Research Article

Practical Use of Amino Acids in Diluent to Sustain Native Chicken Sperm Quality During 5 °C Storage

Khaeruddin1*, Hermawansyah1, Bahri Syamsuryadi1, Andi Kurnia Armayanti1, Abdul Hakim Fattah1, Azmi Mangalisu1, Muhammad Erik Kurniawan1, Dian Yustisia2, Ridha Alamsyah3, Pardi 4

1Program of Animal Science, Faculty of Agriculture, Universitas Muhammadiyah Sinjai, Jl. Teuku Umar No. 8, Sinjai 92611, South Sulawesi, Indonesia; 2Aquatic Resources Management, Faculty of Agriculture, Universitas Muhammadiyah Sinjai, Jl. Teuku Umar No. 8, Sinjai 92611, South Sulawesi, Indonesia; 3Agrotechnology, Faculty of Agriculture, Universitas Muhammadiyah Sinjai, Jl. Teuku Umar No. 8, Sinjai 92611, South Sulawesi, Indonesia; 4Undergraduate students in the Program of Animal Science, Faculty of Agriculture, Universitas Muhammadiyah Sinjai, Jl. Teuku Umar No. 8, Sinjai 92611, South Sulawesi, Indonesia.

Received | December 13, 2025; Accepted | February 05, 2026; Published | April 30, 2026

*Correspondence | Khaeruddin, Program of Animal Science, Faculty of Agriculture, Universitas Muhammadiyah Sinjai, Jl. Teuku Umar No. 8, Sinjai 92611, South Sulawesi, Indonesia; Email: [email protected]

Citation | Khaeruddin, Hermawansyah, Syamsuryadi B, Armayanti AK, Fattah AH, Mangalisu A, Kurniawan ME, Yustisia D, Alamsyah R, Pardi (2026). Practical use of amino acids in diluent to sustain native chicken sperm quality during 5 °C storage. J. Anim. Health Prod. 14(2): 670-677.

DOI | https://dx.doi.org/10.17582/journal.jahp/2026/14.2.670.677

ISSN (Online) | 2308-2801

Copyright: 2026 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Indonesia possesses a high genetic diversity of native chickens; however, their productivity remains relatively low, resulting in continued dependence on imported broiler and layer chickens to meet national demand for meat and eggs. Improving the productivity of native chickens requires advances in nutrition, management practices, and breeding programs (Hidayat and Asmarasari, 2015). Selective breeding is a key strategy to enhance growth performance, survival, fertility, and hatchability, and its effectiveness is strengthened by artificial insemination, which enables directed and efficient mating (Sapkota et al., 2020). Artificial insemination in poultry offers several advantages, including increasing the male-to-female mating ratio, allowing the use of older or injured roosters, preventing preferential mating, and supporting crossbreeding programs (Kharayat et al., 2016). In practical application, cold semen storage is essential for extending the usable duration of ejaculates. Storage at 5°C has been shown to better preserve mitochondrial activity and extend semen shelf life compared to moderate temperatures (Blank et al., 2021). Nonetheless, chicken spermatozoa are highly susceptible to cold-induced damage.

This susceptibility is closely associated with the high content of polyunsaturated fatty acids (PUFAs) in the plasma membrane, which increases vulnerability to oxidative stress and lipid peroxidation (Authaida et al., 2023; Barbarestani et al., 2024). Such oxidative damage compromises membrane integrity, disrupts the acrosome reaction, and ultimately reduces fertilizing capacity. A progressive decline in motility during 24-hour storage at 5°C has also been reported in native chicken semen (Khaeruddin et al., 2024a, c). Therefore, semen diluents require protective components, such as amino acids, which possess antioxidant properties and can reduce intracellular reactive oxygen species (Sangeeta et al., 2015; Davoodian et al., 2017; Koohestanidehaghi et al., 2021).

