (T2), 8 (T3), 24 (T4), and 48 (T5) hours after being stored. Then the straws were warmed in a water bath at 37°C for 15 seconds.

 

Semen Evaluation

Sperm Motility: Sperm motility evaluation used a sample of 10 µL (400×104 sperm cells). Then dropped the semen in a warm glass slide with the coverslip. Then the semen was observed using a light microscope (400x) under five different fields of glass slide view. The motility evaluation was subjectively assessed by percentages on a scale range of 0-100%.

 

Sperm Viability: Sperm viability was assessed by the eosin-nigrosin staining method (Akhlaghi et al., 2014). A mixture of 10 µl semen and 20 µl eosin nigrosine staining was placed on a warm slide object-glass and smear within 15 seconds, dried. The dried smear was observed by counting 200 cells under a light microscope with 400x magnification. Living spermatozoa were characterized by an intact membrane structure and undyed body and dyed body to the dead sperm due to the damaged membrane sperm.

 

Table 1: Percentages of sperm motility KUB chicken spermatozoa stored at 4oC in various diluent treatments within 48 hours of observation

 

Treatment	Observation Time
	T1 (%)	T2 (%)	T3 (%)	T4 (%)	T5 (%)
P0	86.50 ± 2.646x	83.00 ± 2.160x	79.00 ± 3.367x	63.50 ± 10.755x	31.00 ± 8.400x
P1	87.75 ± 1.258x	84.75 ± 2.062x	79.50 ± 4.796x	62.00 ± 7.071x	38.50 ± 10.59x
P2	90.75 ± 1.708x	85.50 ± 4.203x	83.50 ± 2.887x	76.25 ± 5.058y	64.75 ± 4.113y
P3	87.50 ± 2.380x	84.00 ± 1.414x	79.25 ± 3.686x	48.75 ± 8.539z	16.25 ± 4.787z

x, y, z: different superscripts in the same column indicate significant differences (P≤0,05)

 

Table 2: Percentages of sperm viability KUB chicken stored at 4oC in various diluent treatments within 48 hours of observation

 

Treatment	Observation Time
	T1 (%)	T2 (%)	T3 (%)	T4 (%)	T5 (%)
P0	91.75 ± 2.630x	85.25 ± 3.862x	83.75 ± 4.573x	64.00 ± 8.832x	49.25 ± 8.694xy
P1	91.50 ± 3.697x	87.75 ± 2.986x	82.75 ± 4.031x	63.75 ± 2.630x	46.00 ± 6.683y
P2	95.50 ± 2.082x	91.25 ± 1.500x	88.50 ± 3.873x	76.75 ± 2.986y	63.50 ± 2.380z
P3	92.25 ± 1.708x	88.00 ± 2.160x	84.75 ± 2.986x	61.00 ± 13.292x	27.75 ± 10.372x

x, y, z : different superscripts in the same column show significant differences (P≤0.05)

 

Table 3: Percentages of sperm membrane integrity of KUB chicken stored at 4oC in various diluent treatments within 48 hours of observation

 

Treatment	Observation Time
	T1 (%)	T2 (%)	T3 (%)	T4 (%)	T5 (%)
P0	85.50 ± 1.291x	80.25 ± 3.500x	74.75 ± 3.594x	62.25 ± 2.872x	44.50 ± 1.29x
P1	86.25 ± 2.500x	80.75 ± 2.062x	75.50 ± 3.416x	60.75 ± 2.217x	41.75 ± 1.258x
P2	88.50 ± 1.732x	84.25 ± 1.708x	80.25 ± 2.217x	72.75 ± 3.202y	61.25 ± 2.380z
P3	88.25 ± 2.062x	83.50 ± 1.732x	78.00 ± 3.162x	57.50 ± 3.697x	18.50 ± 3.109y

x, y, z : different superscripts in the same column show significant differences (P≤0.05)

 

Membrane Integrity: Membrane integrity examination used the HOST test (hypoosmotic swelling test) which was previously determined by Santiago-Moreno et. (2009) with modification. HOST solution consists of 0.49 g sodium citrate, 0.9 g fructose, and 100 ml distillate water. A 20 μl semen was diluted to 200 μl HOST solution and briefly incubated for 45 minutes at 25°C. The solutions were smeared, fixated with methanol for 10 minutes, washed, and dried. The smear was observed under the light microscope by counting 200 cells of spermatozoa with 400 x magnification. An intact membrane of spermatozoa had swollen and coiled tails while the damaged spermatozoa characterized with straight tails.

