Register      Login
Animal Production Science Animal Production Science Society
Food, fibre and pharmaceuticals from animals
REVIEW

Additives used as stimulative and cryoprotective agents in bovine semen cryopreservation: a systematic review

K. C. Moagi A , T. Chitura https://orcid.org/0009-0002-3818-6755 A * and A. M. Raseona A
+ Author Affiliations
- Author Affiliations

A University of Venda, Department of Animal Science, Private Bag X5050, Thohoyandou 0950, South Africa.

* Correspondence to: Teedzai.Chitura@univen.ac.za

Handling Editor: Joanna Souza-Fabjan

Animal Production Science 65, AN25104 https://doi.org/10.1071/AN25104
Submitted: 3 April 2025  Accepted: 19 July 2025  Published: 7 August 2025

© 2025 The Author(s) (or their employer(s)). Published by CSIRO Publishing

Abstract

Cryopreservation of bovine semen is critical for artificial insemination and genetic preservation, yet a significant remaining challenge is damage to sperm cells during the freezing and thawing process. Recent studies have led to the development of additives designed to shield sperm cells from the detrimental effects of cryopreservation. This systematic review evaluates the effectiveness of cryoprotective and stimulative additives in enhancing the quality of post-thaw bovine semen. A thorough literature search was conducted using Google Scholar, Science Direct, and PubMed databases to identify studies on stimulative and cryoprotective additives used in the cryopreservation of bull semen. The search was conducted using the key terms ‘stimulants bull semen cryopreservation’, ‘cryoprotective additives bull semen cryopreservation’, and ‘additives used in bovine semen cryopreservation’. A total of 57 studies were evaluated, identifying glutathione (2.5–4.0 mM), trehalose (30–100 mM), and melatonin (1–2 mM) as optimal additives for preserving sperm motility, membrane integrity, and reducing oxidative stress. Stimulants such as pentoxifylline (3.60–7.18 mM) improved motility by increasing cyclic adenosine monophosphate levels. Natural additives, including moringa extract and honey, showed similar effectiveness to synthetic additives, with cost and sustainability advantages. Additives such as glutathione, trehalose, and sericin have demonstrated significant effectiveness in preserving sperm membrane integrity and reducing oxidative stress. Critical gaps include dose-dependent toxicity of crocin and melatonin (> 3 mM). Combinatorial approaches such as glutathione + selenium nanoparticles are recommended to mitigate cryodamage. In conclusion, this review emphasizes optimizing additive selection and concentrations based on their specific roles and interactions. These findings guide the development of cost-effective extenders for improving cattle breeding programs globally.

Keywords: antioxidants, biotechnology, cryoprotectants, fertility, livestock, oxidative stress, sperm motility.

References

Abdel-Khalek AE, El-Nagar HA, Ibrahim OM (2016) Effect of leptin and melatonin as protective additives to tris extender on frozen semen quality of buffalo bulls. Journal of Animal and Poultry Production 7(7), 225-231.
| Crossref | Google Scholar |

Ahmed H, Jahan S, Khan A, Khan L, Khan BT, Ullah H, Riaz M, Ullah K (2020) Supplementation of green tea extract (GTE) in extender improves structural and functional characteristics, total antioxidant capacity and in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa. Theriogenology 145, 190-197.
| Crossref | Google Scholar |

Al-Badrany QM, Al-Badry KI, Zalzala SJ, Ibrahim FF, Lateef WY (2020) Effect of different concentration of trehalose or sucrose to tris diluents on some properties of sperms for holstein bulls during dilution, cooling and freezing. Plant Archives 20(1), 1953-1960.
| Google Scholar |

Alam K, Srivastava S, Singh B, Saurabh , Kumar R, Kumar R, Sakhahari DS (2024) Cryopreservation of bovine semen using extract of Cinnamomum zeylanicum. Cryoletters 45(3), 168-176.
| Crossref | Google Scholar |

Ali MM, Banana HJH (2020) Effect of adding n-acetylcystiene and avena sativa extract to tris extender on post-cryopreservative semen characteristics of holstein bulls. Plant Archives 20, 1209-1216.
| Google Scholar |

Ali JS, Hussain SO, Mohammed OA (2022) Comparative study between silymarin (Silybum Marianum), melatonin and a combination of them for improving frozen sperm properties of Holstein Bulls Born in Iraq. International Journal of Health Sciences 6(S3), 12300-12313.
| Crossref | Google Scholar |

Avdatek F, Yeni D, İnanç ME, Çil B, Tuncer BP, Türkmen R, Taşdemir U (2018) Supplementation of quercetin for advanced DNA integrity in bull semen cryopreservation. Andrologia 50(4), e12975.
| Crossref | Google Scholar |

