Effects of supplementation with Mimosa tannin (Acacia mearnsii) extracts on ovine sperm cryopreservation, and viability

Abstract

The increase in human population has placed pressure on livestock industries to increase production. Livestock farming contributes up to 49% of agricultural output in South Africa, with lamb and mutton production increasing in the past decade (Meissner et al., 2013). To ensure cost-effective production practices, farmers use genetic selection and assisted reproductive technologies (ART) to enhance reproductive efficiency and to counter the local deficit and demand (Meissner et al., 2013). Genetic breeding programmes in small ruminants focus on the selection of traits that contribute to improving the production and reproductive efficiency of livestock species (Granleese et al., 2015). The important selected traits especially improve feed conversion, disease resistance, optimal reproductive ability, and the utilisation of ART (Granleese et al., 2015). Selection of animals according to certain traits to specifically improve or optimise production and reproduction efficiency is geared towards improving the genetic composition of the particular animal species, which is vital in food security (Haile et al., 2019). Reproductive performance of male livestock is of economic importance as well as in improving sperm quality and fertility for artificial insemination (AI) programmes which will help minimise losses from genotype (Haile et al., 2019). The addition of antioxidants with very good preservation techniques improves sperm function during storage with good capacitation/fertilization in the female reproductive tract (Ros-Santaella and Pintus, 2021). Plants containing combinations of complex polymeric phenols (tannins) are amongst the most studied phytochemicals because of their biological functions and health-promoting effects (Ros-Santaella and Pintus, 2021; Wurlina et al., 2020). A commercially available tannin-rich extract of black wattle bark, Acacia mearnsii (ATE), comprises a complex mixture of chemicals yet to be identified (Pizzi, 2021). A previous study of supplementation of non-encapsulated tannin extract (TE), and capsulated tannin extract (ETE) improved the reproductive performance of South African mutton merino rams and motivated this study. In the first study, the ATE dried powder underwent ethanol extraction (to recover polyphenolic compounds while excluding water-soluble sugars and salts), yielding MTE_0 (484.28 g, 89.7%), which was fractionated, and antioxidant activity and cytotoxicity of these samples was investigated. For fractionation, part of the MTE_0 extract (1.4 g) was solubilised in methanol and fractionated using column chromatography with Amberlite XAD-16 resin. Following chromatography, the total eluate of 1.24 g (83.8% of MTE_0) was divided into four fractions: MTE_1 (200 mL; 0.52g), MTE_2 (100 mL; 0.02g), MTE_3 (100 mL; 0.63g), and MTE_4 (200 mL; 0.08g) and stored at -20°C. Ultra-performance liquid chromatography coupled with high-resolution mass spectrometry (UPLC-HRMS) analysis was employed to tentatively identify the constituent compounds. In all, six samples (2 extracts: ATE, and MTE_0, with 4 fractions: MTE_1, MTE_2, MTE_3, and MTE_4) were tested. Antioxidant activity was determined by the 2, 2’ -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays which resulted in inhibition concentration (IC50) values of ATE (2.9 × 10⁻³ and 2.4 × 10⁻²), while the extract MTE_0 (1.0 × 10⁻³ and 1.8 × 10⁻²), and fraction MTE_3 (1.4 × 10⁻³ and 2.0 × 10⁻²) had the most active antioxidant capacity amongst the samples when compared to that of ascorbic acid (1.3 × 10⁻³ and 1.2 × 10⁻³). The cytotoxic effects of the extracts and fractions was evaluated on the rat Leydig (LC-540) cells using the AlamarBlue®, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and transmission electron microscopy (TEM) to validate their potential as non-cytotoxic natural antioxidants on the reproductive cells. UPLC-HRMS resulted in the tentative identification of sucrose (341.1084 (-1.8ppm) m/z, [M-H].- in all the extracts, and seven flavonoid compounds were detected in the various fractions, namely gallocatechin (305.0661 (-3.6) m/z, [MH₋], glucosyringic acid (359.0978 (0.8 ppm) m/z, [M-H]-, gallocatechin-(4α→8)-catechin (593.1295 (-3.0 pm) m/z, [M-H]₋₋, fisetinidol-(4α→8)-catechin (577.1346 (1.6ppm) m/z, [M-H].- , arecatannin A1 (865.1980 (0.8ppm) m/z, [M-H]- , fisetinidol-(4α→6')-fisetinidol-(4α→6)-gallocatechin (849.2031 (1.4ppm) m/z, [M-H].