 

DNA Fragmentation: Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) Kit, TMR Red version 12 (Merck, Australia) were being used to evaluate the DNA fragmentation. The spermatozoa smear was fixated 60 minutes, added the permeability solution 0.1% of Triton X-100 in 0.1%, sodium citrate for 2 minutes in ice at 2-8°C, washed with PBS saline (phosphate buffer saline) twice, and dried. A mixture of 50 µl TUNEL was reacted to the smeared, incubated for 60 minutes at 37°C in the dark, and after that gently rinsed with PBS three times. The labeling was performed under a laser confocal microscope with a wavelength of 470-495 nm. Fragmented DNA of spermatozoa will be seen as fluorescent green.

 

Statistical Analysis

Data were analyzed using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test to determine differences between the treatments. A difference of P<0.05 was considered statistically significant. SPSS version 25.0 was used in this study (SPSS Inc., Chicago, IL, USA).

 

RESULT AND DISCUSSION

 

The total average of fresh semen examination on volume, pH, concentration, motility, viability, and abnormalities were 0.26 ± 0.06 ml, 7.18 ± 0.54, 4.24 ± 0.90 x 109 sperm/mL, 92.00 ± 2.47%, 95.00 ± 1.59% and 5.31 ± 1.62%. Otherwise, evaluation of the motility, viability, and membrane integrity after T1, T2, and T3 stored, there were no significant differences between the treatments (P> 0.05), T4 and T5 of the P2 treatment group were significantly higher (P<0.05) while the P3 treatment group was lower (P<0.05) than the other treatment groups (Tables 1, 2, and 3).

 

Results found that the decreasing KUB chicken sperm motility correlates with time at the semen storage. The reaction between the spermatozoa and diluents in cooling storage would be affecting metabolic activity (Lukman et al., 2014). The cold storage was set at 3-5°C and a certain temperature, the motility would decrease as it was caused by the sperm’s utilized energy which derived from glyceryl-phosphorylcholine, fructose, and sorbitol in semen that can be gradually lowered (Iswati et al., 2018). Oxidative-stressed might occur in cold-stored leads to motility decrease and increase the dead sperm (Khan, 2011). Moreover, the level of vitamin C antioxidant in diluent would not affect the motility due to high levels of vitamin C may lead to inhibit the oxidation. However, it could not directly react to the free radicals in a membrane, as vitamin C works outside the cell (Castelini et al., 2000). According to Gecha and Fagan (1992), vitamin C’s protecting ability is at the beginning of fat peroxidation occurred. Furthermore, it does not have a significant effect in the long term of use.

 

Vitamin E and diluent reaction compared to the control group was significantly higher and affecting the sperm. This study found a similar finding that was conducted by Donoghue and Donoghue (1997), vitamin E addition to sperm diluent in turkey able to maintain the spermatozoa motility up to 75% for 48 hours through in vitro storage. Vitamin E can directly bind to free radicals such as peroxyl and alkoxyl so that free radicals will break the bond reaction and form tocopheroxyl radicals at a stable compound. Vitamin E also could interrupt the peroxyl and alkoxyl radicals during the conversion of lipid hydroperoxides that may lead to trigger the peroxidative chain reactions, it prevents damage through the membrane plasm (Bansal et al., 2009).

 

The vitamin E viability showed similar results of Donoghue and Donoghue (1997) on turkey which was stored for 24-48 hours and Cerolini et al. (2000) in pigs who demonstrated that adding α-tocopherol (Vitamin E) to semen extender helped in maintaining the sperm viability by in vitro storage for 48 hours. The longer the semen is being stored, then the higher possibility of spermatozoa mortality increase due to membrane plasm damage. It might disrupt the spermatozoa energy supply and reduce motility. The total amount of dead spermatozoa will affect the living spermatozoa during the storage process (Solihati et al., 2006). Adding Vitamin E to semen extenders tends to improve semen quality in chickens by increasing the viability (Sarica et al., 2007). Vitamin E directly reacts to cells providing a protective effect against free radicals that can maintain the spermatozoa, which is different from vitamin C reacts to the outside cells (Donoghue and Donoghue, 1997). The sperm membrane plasm contains high amounts of unsaturated fatty acids. Thus, it is susceptible to peroxidative damage which is shown by the subsequent loss of membrane integrity. A failure of sperm cell functions will decrease sperm motility (Tabatabaei et al., 2011).

