Effect of adding oat seed aqueous extract in Tri's diluent on some chilled semen characteristics of Awassi rams

Keywords: Awassi rams, Oat seed, Tri's diluent, Semen

Introduction

Enhancing the reproductive efficiency of artificial insemination programs for livestock herds and widening the use of sires with different genetic traits is one of the main priorities of breeders worldwide, as is achieving significant progress in the reproductive performance of females (Mallory et al., 2010; Ayantoye et al., 2025) ^{[1, 2]} and achieving an increase in female fertility (Banana et al., 2021) ^[3], but the greatest increase here depends on the performance of reproductive management of sires (Petrunkina et al., 2011) ^[4]. The field of agricultural research has achieved great success with the need for cryopreservation of reproductive cells and tissues. The quality of semen utilized in the studies was based on preparing a suitable diluent and the amount of added materials (El-Harairy et al., 2016) ^[5]. The dilution of sperm with antioxidants and adding them to diluents of agricultural animals during cooling and freezing protects the sperm from the harmful effects of increased level (ROS-Reactive oxygen species) and improves sperm motility, vitality, and fertility after cooling and freezing (Eidan, 2016) ^[6]. Use of antioxidants from natural sources of plants that produce more than 4000 polyphenolic compounds and phenolic compounds that are good antioxidant sources (Al-Mufarji, 2018) ^[7] has been on the rise recently. Due to their high nutritional value, oats are considered as one of the major feedstuffs in humans and animals (Riely, 2003) ^[8]. Oats contain the good content of polyphenolic compounds that are antioxidant by scavenging reactive nitrogen species and oxygen species (Alemayehu et al., 2023) ^[9]. Consequently, this research was designed to determine the effect of enriching diluent Tri with oat seeds on ew semen traits using the Awassi ram (5 °C) and the storage period (48 and 72 hours).

Materials & methods

Sample collection

Four Awassi rams were sampled on a weekly basis and one ram twice a week for semen. To reduce the variation between the rams, semen from four rams was pooled together to make the semen sample for each replicate. The tests of the semen evaluate and then diluted using Tri's diluent which was prepared in advance according using (Salamon et al., 2000) ^[10] method. The experiment was conducted in four treatments, the first treatment (T₁) was without addition, the second treatment (T₂) was with addition of 1% of oat seed aqueous extract, the third treatment (T₃) was with addition of 2% of oat seed aqueous extract and the fourth treatment (T₄) was with addition of 3% of oat seed aqueous extract. The analyses tests were done after 48 and 72 hours of cooling at 5 °C and the following traits studied.

Motility individual percentage

The motility of individual sperm was estimated using (Chen et al., 2007) ^[11].

The percentage of sperm plasma membrane integrity

The integrity of the sperm plasma membrane was assessed using the swelling assay under reduced osmotic pressure (Jeyendran et al., 1984) ^[12].

The percentage of sperm integrity acrosome

The percentage of integrity acrosome was estimated, and the sperm of abnormal sperm was estimated based on the method of (Hancock) ^[13].

Statistical analysis

The effect of different treatments on the studied traits was analyzed using a statistical analysis program (SAS, 2018) ^[14] in accordance with a completely randomized design (CRD), with the mean separation of the significant differences being performed using the Duncan multiple range test ^[15].

Results & discussion

The results of Table 1 showed significant differences in the individual motility trait, where treatment T₂ was excelled when adding 1% of oat seed aqueous extract to Tri's diluent after cooling for 48 hours, as it recorded (49.00 ± 1.00%) compared to treatments (T₁, T₃ and T₄) as it recorded (43.00 ± 1.22, 44.00 ± 1.00, and 38.00 ± 1.22) respectively. The results also showed the superiority of treatment T₂ in the individual motility trait after cooling for 72 hours, as it recorded (48.00 ± 1.22) compared to treatments (T₁, T₃, and T₄), as it recorded (38.00 ± 1.22, 44.00 ± 1.00, and 33.00 ± 1.22) respectively. As a result of the addition of oat seed aqueous extract in treatment T₂, it is highly possible that a significant increase in individual motility occurred. The occurrence of this is because of the content of oats of active compounds that have antioxidant properties (Agarwal et al., 2021; Khdhr et al., 2024) ^{[16, 17]}. The antioxidant activity of flavonoids that are present in many plants has been shown in a number of previous studies. In the present study, a number of these compounds were investigated for their abilities to attack free radicals derived from reactive oxygen species (Chen et al., 2007) ^[11]. (a) The aqueous extract of oat seeds is an antioxidant which effectively mops up free radicals and reduces any damage to sperm when these free radicals attack its plasma membrane, which in turn leads to decreased sperm motility and reduced fertilizing ability (Majeed et al., 2024) ^[18].