Barakat IAH, Danfour MA, Galewan FAM, Dkhil MA (2015) Effect of various concentrations of caffeine, pentoxifylline, and kallikrein on hyperactivation of frozen bovine semen. BioMed Research International 2015, 948575.
| Crossref | Google Scholar |

Bhardwaz A, Pandey AK, Sharma U, Razzaque WAA, Gupta SK, Kumar S, Kumar S (2016) Effect of cysteine hydrochloride on post-thaw viability and acrosomal integrity of Jersey bull semen. Veterinary Practitioner 17(2), 227-228.
| Crossref | Google Scholar |

Bucak MN, Ataman MB, Başpınar N, Uysal O, Taşpınar M, Bilgili A, Öztürk C, Güngör Ş, Inanc ME, Akal E (2015) Lycopene and resveratrol improve post-thaw bull sperm parameters: sperm motility, mitochondrial activity and DNA integrity. Andrologia 47(5), 545-552.
| Crossref | Google Scholar |

ChaithraShree AR, Ingole SD, Dighe VD, Nagvekar AS, Bharucha SV, Dagli NR, Kekan PM, Kharde SD (2020) Effect of melatonin on bovine sperm characteristics and ultrastructure changes following cryopreservation. Veterinary Medicine and Science 6(2), 177-186.
| Crossref | Google Scholar | PubMed |

Chaudhary KF, Suthar BN, Nakhashi HC, Mohapatra SK (2022) Effect of curcumin as a semen additive on seminal attributes and biochemical parameters of Kankrej bull spermatozoa. Ruminant Science 11(1), 57-62.
| Google Scholar |

Correa F, Ceballos E, Rojano B, Restrepo G, Usuga A (2024) Quality and redox state of bovine sperm cryopreserved with resveratrol use of resveratrol in bovine semen. Reproduction in Domestic Animals 59, e14517.
| Crossref | Google Scholar |

Eidan SM (2016) Effect on post-cryopreserved semen characteristics of Holstein bulls of adding combinations of vitamin C and either catalase or reduced glutathione to Tris extender. Animal Reproduction Science 167, 1-7.
| Crossref | Google Scholar |

El-Sheshtawy RI, El-Nattat WS (2020) Effect of addition of Moringa oleifera extract to tris extender on the preservability of cattle bull semen. International Journal of Veterinary Science 9, 417-420.
| Crossref | Google Scholar |

Eliraqy EZ, Shafik BM, Hussein YS, Alsenosy AA, Abdallah EA, Saad MF (2022) Vital role of garlic extract additive in cryopreservation and freezing of buffalo bulls sperm. Advances in Animal and Veterinary Sciences 10(10), 2133-2141.
| Crossref | Google Scholar |

Elkhawagah AR, Martino NA, Ricci A, Storti V, Rumbolo F, Lange-Consiglio A, Vincenti L (2020) Effect of relaxin on cryopreserved beef bull semen characteristics. Cryobiology 95, 51-59.
| Crossref | Google Scholar |

Fouad WF, Ashour AF (2021) Selenium and zinc as supplements to extenders frozen semen for improving sperm characteristics during cryopreservation. Journal of Animal and Poultry Production 12(2), 71-77.
| Crossref | Google Scholar |

Geng H, Liu X, Shi G, Bai G, Ma J, Chen J, Wu Z, Song Y, Fang H, Wang J (2017) Graphene oxide restricts growth and recrystallization of ice crystals. Angewandte Chemie 129(4), 1017-1021.
| Crossref | Google Scholar |

Gupta S, Kumar A, Mahajan A, Sharma P, Sachan V, Aggrawal J, Yadav S, Saxena A, Kumar Swain D (2022) Curcumin in a tris-based semen extender improves cryosurvival of Hariana bull spermatozoa. Andrologia 54(1), e14255.
| Crossref | Google Scholar |

Hegazy MM, Ibrahim AF, Badawy MI, Awady HG (2020) Antioxidative effect of adding taurine and leptin to tris-extenders on freezing and fertilizing abilities of buffalo-semen. Journal of Animal and Poultry Production 11(7), 261-266.
| Crossref | Google Scholar |

Hussain M, Begum SS, Kalita MK, Ahmed KU, Nath R (2018) Additives used in semen preservation in animals: a short review. International Journal of Chemical Studies 6(5), 354-361.
| Google Scholar |

Hussaini SMH, Zhandi M, Shahneh AZ, Sharafi M, Nejati-Javaremi A, Yousefi A, Emamverdi M, Shehab-El-Deen MAMM, Pastor FM (2017) Effect of tert-butyl hydroquinone on bull semen cryopreservation. CryoLetters 38(5), 372-378.
| Google Scholar |