Amino acids including glutamine, alanine, serine, valine, and glycine play key roles in redox regulation and oxidative detoxification (Ugur et al., 2020) and are present in high concentrations in the seminal plasma of chickens (Santiago-Moreno et al., 2019). Previous studies have demonstrated that amino acids can preserve semen quality in mammals (Tan et al., 2024; Said et al., 2019) and poultry, including the effectiveness of glutamine in cryopreserved rooster semen (Khiabani et al., 2017) and glycine in chilled local chicken semen stored at 5°C for up to 120 hours (Junaedi et al., 2024). In human sperm, histidine consistently supports sperm motility over a long period (Hungerford et al., 2025).

To date, however, no study has directly compared the effects of glycine, glutamine, and histidine in semen diluents for native chickens stored at 5 °C. The novelty of this study lies in the simultaneous evaluation of these three amino acids at different concentrations to determine their distinct effects on motility, viability, membrane integrity, acrosome integrity, DNA stability, and the overall shelf life of chilled semen.

Materials and Methods

Chicken maintenance and semen collection

Six mature Indonesian native roosters aged approximately 10 months were used in this study. The birds were individually housed in cages measuring 50 × 50 × 65 cm and were fed a complete ration containing 20% crude protein at 100 g/bird/day, with drinking water provided ad libitum. Semen collection was performed using the massage method as described by Kucera and Heidinger (2018), which involves massaging the tail region until the tail elevates and the papilla protrudes, followed by ejaculation. The semen was collected into a microtube using a small funnel. Ejaculate from six roosters was pooled in one tube and then divided into 10 tubes based on treatment.

Fresh semen evaluation

Fresh semen was evaluated macroscopically for volume, color, pH, and consistency. The pH was measured using a universal pH indicator strip (MQuant, Merck KGaA, Germany). Microscopic evaluation included sperm concentration, mass movement, motility, viability, and abnormalities. Sperm concentration was determined by diluting semen in 3% NaCl at a 1:500 ratio, placing the diluted sample on a Neubauer hemocytometer (Assistant, Germany), and observing it under a light microscope (Olympus CX33, Japan) at 100× magnification. Sperm abnormalities were assessed using eosin–nigrosin staining and examined at 400× magnification.

Diluent preparation

The diluent was prepared by mixing 90% Ringer’s lactate solution (Widatra Bakti, Indonesia) with 10% egg yolk, followed by centrifugation at 2000 rpm for 30 minutes (Khaeruddin et al., 2024b). The supernatant was collected as the base diluent. Penicillin (1000 IU; Meiji, Indonesia) and streptomycin (1 mg/mL; Meiji, Indonesia) were added, and the pH was adjusted to match semen pH using tris-hydroxymethyl aminomethane. The diluent was divided into 10 tubes, and amino acids were added according to treatment. The treatments consisted of no amino acid (control) and the addition of glycine, glutamine, or histidine (Merck, Darmstadt, Germany) at concentrations of 20, 40, or 60 mM.

Dilution and storage

Semen meeting the quality requirement (motility > 70%) was divided into 10 tubes and diluted at a ratio of 1:5. The diluted semen was then stored at 5°C until all spermatozoa were dead (for longevity evaluation). Semen quality parameters were evaluated after 24 and 48 hours of storage.

Liquid semen evaluation

Liquid semen quality was assessed based on motility, viability, plasma membrane integrity, acrosome integrity, DNA damage, and longevity. Sperm motility was evaluated subjectively under a light microscope at 400× magnification. Viability was assessed using eosin–nigrosin staining (Agarwal et al., 2016) on at least 200 spermatozoa.

Plasma membrane integrity was evaluated using the hypoosmotic swelling test (HOST). In this test, 10 μL of semen was mixed with 100 μl of HOST solution (0.9 g fructose and 0.49 g sodium citrate dissolved in 100 mL distilled water), incubated at 37°C for 30 minutes, and smeared on a slide using eosin–nigrosin. A minimum of 200 spermatozoa were observed at 400× magnification, with membrane-intact sperm identified by tail bending (Najafi et al., 2019; Khaeruddin et al., 2024c).