 

Lipid peroxidation tends to continuously happen (autocatalyst) since the reaction releases different reactive oxygen species (ROS) which could lead to a new reaction of lipid peroxidation. Eventually, it affects the entire membrane plasm of sperm. Lipid peroxidation can alter the membrane function results in metabolism, morphology, motility, and sperm fertility reduction (Prihantoko et al., 2020b). Vitamin E in semen extender roles as an effective membrane-stabilized antioxidant, a natural component of male gametes, and protecting the sperm cell membranes from damage (Tabatabaei et al., 2011), furthermore, vitamin E inhibits oxidative damage through action binding against toxic free radicals, particularly the ROS (Uzochukwu et al., 2020). The result study of vitamin C and the combinations showed no significant effect to maintain the membrane integrity.

 

Table 4: Percentages of fresh sperm DNA fragmentation KUB chicken and stored at 4oC at 48 hours of observation

 

Treatment	Observation Time
	Fresh	T5 (%)
P0	0.875 ± 0.25	2.625 ± 0.478ns
P1	0.875 ± 0.25	2.125 ± 1.030ns
P2	0.875 ± 0.25	2.000 ± 0.816ns
P3	0.875 ± 0.25	2.375 ± 1.108ns

ns: different, not significant (P≥0,05)

 

Evaluation of DNA fragmentation in spermatozoa after 48 hours of storage at 4oC did not show significant DNA damage (Table 4). It is in accordance with the opinion of Gliozzi et al. (2011) who stated that spermatozoa DNA, both the proportion of cell DNA and the proportion of tail DNA did not change after the equilibration time at temperature 4°C, and there was a negative correlation between damaged sperm DNA and sperm motility and membrane integrity. It indicates that the decrease in the value of mortality and integrity of the spermatozoa membrane along with the length of storage time will not affect the DNA damage of the spermatozoa. The main factors involved in the etiology of DNA damage in spermatozoa include oxidative stress, deficiency in natural processes such as chromatin packaging, and failure of apoptosis (Sharma et al., 2004). Oxidative stress provides exposure to DNA which causes more than 20 types of damage to DNA bases resulting in oxidized and fragmented nitrogen bases (Slupphaug et al., 2003).

 

CONCLUSION

 

This study has shown that the level of vitamin E supplement 5 µg/ml to the semen extender would increase the motility, viability, and integrity to the membrane up to 48 hours at 4°C stored. Additions of vitamin C did not affect any sperm quality parameters and the combination of vitamin C and vitamin E had negatives effect on the quality of spermatozoa in KUB chicken at the storage.

 

ACKNOWLEDGEMENTs

 

The author would thank the staff at the Department of Reproduction and Obstetrics, Faculty of Veterinary Medicine, Gadjah Mada University for their cooperation and technical assistance during the research. This study was granted by the Directorate General of Higher Education, Ministry of Education and Culture, Indonesia and Gadjah Mada University.

 

CONFLICT OF INTEREST

 

The authors declare that there are no conflicts of interest in the study publication include the funding and data research.

 

AUTHOR CONTRIBUTION

 

All authors participate from the beginning of this study until the publications are released.

 

REFERENCES

 

• Akhlaghi A, Ahangari YJ, Zhandi M, Peebles E (2014). Reproductive performance, semen quality, and fatty acid profile of spermatozoa in senescent broiler breeder roosters as enhanced by the long-term feeding of dried apple pomace. Anim. Reprod. Sci. 147: 64-73. https://doi.org/10.1016/j.anireprosci.2014.03.006

• Amini MR, Kohram H, Shahaneh AZ, Zhandi M, Sharideh H, Nabi MM (2015a). The effects of different levels of vitamin E and vitamin C in modified Beltsville extender on rooster post-thawed sperm quality. Cell Tissue Bank. 16: 587-592. https://doi.org/10.1007/s10561-015-9506-9

• Amini MR, Kohram H, Zare-Shahaneh A, Zhandi M, Sharideh H, Nabi MM (2015b). The effects of different levels of catalase and superoxide dismutase in modified Beltsville extender on rooster post-thawed sperm quality. Cryobiology. 70: 226-232. https://doi.org/10.1016/j.cryobiol.2015.03.001

• Bansal, Amrit K, Gurmail SB (2009). Antioxidant effect of vitamin E on motility, viability and lipid peroxidation of cattle spermatozoa under oxidative stress. Animal Science Papers and Reports vol. 27 (2009) no. 1, 5-14 Institute of Genetics and Animal Breeding, Jastrzębiec, Poland.