İnanç ME, Çil B, Yeni D, Avdatek F, Orakçı D, Tuncer PB, Türkmen R, Taşdemir U (2019) The effect of green tea extract supplementation in bull semen cryopreservation. Kafkas Universitesi Veteriner Fakultesi Dergisi 25, 703-708.
| Crossref | Google Scholar |

Inyawilert W, Rungruangsak J, Liao Y-J, Tang P-C, Paungsukpaibool V (2021) Melatonin supplementation improved cryopreserved Thai swamp buffalo semen. Reproduction in Domestic Animals 56(1), 83-88.
| Crossref | Google Scholar |

Kapadiya PS, Nakhashi HC, Sutaria TV, Rathod BS, Suthar BN (2017) Melatonin as an additive to shield the hazardous effects of cryopreservation on Kankrej bull semen. The Indian Journal of Animal Reproduction 38(1), 16-19.
| Google Scholar |

Khalil WA, Hassan MAE, El-Harairy MA, Abdelnour SA (2023) Supplementation of thymoquinone nanoparticles to semen extender boosts cryotolerance and fertilizing ability of buffalo bull spermatozoa. Animals 13(18), 2973.
| Crossref | Google Scholar |

Kinkar D, Sarkar D, Debbarma A, Rahman M, Swain SK, Karunakaran M (2020) Review on recent advancement in semen additives for improving cryopreservation of bull semen. Journal of Entomology and Zoology Studies 8, 1493-1505.
| Google Scholar |

Kumar P, Kumar D, Sikka P, Singh P (2015) Sericin supplementation improves semen freezability of buffalo bulls by minimizing oxidative stress during cryopreservation. Animal Reproduction Science 152, 26-31.
| Crossref | Google Scholar |

Kumar G, Saxena A, Yadav DK, Sachan V, Agrawal J, Swain DK (2022) Trehalose supplementation improves membrane integrity and reduces cryocapacitation like changes in cryopreserved Hariana bull semen. Journal of Experimental Zoology, India 25(1), 1169-1172.
| Google Scholar |

Kumar A, Virmani M, Kumar R, Kumar S, Sarthak , Kanwar A, Kumar S, Patil CS (2023) Inclusion of 1,3-dimethylxanthine improved quality parameters of bull semen. The Animal Biology 25, 11-16.
| Crossref | Google Scholar |

Layek SS, Mohanty TK, Kumaresan A, Parks JE (2016) Cryopreservation of bull semen: evolution from egg yolk based to soybean based extenders. Animal Reproduction Science 172, 1-9.
| Crossref | Google Scholar |

Li S, Ren J, Zhang W, Wang B, Ma Y, Su L, Dai Y, Liu G (2023) Glutathione and selenium nanoparticles have a synergistic protective effect during cryopreservation of bull semen. Frontiers in Veterinary Science 10, 1093274.
| Crossref | Google Scholar |

Lone SA (2018) Possible mechanisms of cholesterol-loaded cyclodextrin action on sperm during cryopreservation. Animal Reproduction Science 192, 1-5.
| Crossref | Google Scholar |

Luo X, Wu D, Liang M, Huang S, Shi D, Li X (2023) The effects of melatonin, glutathione and vitamin E on semen cryopreservation of Mediterranean buffalo. Theriogenology 197, 94-100.
| Crossref | Google Scholar |

Malik A (2018) Effects of honey supplementation into the extender on the motility, abnormality and viability of frozen thawed of Bali bull spermatozoa. Asian Journal of Animal and Veterinary Advances 13, 109-113.
| Crossref | Google Scholar |

Mehdipour M, Daghigh Kia H, Nazari M, Najafi A (2017) Effect of lecithin nanoliposome or soybean lecithin supplemented by pomegranate extract on post-thaw flow cytometric, microscopic and oxidative parameters in ram semen. Cryobiology 78, 34-40.
| Crossref | Google Scholar |

Mohammadi T, Hosseinchi Gharehaghaj M, Alaei Novin A (2024) Effects of apigenin and trans-ferulic acid on microscopic and oxidative stress parameters in the semen of water buffalo bulls during cryopreservation. Cryobiology 115, 104868.
| Crossref | Google Scholar |

Mohammed OA, Abdulkareem TA, Ibrahim FF, Al-Zaidi OH, Latif WE, Alwan SH (2020) Effect of adding pentoxifylline and nitric oxide to Tris extender on some post-cryopreserved semen attributes of Holstein bulls. Iraqi Journal of Agricultural Science 51(2), 619-628.
| Crossref | Google Scholar |