- and fisetinidol-(4α→8)-catechin-(6→4α)-fisetinidol (833.2082 (4.0ppm) m/z, [M-H]-. The common flavonoid compounds that were present in both the MTE_0 extract, as well as in the fraction MTE_3 with the most active antioxidant capacity, were gallocatechin-(4α→8)-catechin, arecatannin A1, fisetinidol-(4α→6')-fisetinidol-(4α→6)-gallocatechin and fisetinidol-(4α→8)-catechin-(6→4α)-fisetinidol. In addition, cytotoxicity evaluation on rat Leydig cells (LC-540) demonstrated favourable safety profiles, with IC₅₀ values ranging from 8.21-58.04 µg/mL for extracts and fractions (MTT and AlamarBlue® assays), substantially higher than the positive control doxorubicin (0.60 µg/mL). Transmission electron microscopy revealed no ultrastructural damage at functional antioxidant concentrations, with fraction MTE_1 (IC₅₀ 58.04 µg/mL) exhibiting the lowest cytotoxicity. In the second experiment, ejaculates (n=6) of matured merino ram live body weight (LBW) of 120 kg were added to the extract and gallocatechin with OptidylTM, a commercial extender for chilled semen storage at different concentrations (0, 12.5, 25, 50, and 100 µM) of MTE_0, and gallocatechin and stored for 10 days at 4 to 10°C. The aliquots of MTE_0 and gallocatechin-treated spermatozoa were assessed for viability, acrosomal integrity, mitochondrial membrane potential, and capacitation status at the end of incubation. The kinematics parameters were evaluated by computer-assisted sperm analysis (CASA) with the trial performed in triplicate. Data were analysed by a one-way ANOVA procedure of IBM SPSS 21.0 for Windows. In chilled semen stored at 4-10°C for 10 days, gallocatechin at 25 µM significantly (p<0.001) improved the live spermatozoa proportions (32.38±0.55%; 19.54±0.26%), while affecting ROS levels (35.38±0.55%; 25.60±0.55%) and capacitation status (31.48±0.81%; 20.46±0.81%) compared to control. In the third experiment, 6 adult (3 ± 5 years) Merino rams with average LBW (116kg) (n=6) were used. The rams were kept in individual pens at a commercial semen station (RAMSEM) in Bloemfontein. The ejaculates were collected by trained attendants with artificial vaginas (AV) twice weekly for 3 weeks. Immediately, ejaculates were transferred to a water bath at 37°C. Ejaculates with at least 1 mL per ejaculate, creamy fresh semen of an average of 2.3 x 109 sperm mL-1, with 60% live sperm, and mass motility of 3-4 out of 5 scales were used for the experiment. This was extended to a final concentration of 13 X 106 sperm mL-1 with the tris-based extender Optidyl TM (Biovet, France). This was pooled and divided into five aliquots (0, 12.5, 25, 50, and 100 µM). Immediately after the collection, we determined the volume, the mass motility, and the percentage of live sperm of the fresh semen. For each aliquot, a concentration of gallocatechin (0, 12.5, 25, 50, and 100 µM) was added before freezing, and stored inside liquid nitrogen tanks. The spermatozoa were assessed for viability, acrosomal integrity, mitochondrial membrane potential, and capacitation status at the end of incubation. The motility parameters were evaluated by CASA with the trial performed in triplicate and the data were analysed by ANOVA. In conclusion, the review was the first to recommend the addition of tannin-rich extract or compounds into semen destined for cryopreservation, exploiting their diverse effects on biological antioxidation (Liman et al., 2022). Furthermore, it was recommended that future studies be conducted on the biological activities of tannins by determining their chemical structure, biological activities, and structural activity relationships so that potential applications can be explored (Liman et al., 2022). The initial laboratory-based experiment was the first to fractionate ATE and explore its antioxidant activity, and constituents were tentatively identified using UPLC-HRMS. It was concluded that gallocatechin is a promising candidate antioxidant for use in semen during preservation. Gallocatechin has a higher number of hydroxyl groups and greater polymerization that afforded the greatest antioxidation activities. In this study, gallocatechin addition to chilled semen recorded a significant (p ≤ 0.001) positive effect compared to the MTE_0 extract). The addition of gallocatechin at 25 µM resulted in significantly (p ≤ 0.001) improved proportions of live spermatozoa (32.38 ± 0.55: CTL 19.54 ± 0.26 %), modified ROS (35.38 ± 0.55: CTL 25.60 ± 0.55 %), and capacitation status (31.48 ± 0.81: CTL 20.46 ± 0.81 %) compared to control (CTL). On the contrary, in a further experiment, gallocatechin at 100 µM significantly affected (p ≤ 0.001) kinematic parameters such as average path velocity (VAP) (97.29 ± 2.04: CTL 89.31 ± 1.54 %), progressive velocity (VSL) (80.95 ± 2.18: CTL 72.36 ± 2.03) and beat cross frequency (BCF) (36.27 ± 0.39; CTL 34.06 ± 0.57%) of cryopreserved semen compared with control. Gallocatechin addition to semen resulted in a significant (p≤0.001) positive effect compared with the MTE_0 extract. Thus, gallocatechin addition to ovine semen significantly reduces semen deterioration following cryopreservation. The findings in the second experiment are an important fertility indicator, while the findings in the third experiment demonstrated improved post-thawed ovine semen quality and longevity. This study is the first to introduce gallocatechin as an efficient antioxidant additive to ovine semen to improve its quality during storage, assisting with improving post-thaw ovine semen quality and longevity. Future studies to elucidate the mechanism of anti-oxidative stress action of gallocatechin and its derivatives on semen motility and longevity are recommended.