Acrosome integrity was examined using Coomassie Brilliant Blue (CBB) staining. Liquid semen was smeared on a slide, fixed in 5% formalin, air-dried, incubated at 37°C for 30 minutes, washed, air-dried, stained in 0.25% CBB (R250; BBI Life Sciences, Canada) dissolved in 10% glacial acetic acid and 25% methanol for 5 minutes, rinsed, and observed under a microscope at 1000× magnification with oil immersion (Silyukova et al., 2022; Khaeruddin et al., 2024c).

DNA damage was assessed using Toluidine Blue staining following Rui et al. (2017). Semen smears were air-dried, fixed in 96% ethanol–acetone (1:1) at 4°C for 30 minutes, air-dried for 30 minutes, hydrolyzed in 0.1 N HCl for 5 minutes at 4°C, rinsed three times with running water, air-dried, stained with Toluidine Blue for 20 minutes, rinsed, and dried. At least 200 spermatozoa were evaluated at 400× magnification; deep blue coloration indicated DNA damage, while light blue indicated intact DNA. Sperm longevity was recorded from the first day of storage until all spermatozoa lost viability.

Data analysis

The experiment used a completely randomized design (CRD) consisting of 10 treatments (types and concentrations of amino acids) with five replications (five separate semen collection days). Data were analyzed using Analysis of Variance (ANOVA) in SPSS version 25, followed by Duncan’s Multiple Range Test when significant differences were detected.

RESULTS

Characteristics of fresh semen

Table 1 shows that the characteristics of fresh semen from native chickens fall within normal physiological ranges. The ejaculate volume (0.22 ± 0.04 mL), slightly alkaline pH (7.85 ± 0.22), milky appearance, and thick consistency indicate good semen quality. The high sperm concentration (5.58 ± 0.72 × 10⁹/mL) and strong mass movement (++/+++) further reflect vigorous sperm activity, confirming that the samples were suitable for dilution and subsequent storage.

 

Table 1: Characteristics of fresh semen from native chickens.

Variable	Mean ± SEM
Volume (mL)	0.22±0.04
pH	7.85±0.22
Color	Milky
Consistency	Thick
Sperm concentration (109/mL)	5.58±0.72
Mass movement	++/+++
Motility (%)	80.00±2.89
Viability (%)	98.07±0.25
Abnormality (%)	3.65±0.36

 

Microscopic evaluations also demonstrated excellent semen quality, as indicated by high motility (80.00 ± 2.89%), very high viability (98.07 ± 0.25%), and a low abnormality rate (3.65 ± 0.36%). Overall, these values confirm that the initial semen used in this study was of optimal quality, ensuring that any changes observed during cold storage can be attributed primarily to the effects of the amino acid treatments rather than to poor initial semen condition.

Motility

Figure 1 shows that glycine and glutamine effectively maintained sperm motility during 24- and 48-hour storage at 5°C. At 24 hours, glycine at 40 mM and all glutamine concentrations produced the highest motility values, significantly outperforming the control. In contrast, histidine at 60 mM caused a marked decline in motility, indicating a detrimental effect at high concentrations.

 

After 48 hours, motility decreased across all treatments due to extended storage, yet glycine at 40 mM continued to provide the strongest protective effect. Glutamine also maintained higher motility than the control, whereas histidine at 60 mM again resulted in the lowest motility. Overall, glycine particularly at 40 mM was the most effective additive, while high-dose histidine negatively affected sperm performance.

Viability

Figure 2 shows that both glycine and glutamine help protect rooster sperm from the stress of cold storage, allowing the cells to remain highly viable even after 24 and 48 hours at 5°C. Their presence in the diluent appears to support the sperm’s resilience, keeping survival rates consistently high and comparable to fresh conditions. In contrast, histidine especially at 40 and 60 mM seems to overwhelm the cells, sharply reducing their ability to survive the cold. This contrast highlights how some amino acids nurture and preserve sperm vitality, while others, when given in excess, may instead compromise their stability.