T₁ = control treatment with Tris dilutes without antioxidant addition, T₂ = Tris dilute + 1% oat seed extract, T₃ = Tris dilute + 2% oat seed extract, T₄ = Tris dilute + 3% oat seed extract. Significant differences exist between means with the same letters within the same column. * (Taking values less than or equal to 0.05 results in a P value from NULL to 0.05.)

Table 2 revealed the performance of treatments in percentage plasma membrane of sperm integrity, where the treatment T₂ by adding 1% oat seed aqueous extract after 48 hours cooling showed significant difference as it recorded (79.00 ± 1.00) whereas the T₁, T₃ and T₄ treatments recorded (77.00 ± 1.22, 78.00 ± 1.00 and 77.00 ± 1.22) respectively. The results also revealed that the percentage of sperm plasma membrane integrity was superior in T₂ (79.00 ± 1.22) than in T₃, T₄ and T₁ (77.00 ± 1.00, 76.00 ± 1.22 and 75.00 ± 1.55%) respectively after cooling for 72 hours. This higher percentage of integrity of the sperm plasma membrane in treatment T₂ could be explained by the use of the oat seed aqueous extract. The lipids are large molecule, easy to be peroxidated, and present in the sperm plasma membrane as polyunsaturated fatty acids (PUFA) of which the number of carbon-carbon double bonds is > 2. The components of the sperm plasma membrane that contribute to membrane flexibility are the fatty acids (PUFA) (Leahy et al., 2011; Essa et al., 2025) ^{[19, 20]}. Unsaturated fatty acids (PUFA) are known to trigger a chain of Chemical Reactions, termed as lipid peroxidation, which ultimately leads to a defect in the membrane mechanics resulting in a reduction of membrane enzymes and channel ions activity and consequently a loss of membrane elasticity, which further hinders the membrane mechanics required for the process of fertilization (Agarwal et al., 2006) ^[21]. Lipid peroxidation (LPO) occurs in sperm which is self-replicating and is stopped by antioxidants: antioxidants in oats would work to reduce and minimise the impact of harmful free radicals (Li et al., 2017) ^[22].

T₁ = control treatment with Tris dilutes without antioxidant addition, T₂ = Tris dilute + 1% oat seed extract, T₃ = Tris dilute + 2% oat seed extract, T₄ = Tris dilute + 3% oat seed extract. Means with letters different within the same column are significantly different from each other. * (P ≤ 0.05).

The results of Table 3 showed significant differences in the percentage sperm of acrosome integrity, as treatment T₂ was excelled when adding 1% of oat seed aqueous extract to Tri's diluent after 48 hours of cooling, as it recorded (89.00 ± 1.20%) compared to treatments (T₃, T₄ and T₁), as it recorded (88.00 ± 1.10, 87.00 ± 1.20, and 86.00 ± 1.19%) respectively. The results also showed superiority of treatment T₂ in the percentage of sperm acrosome integrity after 72 hours of cooling, as it recorded (89.00 ± 1.30%) compared to treatments (T₁, T₃, and T₄), as it recorded (87.00 ± 1.15, 87.00 ± 1.05, and 86.00 ± 1.11%) respectively. The significant increase in the percentage of acrosome integrity of sperm in treatment T₂ may be attributed to the addition of oat seed aqueous extract. The result of the current study can be interpreted that the integrity of the acrosome is related to the integrity of the plasma membrane sperm, as the sperm plasma membrane in turn protects the healthy cells in the acrosome vesicle at the tip in the head so that the acrosome vesicle remains intact and increases the protection of the acrosome protective cover (Rizal et al., 2003; Al-ani et al., 2023) ^{[23, 24]}. Oat extract acts as an antioxidant by activating the role of glutathione, a strong antioxidant in seminal plasma, which works to reduce the damage of oxidative factors and improve the qualities of semen (Rushworth et al., 2013) ^[25].