• Bréque C, Surai P, Brillard JP (2003). Roles of antioxidants on prolonged storage of avian spermatozoa in vivo and in vitro. Mol. Reprod. Dev. 66, 314-323. https://doi.org/10.1002/mrd.10347

• Castelini C, P. Lattaioli, M. Bernardini, A. Dal B (2000). Effect of Dietary α-tocopheryl Acetate and Ascorbic Acid on Rabbit Semen Stored at 5oC. Theriogenology. 54 : 523 – 533 https://doi.org/10.1016/S0093-691X(00)00367-8

• Cerolini S, Maldjian A, Surai P, Noble R (2000). Viability susceptibility to peroxidation and fatty acid composition of boar semen during liquid storage. Anim. Reprod. Sci. 58: 99-111. https://doi.org/10.1016/S0378-4320(99)00035-4

• Cerolini S, Zaniboni L, Maldjian A, Gliozzi T (2006). Effect of docosahexaenoic acid and α- tocopherol enrichment in chicken sperm on semen quality, sperm lipid composition and susceptibility to peroxidation. Theriogenology. 66: 877-886. https://doi.org/10.1016/j.theriogenology.2006.02.022

• De Lamirande E, Jiang H, Zini A, Kodama H, Gagnon C (1997). Reactive oxygen species and sperm physiology. Rev. Reprod. 2: 48–54. https://doi.org/10.1530/ror.0.0020048

• Donoghue AM, Donoghue DJ (1997). Effects of water and lipid soluble antioxidants on turkey sperm viability, membrane integrity, and motility during liquid storage. Poult. Sci. 76:1440–1445. https://doi.org/10.1093/ps/76.10.1440

• Erwan E (2020). Effects of substitution of corn for commercial ration on performance and plasma cholesterol in KUB chicken. Online J. Anim. Feed Res., 10(4): 185-190. https://doi.org/10.51227/ojafr.2020.26

• Gecha OM, JM Fagan (1992). Protective Effectof Ascorbic Acid on Breakdown of Proteins Exposed to Hydrogen Peroxide in Chicken Skeletal Muscle. J. Nutr. 122 : 2087 – 2093. https://doi.org/10.1093/jn/122.11.2087

• Gliozzi TM, L Zaniboni, S Cerolini (2011). DNA fragmentation in chicken spermatozoa during cryopreservation. Theriogenology. 75 1613–1622. https://doi.org/10.1016/j.theriogenology.2011.01.001

• Hu J-H, Tian W-Q, Zhao X-L, Zan L-S, Wang H, Li Q-W, XinY-P (2010). The cryoprotective effects of ascorbic acid supplementation on bovine semen quality. Anim. Reprod. Sci. 121: 72–77. https://doi.org/10.1016/j.anireprosci.2010.04.180

• Iswati Isnaini N, Susilawati T (2018). Effect of addition of glutathione in diluent ringer’s on spermatozoa quality of domestic chicken during cold storage Asian Jr. Microbiol. Biotech. Env. Sc. 20 (1) 12-20.

• Jeong YJ, Kim MK, Song HJ, Kang EJ, Ock SA, Kumar M., Balasubramanian S, Rho GJ (2009). Effect of alphatocopherol supplementation during boar semen cryopreservation on sperm characteristics and expression of apoptosis related genes. Cryobiology. 58: 181–189. https://doi.org/10.1016/j.cryobiol.2008.12.004

• Khan RU (2011). Antioxidant and Poultry Semen Quality. World’s Poult. Sci. J. 67 : 298-308. https://doi.org/10.1017/S0043933911000316

• Lukman HY, Busono W, Wahyuningsih S, Suyadi (2014). Sperm Motility and Viability after aTocopherol Dilution in Tris Aminomethane-Base Extender During Cold Storage in Bali Bull. International J. Chem. Tech. Res. 6 (14) : 5726-5732.