 

Membrane integrity

Figure 3 shows that the plasma membrane integrity of rooster sperm remained consistently high after 24 and 48 hours of storage at 5°C. Most treatments including the control as well as glycine and glutamine at various concentrations maintained similar levels of membrane stability, indicating that the sperm cells were able to preserve their structural integrity during cold storage. Although histidine at higher concentrations produced a slight reduction in membrane integrity, the values were still within an acceptable range. Overall, these results highlight that the diluent, whether supplemented with amino acids or not, was effective in maintaining the fundamental integrity of sperm membranes throughout the storage period.

Acrosome intact

Figure 4 shows that sperm acrosome integrity remained well preserved during cold storage at 5°C, both after 24 and 48 hours, regardless of amino acid supplementation. All treatments, including the control, glycine, glutamine, and histidine at different concentrations, maintained similarly high percentages of intact acrosomes, with only a slight decline after 48 hours. These findings indicate that the acrosome is relatively resistant to cold-induced stress and that the addition of amino acids does not markedly influence acrosomal stability during short-term storage.

 

 

DNA damage

Figure 5 shows that sperm DNA damage remained relatively low after 24 hours of storage and increased moderately after 48 hours at 5°C. At 24 hours, supplementation with glycine and glutamine tended to result in lower DNA damage compared with the control, indicating a protective effect on sperm genetic material. In contrast, histidine particularly at higher concentrations—showed a tendency toward increased DNA damage. After 48 hours of storage, DNA damage increased across all treatments as a consequence of prolonged cold exposure; however, glycine and glutamine continued to maintain lower levels of DNA damage than histidine. These results indicate that glycine and glutamine are more effective in preserving sperm DNA integrity during cold storage, whereas high concentrations of histidine are less favorable for maintaining genetic stability.

 

Longevity

Figure 6 shows that glycine consistently maintained sperm longevity during storage at 5°C, with survival times comparable to or slightly longer than the control across all tested concentrations. Glutamine provided moderate support, allowing sperm to remain viable for a shorter period, while histidine markedly reduced sperm longevity, particularly at 40 and 60 mM. These findings highlight glycine as the most favorable amino acid for sustaining sperm survival during cold storage, whereas high concentrations of histidine compromise the ability of sperm cells to survive over time.

 

DISCUSSION

This study aims to investigate the effect of adding glycine, glutamine, and histidine at multiple concentrations (20 mM, 40 mM, and 60 mM), to diluents on motility, viability, plasma membrane integrity, intact acrosome, DNA damage, and longevity of native Indonesian chicken sperm stored for 24 hours and 48 hours at 5°C. In this study, glycine did not show any difference in sperm quality compared to glutamine. These results correspond with the study on Sumba-Ongole cattle by Said et al. (2019), who reported that at the same concentration, glycine and glutamine did not produce any difference in sperm motility after cooling.

The addition of 40-60 mM histidine had no beneficial effect in this study, leading to lower sperm motility and viability. The result is consistent with the report by Matás et al. (2008), where the addition of L-histidine to diluent caused a significant decrease in boar sperm motility after 120 hours of storage. Furthermore, histidine was shown to have a negative effect on viability in goldfish sperm (Lahnsteiner, 2009).

The mechanism by which histidine caused the decrease in motility remains unclear, as no previous reports have described its negative effects on sperm. However, this may explain the effects in other cell types, such as in bacterial cells, where the addition of L-histidine increases the formation of intracellular hydroxyl radicals in Escherichia coli (Nagao et al., 2018). Based on observation, excessive hydroxyl radicals can cause cell damage. Rauen et al. (2007) reported that hepatocyte cell culture media containing high concentrations of L-histidine caused hepatocyte cell inactivation in 3 hours with the occurrence of lipid peroxidation. In mammalian cells such as Chinese hamster ovary, L-histidine significantly increases H₂O₂ toxicity, causing more DNA double-strand breaks and severe mitochondrial damage. This implies that excess histidine worsens the effects of free radicals already present (Guidaerlli, 1995).