The results of Table 3 indicated that there was significant difference between the percentage of sperm with intact acrosome after 48 hours of cooling in treatment T₂ (89.00 ± 1.20) as it excelled over treatments (T₃, T₄ and T₁) with (88.00 ± 1.10, 87.00 ± 1.20, and 86.00 ± 1.19) respectively. It was also observed that the percentage of sperm acrosome integrity after 72 hours of cooling indicated that the treatment T₂ recorded the higher percentage (89.00 ± 1.30%) compared to T₁, T₃ and T₄ (87.00 ± 1.15, 87.00 ± 1.05 and 86.00 ± 1.11%) respectively. The percentage of the sperm that were acrosome intact in treatment T₂ can be concluded due to the addition of oat seed aqueous extract. The result of the current study can be interpreted that the integrity of the acrosome is related to the integrity of the plasma membrane sperm, as the sperm plasma membrane in turn protects the healthy cells in the acrosome vesicle at the tip in the head so that the acrosome vesicle remains intact and increases the protection of the acrosome protective cover (Rizal et al., 2003; Al-ani et al., 2023) ^{[23, 24]}. It has antioxidant activity as it increases the activity of glutathione, a potent antioxidant in seminal plasma, which will help to reduce the damage caused by oxidative factors and improve the characteristics of semen (Rushworth et al., 2013) ^[25].

T₁ = control treatment with Tris dilutes without antioxidant addition, T₂ = Tris dilute + 1% oat seed extract, T₃ = Tris dilute + 2% oat seed extract, T₄ = Tris dilute + 3% oat seed extract. Means with letters different within the same column are significantly different from each other. * (P ≤ 0.05).

Table 4 results indicated that the percentage of total sperm abnormalities decreased significantly after addition of 1% of oat seed aqueous extract to Tri's diluent after 48 hours cooling in the treatment T₂ as it recorded 0.1 ± 9.01 while the T₁, T₃ and T₄ recorded 11.50 ± 0.2, 10.10 ± 0.7 and 10.01 ± 0.6%, respectively. The results also indicated that after 72 hours cooling treatment T₂ had reduced the percentage of total sperm abnormality (11.90 ± 0.1%) compared to treatments (T₁, T₃, and T₄), as it recorded (11.80 ± 0.5, 10.3 ± 0.9, and 10.2 ± 0.8%) respectively. This marked downfall in the percentage of total abnormality in the sperm in treatment T₂ may be due to the inclusion of oat seed aqueous extract. Oxidative stress damaging the membranes during the cryopreservation and freezing process is responsible for the damage to the membranes in sperm. Raised levels of lipid oxidation can cause abnormalities in sperm. Sperms can become abnormal as a result of lipid oxidation. Semen trait improvement during cryopreservation can be obtained by using OAE in the semen diluent, while OAE as an antioxidant can protect the sperm from damage caused by the oxidative stress during cryopreservation as evidenced by semen trait improvement such as reduction in the incidence of sperm abnormalities (Alemayehu et al., 2023; Duncan, 1955) ^{[9, 15]}.

T₁ = control treatment with Tris dilutes without antioxidant addition, T₂ = Tris dilute + 1% oat seed extract, T₃ = Tris dilute + 2% oat seed extract, T₄ = Tris dilute + 3% oat seed extract. Means with letters different within the same column are significantly different from each other. * (P ≤ 0.05).

Conclusions

The results obtained in this study showed that the addition of 1% of aqueous extract of oat seeds improved some traits of the semen of Awassi rams at the post-cooled phase including individual movement, acrosome integrity, and plasma membrane integrity of sperm, along with a decrease in a total sperm abnormality percentage.