Motemani M, Chamani M, Sharafi M, Masoudi R (2017) Alpha-tocopherol improves frozen-thawed sperm quality by reducing hydrogen peroxide during cryopreservation of bull semen. Spanish Journal of Agricultural Research 15(1), e0401.
| Crossref | Google Scholar |

Mráčková M, Vojtěchovská L, Andrlová P, Krisová Š, Sedlinská M (2021) Influence of pentoxifylline and caffeine on stallion epididymal sperm motility after thawing. Acta Veterinaria Brno 90(3), 271-276.
| Crossref | Google Scholar |

Nadri T, Towhidi A, Zeinoaldini S, Riazi G, Zhandi M, Sharafi M (2020) Supplemental glutathione improves post-thaw quality of holstein bulls sperm in a nanomicelle based extender. Iranian Journal of Applied Animal Science 10(4), 615-622.
| Google Scholar |

Nain D, Mohanty TK, Dewry RK, Bhakat M, Nath S, Gupta VK, Parray MA (2023) Butylated hydroxytoluene (BHT) improves the post-thaw semen quality in low-dose sperm cryopreservation in Murrah buffalo bull. Cryoletters 44(1), 57-64A.
| Crossref | Google Scholar |

Ozimic S, Ban-Frangez H, Stimpfel M (2023) Sperm cryopreservation today: approaches, efficiency, and pitfalls. Current Issues in Molecular Biology 45(6), 4716-4734.
| Crossref | Google Scholar |

Panwar S, Singh AK, Kumar A, Honparkhe M, Ratta NS, Singh P (2024) Effect of cysteine supplementation in semen extender on freezability and fertility of Murrah buffalo semen. The Indian Journal of Animal Sciences 94(6), 545-552.
| Crossref | Google Scholar |

Patel A, Saxena A, Swain DK, Yadav D, Yadav SS, Kumar A, Kumar A (2015) Effect of supplementation of butylated hydroxytoluene on post-thaw sperm viability, motility and membrane integrity of Hariana bulls. Veterinary World 8(6), 808-812.
| Crossref | Google Scholar |

Patel TM, Dhami AJ, Chaudhari DV, Pathan MM (2019) Influence of antioxidant sericin in tris extender on oxidative markers during cryopreservation (-196°C) of bovine semen. Indian Journal of Veterinary Sciences & Biotechnology 15(2), 34-37.
| Crossref | Google Scholar |

Pinto SCC, Souza FA, Arruda RP, Alves MBR, Batissaco L, Garcia-Oliveros LN, Passarelli MS, Carneiro LC, Celeghini ECC (2023) Impact of adding different concentrations of IGF-I and insulin to the semen extender on bull sperm quality post-cryopreservation. Arquivo Brasileiro de Medicina Veterinária e Zootecnia 75, 771-786.
| Crossref | Google Scholar |

Priyanto L, Herdis , Santoso , Sitaresmi PI, Priyatno TP, Anwar RI, Mahari DA, Lupitasari FBI, Surachman M, Darmawan IWA, Abrar A, Setiawan A (2024) The effects of various high dosage of α-Tocopherol and ascorbic acid in Tris egg yolk extender on post-thawed sperm quality in tropical Brahman bulls. Pakistan Journal of Zoology 56, 2001-2500.
| Crossref | Google Scholar |

Putranti OD, Adriani L, Soeparna S, Lestari TD (2019) Effect of caffeine on motility of epididymis spermatozoa of Bali bull in slaughterhouse Cibinong. Chalaza Journal of Animal Husbandry 4(2), 44-47.
| Crossref | Google Scholar |

Raheja N, Choudhary S, Grewal S, Sharma N, Kumar N (2018) A review on semen extenders and additives used in cattle and buffalo bull semen preservation. Journal of Entomology and Zoology Studies 6(3), 239-245.
| Google Scholar |

Salman A, Caamaño JN, Fernandez-Alegre E, Hidalgo CO, Nadri T, Tamargo C, Fueyo C, Fernandez A, Merino MJ, Martínez-Pastor F (2021) Supplementation of the BIOXcell extender with the antioxidants crocin, curcumin and GSH for freezing bull semen. Research in Veterinary Science 136, 444-452.
| Crossref | Google Scholar |

Sapanidou V, Lavrentiadou SN, Errico M, Panagiotidis I, Fletouris D, Efraimidis I, Zervos I, Taitzoglou I, Gasparrini B, Tsantarliotou M (2022) The addition of crocin in the freezing medium extender improves post-thaw semen quality. Reproduction in Domestic Animals 57(3), 269-276.
| Crossref | Google Scholar |