Based on observation, sperm quality declined during 48 hours of storage. Blank et al. (2021) reported reduced motility associated with decreased mitochondrial activity. Prolonged storage increased acrosome reaction values through elevated amidase activity and raised ROS levels, leading to lipid peroxidation (Ratchamak et al., 2025), oxidative stress, mitochondrial membrane potential loss, impaired ATP production, and reduced motility (Słowińska et al., 2018). In this study, glycine treatment led to motility of 42% after 24 hours and 30% after 48 hours, lower than values reported by Junaedi et al. (2024) for KUB chicken sperm (67.5% and 60% with egg yolk lactate Ringer diluent containing 60 mM glycine). Motility at 24 hours was also below reports for Thai native chicken sperm (81.96% with IGGKPh containing serine and 65.1% with BPSE containing vitamin B12) and at 48 hours below the 61.9% reported by Kheawkanha et al. (2023) and Suwimonteerabutr et al. (2024), respectively. Viability after 24 hours with glycine and glutamine treatments approached the 85.98% reported by Kheawkanha et al. (2023) in Thai native chicken sperm with IGGKPh containing serine. It was higher than values for Lohman Brown sperm (76.59% with EM diluent) (Tvrdá et al., 2023), Thai native sperm (70.1% with BPSE) (Suwimonteerabutr et al., 2024), and Ross 308 sperm (60.5% with Lake diluent) (Alipour-Jenaghard et al., 2023). After 48 hours, viability with glycine and glutamine treatments remained higher than previous reports, at 67.9% (Suwimonteerabutr et al., 2024) and 30 (Alipour-Jenaghard et al., 2023).

Plasma membrane integrity in this research was higher than previously reported, with 61.5% after 24 hours of storage and 31.5% after 48 hours in Ross 308 Breeder chicken sperm using Lake diluent (Alipour-Jenaghard et al., 2023). In comparison, Suwimonteerabutr et al. (2024) reported 62.9% after 24 hours and 59.9% after 48 hours in Thai native chicken sperm using BPSE diluent. The proportion of intact acrosomes at 24 hours of storage was similar to the results of Tvrdá et al. (2023), who reported 81.91% in Lohman Brown chicken sperm using EM diluent, but lower than the values reported by Suwimonteerabutr et al. (2024), which were 96.8% at 24 hours and 95.8% at 48 hours in Thai native chicken sperm using BPSE diluent. The percentage of sperm with intact DNA in this research was also considerably higher than that reported by Tvrdá et al. (2023) in Lohman Brown chicken sperm using EM diluent.

CONCLUSION

In conclusion, the addition of various types and concentrations of amino acids affects the motility, viability, and longevity of native Indonesian chicken sperm during storage at 5°C. The use of glycine and glutamine at all concentrations (20, 40, and 60 mM), as well as 20 mM histidine in the diluent did not show differences in sperm motility and viability compared to without amino acids. Meanwhile, approximately 40-60 mM histidine decreased sperm motility, viability, and longevity.

ACKNOWLEDGEMENT

This study received financial support from the Institute for Research and Community Service, Universitas Muhammadiyah Sinjai.

NOVELTY STATEMENT

This study was the first to use a diluent containing glutamine and histidine in liquid-stored chicken semen. This study examined the effect of adding amino acids to the diluent on the longevity of chicken sperm stored at 5°C. The results showed that 40-60 mM histidine reduced chicken sperm quality during storage.

AUTHOR’s CONTRIBUTION

KK: Research design, conducting research, data analysis, writing the manuscript. HH: Literature search, conducting research. BS AHF and AK: Methodology and supervision AM, MEK, DY and RA: Editing and revising the manuscript. P: Assisting in conducting research.