• Moghbeli M, Kohram H, Zare-Shahaneh A, Zhandi M, Sharafi M, Nabi MM, Zahedi V, Sharideh H (2016). Are the optimum levels of the catalase and vitamin E in rooster semen extender after freezing-thawing influenced by sperm concentration? Cryobiology. 72 (2016): 264e268. https://doi.org/10.1016/j.cryobiol.2016.03.008

• Padhi MK (2016). Importance of indigenous breeds of chicken for rural economy and their improvements for higher production performance. Scientifica. 2016:1-9. DOI: http://dx.doi.org/10.1155/2016/2604685

• Partyka A, Łukaszewicz E, Niżański W (2012). Lipid peroxidation and antioxidant enzymes activity in avian semen. Anim. Reprod. Sci. 134: 184-190. https://doi.org/10.1016/j.anireprosci.2012.07.007

• P Peruma, J K Chamuah, C Rajkhowa (2013). Effect of catalase on the liquid storage of mithun (Bos frontalis) semen. Asian Pacific J. Reprod. 2(3): 209-214. https://doi.org/10.1016/S2305-0500(13)60148-7

• Prihantoko K D, F Yuliastuti, H Haniarti, A Kusumawati, D T Widayati, A Budiyanto (2020a) The Effect of Genistein on the Plasma Membrane Integrity of Frozen Ongole Grade Bull Semen Based on Skim Milk – Soy Lecithin Extender. IOP Conf. Ser.: Earth Environ. Sci. 465 012054. https://doi.org/10.1088/1755-1315/465/1/01205

• Prihantoko K D, F Yuliastuti, H Haniarti, A Kusumawati, D T Widayati, A Budiyanto (2020b). The Acrosome Integrity Examination of Post-thawed Spermatozoa of Several Ongole Grade Bull in Indonesia Using Giemsa Staining Method. IOP Conf. Ser.: Earth Environ. Sci. 478 012042. https://doi.org/10.1088/1755-1315/478/1/012042

• Santiago Moreno J, Castaño C, Coloma M, Gómez Brunet A, Toledano Díaz A, López Sebastián A, Campo J (2009). Use of the hypo-osmotic swelling test and aniline blue staining to improve the evaluation of seasonal sperm variation in native Spanish free-range poultry. Poult. Sci. 88: 2661-2669. https://doi.org/10.3382/ps.2008-00542

• Sarica S, Corduk M, Suicmez M, Cedden F, Yildirim M, Kilinc K (2007). The effects of dietary L-carnitine supplementation on semen traits, reproductive parameters, and testicular histology of Japanese quail breeders. J. Appl. Poult. Res. 16:178–186. https://doi.org/10.1093/japr/16.2.178

• Sharma RK, Said T, Agarwal A (2004). Sperm DNA damage and its clinical relevance in assessing reproductive outcome. Asian J. Androl. 2004;6:139–48.

• Slupphaug G, Kavli B, Krokan HE (2003). The interacting pathways for prevention and repair of oxidative DNA damage. Mutat ResFund Mol. Mech. Mutag. 2003;531:231–51. https://doi.org/10.1016/j.mrfmmm.2003.06.002

• Solihati N, Pinnezak A, Idi R, Setiawan R, Asmara IY, Sujana BI (2006). The Storage Time Effect of The Local Chicken Chilled Semen at 5°C on Fertility and Fertile Period of Sperm. Sperma. J. Ilmu Ternak. 6 (1) : 7-11. https://doi.org/10.24198/jit.v6i1.2258

• Tabatabaei Shiva, B, Rooz A, Ayen, Esmail (2011). Effects of Vitamin E Addition to Chicken Semen on Sperm Quality During in Vitro Storage of Semen. Vet. Res. Forum. 2: 103-111.

• Tabatabaei S (2012). Effect of ascorbic acid on chicken semen quality during liquid storage. Comp. Clin. Pathol. 21:621–626. https://doi.org/10.1007/s00580-010-1145-8

• Uzochukwu IE, Amaefule BC, Ugwu SOC (2020). Effect Of Dietary Supplementation Of Vitamins C And E On The Semen Quality Of Local Turkeys. Journal of Tropical Agriculture, Food, Environ. Extension. 18:(3) (January 2020) pp. 25 - 30. https://doi.org/10.4314/as.v19i1.4

• Wen Chaoliang, Chunning Mai, Bin Wang, Junying Li, Congjiao Sun, Ning Yang (2020). Detrimental effects of excessive fatty acid secretion on female sperm storage in chickens. J. Anim. Sci. Biotechnol. 11: 26 (2020). https://doi.org/10.1186/s40104-020-0432-8