Shah N, Singh V, Yadav HP, Verma M, Chauhan DS, Saxena A, Yadav S, Swain DK (2017) Effect of reduced glutathione supplementation in semen extender on tyrosine phosphorylation and apoptosis like changes in frozen thawed Hariana bull spermatozoa. Animal Reproduction Science 182, 111-122.
| Crossref | Google Scholar |

Sharafi M, Borghei-Rad SM, Hezavehei M, Shahverdi A, Benson JD (2022) Cryopreservation of semen in domestic animals: a review of current challenges, applications, and prospective strategies. Animals 12(23), 3271.
| Crossref | Google Scholar |

Shokry DM, Badr MR, Sakr A-AM, Elmesiry AM, Assy MM, Rawash Z, Abd Eldaim MA (2024) Enhancement potential of Moringa oleifera leaves extract on buffalo bull cryopreserved semen quality and fertilization capacity. Animal Reproduction Science 262, 107414.
| Crossref | Google Scholar |

Silva ECB, Cajueiro JFP, Silva SV, Guerra MMP (2021) Plant extracts and cryopreservation: potentials and challenges in sperm preservation. Cryobiology 98, 25-32.
| Google Scholar |

Singh AK, Saxena A, Swain DK, Yadav B, Kumar A, Kumar M, Sachan V, Agrawal JK (2024) Cryoprotective assessment of lycopene in tris-based extender on freezability of bull semen. Indian Journal of Animal Research 1, 6.
| Crossref | Google Scholar |

Thumar HK, Patel MR, Nakhashi HC, Suthar BN, Sutaria TV, Chauhan PM, Desai SP (2017) Comparative efficacy of melatonin and trehalose additives on enzyme leakage during cryopreservation of kankrej bull semen. International Journal of Agriculture Sciences 9, 4940-4942.
| Google Scholar |

Ugur MR, Saber Abdelrahman A, Evans HC, Gilmore AA, Hitit M, Arifiantini RI, Purwantara B, Kaya A, Memili E (2019) Advances in cryopreservation of bull sperm. Frontiers in Veterinary Science 6, 268.
| Crossref | Google Scholar |

Verma AK, Kumar R, Srivastava S, Alam K, Kumar R, Sakhahari DS, Verma SK (2023) Methionine as a semen additive to improve Murrah buffalo (Bubalus bubalis) semen cryopreservation. The Indian Journal of Veterinary Sciences and Biotechnology 19(6), 39-43.
| Google Scholar |

Wang M, Wu S, Yang B, Ye M, Tan J, Zan L, Yang W (2023) Grape seed proanthocyanidins improve the quality of fresh and cryopreserved semen in bulls. Animals 13(17), 2781.
| Crossref | Google Scholar |

Woelders H, Matthijs A, Engel B (2017) Effect of freezing rate and concentration of cryoprotectant on survival and fertility of frozen-thawed bull sperm. Theriogenology 88, 1-10.
| Google Scholar |

Yadav DK, Kumar A, Gupta S, Sharma P, Kumar G, Sachan V, Yadav B, Yadav S, Saxena A, Swain DK (2023) Antioxidant additive melatonin in tris-based egg yolk extender improves post-thaw sperm attributes in Hariana bull. Animal Reproduction Science 251, 107214.
| Crossref | Google Scholar |

Yadav BK, Gangwar K, Agrawal JK, Sachan V, Yadav B, Kumar A, Saxena A (2024) Effect of carnitine on physico-morphological properties of cryopreserved hariana bull spermatozoa. The Indian Journal of Veterinary Sciences and Biotechnology 20(3), 116-119.
| Google Scholar |

Yangngam Y, Chapanya S, Vongpralub T, Boonkum W, Chankitisakul V (2021) Effect of semen extender supplementation with sericin on post-thaw dairy bull sperm quality and lipid peroxidation. Czech Journal of Animal Science 66(1), 13-20.
| Crossref | Google Scholar |

Yeni D, Gülhan MF, İnanç ME, Avdatek F, Güngör Ş, Türkmen R, Tuncer PB, Tasdemir U (2021) The cryoprotective potential of propolis supplemented in frozen-thawed bull semen; biochemical and physiological findings. Polish Journal of Veterinary Sciences 25, 5-12.
| Crossref | Google Scholar |

Yimer N, Muhammad N, Sarsaifi K, Rosnina Y, Wahid H, Khumran AM, Kaka A (2015) Effect of honey supplementation into Tris extender on cryopreservation of bull spermatozoa. Malaysian Journal of Animal Science 18, 47-54.
| Google Scholar |