Generative AI and AI-assisted technology statement

The authors declare that no generative AI or AI-assisted technologies were used in the preparation of this manuscript.

Conflict of interest

The authors have declared no conflict of interest.

REFERENCES

Agarwal A, Gupta S, Sharma, R (2016). Eosin-nigrosin staining procedure. In: Agarwal A, Gupta S, Sharma R, editors. Andrological evaluation of male infertility: A laboratory guide. Switzerland: Springer; pp. 73-77. https://doi.org/10.1007/978-3-319-26797-5_8

Alipour-Jenaghard P, Daghigh-Kia H, Masoudi R (2023). Preservation of the quality and fertility potential of post-thawed rooster sperm using MitoQ. Theriogenology, 208: 165-170. https://doi.org/10.1016/j.theriogenology.2023.06.014

Authaida S, Ratchamak R, Boonkum W, Chankitisakul V (2023). Increasing sperm production and improving cryosurvival of semen in aged Thai native roosters as affected by selenium supplementation. Anim. Biosci., 36(11): 1647. https://doi.org/10.5713/ab.23.0079

Barbarestani SY, Samadi F, Zaghari M, Khademian S, Pirsaraei ZA, Kastelic JP (2024). A review of antioxidant strategies to improve reproduction in aging male broiler breeders. GeroScience, 47(1): 573-589. https://doi.org/10.1007/s11357-024-01363-1

Blank MH, Ruivo LP, Novaes GA, Lemos EC, Losano JD, Siqueira AF, Pereira RJ (2021). Assessing different liquid-storage temperatures for rooster spermatozoa. Anim. Reprod. Sci., 233: 106845. https://doi.org/10.1016/j.anireprosci.2021.106845

Davoodian N, Kadivar A, Ahmadi E, Mohebbi A (2017). Effects of two amino acids on motion parameters and enzymatic antioxidant activity of freeze-thawed stallion spermatozoa. J. Equine Vet. Sci., 59: 49–56. https://doi.org/10.1016/j.jevs.2017.09.007

Guidarelli A, Sestili P, Cossarizza A, Franceschi C, Cattabeni F, Cantoni O (1995). Evidence for dissimilar mechanisms of enhancement of inorganic and organic hydroperoxide cytotoxicity by L-histidine. J. Pharmacol. Exp. Ther., 275(3): 1575-1582. https://doi.org/10.1016/S0022-3565(25)12220-9

Hidayat C, Asmarasari SA (2015). Native chicken production in Indonesia: A review. Jurnal Peternakan Indonesia, 17(1): 1-11. https://doi.org/10.25077/jpi.17.1.1-11.2015

Hungerford AJ, Harrison N, Bakos HW, Aitken, RJ (2025). Development of an improved medium for the preservation of human spermatozoa. J. Assist. Reprod. Genet., 42: 2167-2180. https://doi.org/10.1007/s10815-025-03525-2

Junaedi J, Isnaini N, Natsir MH, Susilawati T (2024). Supplementation of glycine and glucose into egg yolk lactated ringer diluent on the quality of local chicken semen stored at 5 oC for 120 hours. J. Med. Vet., 7(1): 105-122. https://doi.org/10.20473/jmv.vol7.iss1.2024.105-122

Khaeruddin K, Ciptadi G, Yusuf M, Fattah AH, Syamsuryadi B, Wahjuningsih S (2024a). The quality of Gaga roosters semen during cold storage using a diluent supplemented with sorbitol. Trop. Anim. Sci. J., 47(4): 436-447. https://doi.org/10.5398/tasj.2024.47.4.436

Khaeruddin K, Ciptadi G, Yusuf M, Hermawansyah H, Sahiruddin S, Wahjuningsih S (2024b). Effect of addition of glucose or fructose in three types of crystalloid fluid on spermatozoa quality of Gaga’ chicken from South Sulawesi, Indonesia. Pak. J. Zool., 56(5): 2441-2448. https://doi.org/10.17582/journal.pjz/20211128011130

