Evaluation of frozen-thawed rooster sperm quality parameters in extender containing L-cysteine

Cryopreservation of the sperm causes irreversible damage to the sperm cell. The main reason of abnormal spermatozoa during storage at low temperatures are the occurrence of lipid peroxidation (LPO), production of reactive oxygen species and antioxidants imbalances (Tuncer et al. 2010). Also, factors such as the formation of ice crystals, production of reactive oxygen species, temperature changes, lipid peroxidation, changes in membrane composition, chemical toxicity due to cryoprotectants and osmotic stress, reduce the quality of sperm after thawing (Barbas and Mascarenhas 2009). The addition of antioxidants to the diluent of bird sperm during the freezing process reduces damage to sperm and maintains motility and viability rate of sperms after its thawing, which might affect the development of artificial insemination in birds. Cysteine is a sulfur-containing amino acid with antioxidant properties that can easily penetrate the cell membrane and increases the biosynthesis of intracellular glutathione and eliminates the free oxygen radicals (Kaeoket et al. 2010, Sariozkan et al. 2009). In recent years, researchers found that the use of cysteine as an antioxidant in the freezing of various mammalian sperm (Bucak et al. 2009; Tuncer et al. 2010) improve the mobility and the viability of frozen-thawed sperm. In this study, after two weeks of adaptation period, semen was collected from eight mature roosters (Ross 308). Initial semen assessments such as volume, progressive motility, concentration, viability, and percentage of abnormal sperm were conducted in the laboratory. Next, the samples with standard quality were split into four equal aliquots and diluted (1:30; v/v) with basic extender supplemented by different concentrations of cysteine (2.5, 5 and 7.5 Mm) at 37 ˚C. In this experiment, freezing procedure was conducted in two steps. So that the 3ml of extender containing different concentrations of cysteine and semen samples, were cooled slowly at 5◦C for 2 h to reach thermal equilibrium. Then, 1 ml of the semen extender (precooled to 5◦C) was added to the semen (extender plus semen) to provide a final concentration of 100 × 106 sperm/ml and 8% glycerol at a temperature of 5℃. Immediately after 1 h the sample was loaded into 0.25 mL straws (IMV, L’Aigle, France). Then, the straws were frozen in liquid nitrogen vapor, 4 cm above the liquid nitrogen, for 7 min, and plunged into liquid nitrogen for storage. After storing, the samples were evaluated twice with an interval of 15 days and frozen straws were thawed individually at 37℃ for 30 min in a water bath and then evaluated individually. The quality of sperms in the present work was evaluated after keeping them for 15 and 30 days in liquid nitrogen. The results of this study showed that the sample diluted with 2.5 mM of cysteine indicate the highest viability performance in keeping for 15 days in the liquid nitrogen compared to the control group and samples with other dosages of this amino acid (P<0/05). The viability of the sperms diluted for 30 days is considerably lower than that of those diluted during 15 days. The integrity of the membrane of sperms diluted with 5 mM of cysteine stored in liquid nitrogen for 15 days indicate the best performance compared to the samples with other dosages of amino acid and the control group (Table 3). Based on the Table 3, the minimum abnormality of sperms was seen in the group treated with 7.5 mM of cysteine for 15 days (18.3%) while the maximum sperm abnormality was for the sperm in control group of day 30 (30.6%). Moreover, results of table 4 indicated that the samples with 2.5 mM of cysteine had the best performance in terms of total and progressive motilities among other treatments and control group
(P<0/05). It has been reported that sperm storage in the presence of cysteine improves sperm motility after freezing. In this experiment, cysteine increased the viability and motility of sperm compared to the control group, which were consistent with the results of previous studies on boar (Kaeoket et al. 2010), ram (Coyan et al. 2011), dog (Michael et al. 2010) Cow (Topraggaleh et al. 2014) and Goat (Memon et al. 2012). Moreover, the results of this study was agree with Partyka and et al. 2013 that proposed the cysteine (5 mM) improves the integrity of the rooster sperm membrane. Bucak et al. (2009) reported that the addition of cysteine (5 mM) and terhalose (50 mM) increased the amount of live and motile ram sperm after freezing-thawing process. According to the results of Ahmadian et al. (2014), the low level of cysteine in the thrice-based diluent provides the best response in the process of freezing-thawing of ram sperm, which is consistent with the results of our research.