Effects of Costmary (Tanacetum balsamita L.) extract on some of quality parameters of Moghani ram sperm after freezing and thawing

The Artificial insemination technique is based on sperm cryopreservation that induces irreversible damages to sperm (Purdy 2006), which may result in loss of sperm motility, viability, plasma membrane integrity, and ultimately male fertility (Baghshahi et al. 2014). Physical and chemical damages during cryopreservation are associated with significant amounts of production of reactive oxygen species (ROS) and lipid peroxidation of the phospholipids in the membrane by free radicals (Chatterjee et al. 2001). The removal of ROS is catalyzed by antioxidant enzymes such as glutathione peroxidase (GSH-PX), superoxide dismutase (SOD) and catalase (CAT). Numerous non-enzymatic defenses (vitamin C, vitamin E, and glutathione (GSH)) are also employed to provide protection. An imbalance between free radical production and their removal results with ageing allowing progressive damage to occur. Therefore, for protecting the sperm against oxidative damage, numerous researchers have investigated the effects of a various synthetic and natural antioxidants on spermatozoa during cryopreservation processes (Malo et al. 2010). Various plant products contain antioxidant compounds such as flavonoids, tannins, coumarins, xanthons, phenolics, lignans and terpenoids. For this reason, there is a growing interest in using them as natural antioxidants. Several studies have shown that the use of herbal antioxidants during the freezing-thawing process of sperm had positive effects on sperm quality (Daghigh Kia et al. 2016; Vahedi et al. 2018). The herb of costmary (Tanacetum balsamita L.) contains phenolic compounds, such as flavonoids and phenolic acids (Shahhoseini et al. 2019). It has been shown that costmary exhibit antioxidant effects due to phenolic compounds (Bączek et al. 2017).

The aim of current study was to evaluate the effect of Costmary extract as a natural antioxidant on post-thawed ram sperm quality.

This study was performed at the Iranian Moghani sheep Breeding Center located in Jafarabad city, Province Ardebil, Iran. Four mature and fertile rams (3-4 years old, mean live weight of 70±4.2 kg), were used in this study. Ejaculates were collected twice a week for 8 weeks by an artificial vagina (42-43°C). Only samples containing spermatozoa with greater than 70% motility were accepted for experiment. To eliminate individual differences, semen samples were pooled and processed for extending. The pooled ejaculate was diluted (37 ◦C) using egg yolk-citrate extender containing different concentrations of Tanacetum balsamita extract (0, 2, 4, 8, 12, and 16 mL/dL). Diluted semen samples were aspirated into 0.25 ml straws and equilibrated at 4°C for 3 h. After equilibration, the straws were placed on liquid nitrogen (LN2) vapor for 8 min, then plunged into liquid nitrogen, and stored in a liquid nitrogen tank until thawed and used for evaluation of sperm parameters. The frozen straws were thawed individually in a water bath (37 ◦C) for 30 s for evaluation. A computer-assisted sperm analysis (HFT CASA, Hooshmand Fanavar Tehran Co, Iran) was used to analyze sperm motility and velocity characteristics. Sperm viability was assessed using a modification of the eosin-nigrosin staining method described by Evans and Maxwell (1987). Sperm membrane functionality was evaluated using the hypoosmotic swelling test (HOST) (Revell and Mrode, 1994). For the assessment of the sperm morphology abnormalities, at least three drops of each sample were added to Eppendorf tubes containing 1 ml of Hancock solution (62.5 ml formalin (37%), 150 ml sodium saline solution, 150 ml buffer solution and 500 ml bi-distilled water). The prepared slides were assessed by phase-contrast microscopy using a 400× magnification. All data were analyzed by completely randomized design using the GLM procedure of SAS version 9.1 (SAS Institute, 2004).

Samples cryopreserved in 8 and 12 mL/dL Tanacetum balsamita extract had higher total motility and progressive motility compared to the control group (p < 0.05). The percentage of VSL, VCL and VAP were higher (p < 0.05) in the extender containing 8 and 12 mL/dL extract compared to control and 16 mL/dL groups. LIN parameter was higher (p < 0.05) in 8 mL/dL compared to 16 mL/dL extract (46.83±3.82 vs. 40.52±3.23). For parameter STR, the highest value (p < 0.05) was observed at 8 and 12 mL/dL of extract (81.09±7.56% and 80.27±7.18%, respectively). The highest (p < 0.05) percentage of sperm viability and plasma membrane integrity were observed in groups containing 8 and 12 mL/dL extract. Percentage of acrosome abnormality was higher (p < 0.05) in 12 mL/dL extract groups (21.25%) compared to control and 2 mL/dL extract groups (26.70% and 27.23%, respectively). Some studies have reported that herbal antioxidants reduce the free radicals following the freeze–thawing process (Ashrafi et al. 2013). In the present study, treatment of costmary extract resulted in a significant improvement in motility parameters, viability and membrane integrity of frozen-thawed ram sperm. The main constituents found in the herb of costmary extract are polyphenolic compounds, such as flavonoids and phenolic acids (Faraloni, 2018). Among flavonoids there are mainly glycosides of luteolin, apigenin and quercetin while phenolic acids are represented mainly by chlorogenic, caffeic and dicaffeoylquinic acids. The attacks of ROS during cryopreservation lead to reduction of oxygen and it is related to lipids peroxidation of the sperm membranes that destroys the structure of the lipid matrix. Flavonoids increase membranes integrity by preventing of free radicals production and lipid peroxidation in the membrane that induce oxidative damage to the membrane components (Daghigh Kia et al. 2016). Therefore, costmary extract may play a protective role against oxidative damage and scavenge produced free radicals from cells.

In conclusion, this study showed that supplementation of extender with 12 mL/dL Tanacetum balsamita L. extract has a beneficial effect on the quality of frozen-thawed ram semen.