Khaeruddin K, Ciptadi G, Yusuf M, Udrayana SB, Iswati I, Wahjuningsih S (2024c). Effectiveness of butylated hydroxytoluene in maintaining the quality of Gaga chicken sperm in liquid storage for 72 hours. Adv. Anim. Vet. Sci., 12(2): 371-380. https://doi.org/10.17582/journal.aavs/2024/12.2.371.380

Kharayat NS, Chaudhary GR, Katiyar R, Balmurugan B, Patel M, Uniyal S, Raza M, Mishra GK (2016). Significance of artificial insemination in poultry. Res. Rev. J. Vet. Sci. Technol., 5(1): 1-5.

Kheawkanha T, Chankitisakul V, Thananurak P, Pimprasert M, Boonkum W, Vongpralub T (2023). Solid storage supplemented with serine of rooster semen enhances higher sperm quality and fertility potential during storage at 5 °C for up to 120 h. Poult. Sci., 102(6): 102648. https://doi.org/10.1016/j.psj.2023.102648

Khiabani AB, Moghaddam G, Kia HD (2017). Effects of adding different levels of glutamine to modified Beltsville extender on the survival of frozen rooster semen. Anim. Reprod. Sci., 184: 172-177. https://doi.org/10.1016/j.anireprosci.2017.07.013

Koohestanidehaghi Y, Torkamanpari M, Shirmohamadi Z, Lorian K, Vatankhah M (2021). The effect of cysteine and glutamine on human sperm functional parameters during vitrification. Andrologia, 53(1): e13870. https://doi.org/10.1111/and.13870

Kucera AC, Heidinger BJ (2018). Avian semen collection by cloacal massage and isolation of DNA from sperm. J. Vis. Exp., 132: e55324. https://doi.org/10.3791/55324-v

Lahnsteiner F (2009). The role of free amino acids in semen of rainbow trout Oncorhynchus mykiss and carp Cyprinus carpio. J. Fish. Biol., 75(4): 816-833. https://doi.org/10.1111/j.1095-8649.2009.02317.x

Masoudi R, Sharafi M, Pourazadi L (2019). Improvement of rooster semen quality using coenzyme Q10 during cooling storage in the Lake extender. Cryobiology, 88: 87-91. https://doi.org/10.1016/j.cryobiol.2019.03.003

Matás C, Gardon JC, Garcia-Vazquez FA, Pacchini S, Ducci M (2008). Does the supplementation of the boar semen extender with carnosine, L-histidine, and taurine preserve sperm function during storage? Reprod. Fertil. Dev., 20(1): 192. https://doi.org/10.1071/RDv20n1Ab225

Nagao T, Nakayama-Imaohji H, Elahi M, Tada A, Toyonaga E, Yamasaki H, Okazaki K, Miyoshi H, Tsuchiya K, Kuwahara T (2018). L-histidine augments the oxidative damage against Gram-negative bacteria by hydrogen peroxide. Int. J. Mol. Med., 41(5): 2847-2854. https://doi.org/10.3892/ijmm.2018.3473

Najafi A, Taheri RA, Mehdipour M, Martínez-Pastor F, Rouhollahi AA, Nourani MR (2019). Improvement of post-thawed sperm quality in broiler breeder roosters by ellagic acid-loaded liposomes. Poult. Sci., 98(1): 440-446. https://doi.org/10.3382/ps/pey353

Neamah HJ (2022). Investigation of amino acids effect to reduce oxidation stress and improve ram’s semen quality: A review study. Dijlah J. Agric. Sci., 1(1): 103-109.