Supplementary materials

There are no supplementary materials.

Author contributions

M. K. H. AL-dulaimi: methodology, writing original draft preparation; M. K. A Al-juhaishi writing; H. R. Alnajm review and editing; P.R Tapaloaga; A. Javanmard and D. Tapaloaga statistical analysis. The manuscript was reviewed by all authors.

Funding

This research did not receive any external funding.

Institutional review board statement

The study was carried out based on the protocol approved by the Al-Musaib Technical College, Al-Furat Al-Awsat Technical University, Department of Animal Production Techniques.

Data availability statement

Data is available upon request.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgments

Thanks to the Dean of the College Head of Al-Musaib Technical College, Al-Furat Al-Awsat Technical University, Iraq, Department of Animal Production Techniques and all who assisted in this study.

References

1. 1.Mallory DA, Wilson DJ, Busch DC, Ellersieck MR, Smith MF, Patterson DJ. Comparison of long-term progestin-based estrus synchronization protocols in beef heifers. J Anim Sci. 2010;88(11):3568–3578.
2. 2.Ayantoye JO, Kolachi HA, Zhang X, Shahzad M, Kandil OMT, Wan P, Zhao X. Advances in Timed Artificial Insemination: Integrating Omics Technologies for Enhanced Reproductive Efficiency in Dairy Cattle. Animals. 2025;15(6):816.
3. 3.Banana JH H, Neamah HJ, Saieed AY. Evaluation of adding aqueous extract of Opuntia Ficus Indica leaves to Tris extender on the criteria of Awassi rams semen stored at cooling. Indian Journal of Forensic Medicine & Toxicology. 2021;15(3):3164-3169.
4. 4.Petrunkina AM, Harrison RA. Cytometric solutions in veterinary andrology: developments, advantages, andlimitations. Cytometry A. 2011;79(5):338–348.
5. 5.El-Harairy MA, Abdel-Khalek AE, Khalil WA, Khalifa E, El-Khateeb AY, Abdulrhman AM. Effect of aqueous extracts of Moringa oleifera leaves or Arctium lappa roots on lipid peroxidation and membrane integrity of ram Sperm preserved at cool temperature. J. Anim. Poult. Prod., Mansoura Univ. 2016;7(12):467-473.
6. 6.Eidan SM. Effect on post-cryopreserved semen traits of Holstein bulls of adding combinations of vitamin C and either catalase or reduced glutathione to Tris extender. Anim. Reprod. Sci. 2016;167:1-7.
7. 7.Al-Mufarji Isser Hamid Salman. Effect of adding alcoholic extract of Ferula hermonis and some antioxidants to Tri's diluent on the quality of chilled and frozen semen in Holstein bulls. Master's thesis, College of Agriculture - University of Diyala. 2018.
8. 8.Riely E. The Chef’s Companion. 3rd ed. New Jersey: Wiley and Sons, Inc; 2003.
9. 9.Alemayehu GF, Forsido SF, Tola YB, Amare E. Nutritional and Phytochemical Composition and Associated Health Benefits of Oat (Avena sativa) Grains and Oat-Based Fermented Food Products. TheScientificWorldJournal. 2023;2023:2730175.
10. 10.Salamon S, Maxwell WM. Storage of ram semen. Animal reproduction science. 2000;62(1-3):77–111.
11. 11.Chen CYO, Milbury PE, Collins FW, Blumberg JB. Avenanthramides are bioavailable and have antioxidant activity in humans after acute consumption of an enriched mixture from oats. Journal oF Nutrition. 2007;137:1375-1382.