Ratchamak R, Authaida S, Koedkanmark T, Saiyamanon H, Boonkum W, Chankitisakul V (2025). Optimization of rooster semen preservation: a comparative study of extender types and antioxidant supplementation strategies during cold storage. Poult. Sci., 104(6): 105137. https://doi.org/10.1016/j.psj.2025.105137

Rauen U, Klempt S, De Groot H (2007). Histidine-induced injury to cultured liver cells, effects of histidine derivatives and of iron chelators. Cell Mol. Life Sci., 64(2): 192-205. https://doi.org/10.1007/s00018-006-6456-1

Rui BR, Angrimani DS, Losano JDA, de Cássia Bicudo L, Nichi M, Pereira RJ (2017). Validation of simple and cost-effective stains to assess acrosomal status, DNA damage and mitochondrial activity in rooster spermatozoa. Anim. Reprod. Sci., 187: 133-140. https://doi.org/10.1016/j.anireprosci.2017.10.017

Said S, Setiorini S, Adella M, Sari I, Fathaniah N, Maulana T (2019). Effect of addition of selected amino acids in semen extender on quality and DNA stability of frozen-thawed Sumba Ongole bull spermatozoa. J. Ilmu Ternak dan Vet., 24(1): 15-21. https://doi.org/10.14334/jitv.v24i1.1873

Sangeeta S, Arangasamy A, Kulkarni S, Selvaraju S (2015). Role of amino acids as additives on sperm motility, plasma membrane integrity and lipid peroxidation levels at pre-freeze and post-thawed ram semen. Anim. Reprod. Sci., 161: 82-88. https://doi.org/10.1016/j.anireprosci.2015.08.008

Santiago-Moreno J, Bernal B, Perez-Cerezales S, Castano C, Toledano-Díaz A, Esteso MC, Gutiérrez-Adán A, López-Sebastián A, Gil MG, Woelders H, Blesbois E (2019). Seminal plasma amino acid profile in different breeds of chicken: Role of seminal plasma on sperm cryoresistance. PLoS One, 14(1): e0209910. https://doi.org/10.1371/journal.pone.0209910

Sapkota S, Kolakshyapati MR, Devkota NR, Bhattarai N, Gorkhali NA (2020). Selective breeding to improve productive and reproductive performances and survivability of indigenous Sakini chicken. J. Nepal Agric. Res. Counc., 6: 62-69. https://doi.org/10.3126/jnarc.v6i0.28116

Silyukova Y, Fedorova E, Stanishevskaya O (2022). Influence of technological stages of preparation of rooster semen for short-term and long-term storage on its quality characteristics. Curr. Issues Mol. Biol., 44(11): 5531-5542. https://doi.org/10.3390/cimb44110374

Słowińska M, Liszewska E, Judycka S, Konopka M, Ciereszko A (2018). Mitochondrial membrane potential and reactive oxygen species in liquid stored and cryopreserved turkey (Meleagris gallopavo) spermatozoa. Poult. Sci., 97(10): 3709-3717. https://doi.org/10.3382/ps/pey209

Suwimonteerabutr J, Yamsrikaew U, Damthongsen K, Suksirisamphan T, Leeniwa P, Lawanyakul P, Nuntapaitoon M (2024). Improving the quality of chilled semen from Thai native chicken using phosphorus and vitamin B12 supplementation in semen extender. Poult. Sci., 103(1): 103262. https://doi.org/10.1016/j.psj.2023.103262

Tan M, Zhao Y, Ren L, Li C, Cai J, He B (2024). Methionine improves boar sperm quality by promoting mitochondrial translation during liquid Storage. Animals, 14(15): 2227. https://doi.org/10.3390/ani14152227

Tvrdá E, Petrovičová M, Ďuračka M, Benko F, Slanina T, Galovičová L, Kačániová M (2023). Short-term storage of rooster ejaculates: Sperm quality and bacterial profile differences in selected commercial extenders. Antibiotics, 12(8): 1284. https://doi.org/10.3390/antibiotics12081284

Ugur MR, Dinh T, Hitit M, Kaya A, Topper E, Didion B, Memili E (2020). Amino acids of seminal plasma associated with freezability of bull sperm. Front. Cell Dev. Biol., 7: 347. https://doi.org/10.3389/fcell.2019.00347