12. 12.Jeyendran RS, Vander van HH, Perez-Pelaez M, Crabo B, Zaneveld LJD. Development of an assay to assess thefunctional integrity of the human sperm membrane and its relationship to other semen traits. J. Reprod. Fertile. 1984;70:219-228.
13. 13.Hancock J. A staining technique for the study oftemperature shockin semen. Nature. 1951;167:323-324.
14. 14.SAS. Statistical Analysis System, User's Guide. Statistical. Version 9.6th ed. Cary, N.C. USA: SAS. Inst. Inc; 2018.
15. 15.Duncan DB. Multiple range and multiple F tests. Biometrics. 1955;11:1–42.
16. 16.Agarwal A, Leisegang K, Majzoub A, Henkel R, Finelli R, Panner Selvam MK, Tadros N, Parekh N, Ko EY, Cho CL, Arafa M, Alves MG, Oliveira PF, Alvarez JG, Shah R. Utility of Antioxidants in the Treatment of Male Infertility: Clinical Guidelines Based on a Systematic Review and Analysis of Evidence. The world journal of men's health. 2021;39(2):233–290.
17. 17.Khdhr D, Karim K. CYTOGENETIC STUDY OF THE HAMDANI SHEEP BREED OF THE IRAQI KURDISTAN REGION. Anbar Journal of Agricultural Sciences. 2024;22(2):913-924.
18. 18.Majeed A, Mohammed T, Ahmed A. EFFECT OF ADDING NATURAL AGAINST SYNTHETIC ANTIOXIDANTS ON OVINE OOCYTE MATURATION, MALONDIALDEHYDE LEVELS, AND IN VITRO FERTILIZATION. Anbar Journal of Agricultural Sciences. 2024;22(2):1070-1081.
19. 19.Leahy T, Gadella BM. Sperm surface changes and physiological consequences induced by sperm handling and storage. Reproduction. 2011;142:759–778.
20. 20.Essa A, Mohammed T, Al-Anbari N. POLYMORPHISMS AND ALLELE FREQUENCY OF BMPR1B GENES IN LOCAL AWASSI SHEEP. Anbar Journal of Agricultural Sciences. 2025;23(1):64-76.
21. 21.Agarwal A, Prabakaran SA, Allamaneni S. What an andrologist / urologist should know about free radicals and why. Urology. 2006;67:2–8.
22. 22.Li X, Li M, Ling A, Hu X, Ma Z, Liu L, Li Y. Effects of genotype and environ-ment on avenanthramides and antioxidant activity of oats grown in northwestern China. Journal of Cereal Science. 2017;73(1):130–137.
23. 23.Rizal M, Teoliher MR, Yusuf TL, Purwantara B, Situmorang P. Media Kedokteran Hewan 1979. 2003.
24. 24.Al-ani H, Asker A, Hseen N. THE EFFECT OF USING ANABASIS ARTICULATE EXTRACT AND MELATONIN HORMONE LOCALLY IN VAGINAL SPONGES ON THE REPRODUCTIVE PERFORMANCE OF LOCAL IRAQI EWES. Anbar Journal of Agricultural Sciences. 2023;21(1):214-223.
25. 25.Rushworth GF, Megson IL. Existing and potential therapeutic use for N- acetylcysteine: the need for conversion to intracellular glutathione for antioxidant benefits. Pharmacology and Therapeutics. 2013;141(2):150–159.
26. 26.Abdul Karim, Talal Anwar, Mohammed, Omar Adel, Shabr, Abdullah Mohammed Hassan, Ibrahim, Faris Faisal, Latif, Wafaa Yadam. Effect of adding carnitine and inositol to Tri's diluent on the quality of semen after freezing in Holstein bulls. Anbar Journal of Veterinary Sciences. 2016;9:8-18.
27. 27.Al-Mohammedy A, Abdul-Rahaman Y. THE IMPACT OF BIOTIN AND HCG ON THE LEVELS OF ESTRADIOL-17Β, PROGESTERONE AND SHEEP PREGNANCY ASSOCIATED PLASMA PROTEIN B (PAPPB) IN LOCAL IRAQI EWES. Anbar Journal of Agricultural Sciences. 2024;22(1):106-114.
28. 28.Engdawork A, Belayhun T, Aseged T. The role of reproductive technologies and cryopreservation of genetic materials in the conservation of animal genetic resources. Ecological Genetics and Genomics. 2024;31:100250.