نانوامولسیون عصاره گیاهی رازیانه روی صفات اسپرم قوچ در نگهداری درشرایط سرد

Sperm preservation is essential for utilizing rams with superior genetics, and this process is achieved by reducing or halting metabolic activities. Due to the high content of unsaturated fatty acids, ram sperm is highly susceptible to oxidative stress. Short and long-term sperm storage can significantly enhance the success rate of artificial insemination (AI), offering numerous advantages over natural mating. This preservation is made possible by using various additives.The natural storage duration of ram sperm without protective agents is quite limited. However, when preserved in extenders containing additives such as egg yolk, sperm can be maintained for up to 24 hours at refrigeration temperatures. Nevertheless, cold storage for several days can improve sperm quality by enhancing motility, viability, and membrane integrity. Artificial insemination (AI) is one of the most effective methods for genetic improvement in livestock, but it still faces limitations in sheep due to variable results, as the success of this method depends on various factors (Farahavar et al, 2023). Compared to traditional methods, laparoscopic AI (lone et al, 2022) provides greater precision and improves fertilization success. The first successful artificial insemination was performed on a dog in 1784 by an Italian physiologist, and the technique was later adapted for cattle (Zamiri et al, 2016). Today, AI is widely employed across various livestock species, including cattle, sheep, goats, pigs, and horses (Nategh Kandroud et al, 2021). This method facilitates the dissemination of superior genetic traits, enabling the simultaneous insemination of multiple ewes using sperm from a single ram, which is not feasible through natural mating. Additionally, AI helps prevent transmitting sexually transmitted diseases (Zamiri et al, 2016). The success of AI relies on accurate sperm collection, evaluation, and the appropriate use of extenders and preservatives (Sultanpour et al, 2011). This study was conducted at the Khalatpoushan educational and research station of the university of Tabriz. During the experiment, the rams were kept in a sheltered facility. Semen was collected from four rams under natural conditions, and the rams were adapted to semen collection using an artificial vagina two weeks before the start of the experiment. For semen collection, a mating pen and an artificial vagina were used. The artificial vagina was filled with water at 45-50°c, and semen collection was carried out under hygienic conditions. The samples were quickly transferred to the laboratory for evaluation. A total of 20 ejaculates were collected during the breeding season. Fresh semen samples were evaluated for volume, wave motion, progressive motility, non - progressive motility, percentage of live and abnormal sperm, sperm concentration, and pH using routine methods. Samples selected for the treatments had a volume greater than 1.5 ml, progressive motility above 70%, and abnormalities less than 15%.
To prepare the extender, 80 ml of a solution containing 7.5 gr tris, 250 mg fructose, 1 gr citric acid, 10,000 units of penicillin, and 100 mg streptomycin was mixed with 20 ml egg yolk. Each semen sample was diluted at a ratio of 1:7 with the extender solution, and for each treatment, four microtubes (0.5 ml each) were filled.
The treatments were as follows:
Control group: base extender without fennel nanoemulsion.
Treatment 1: 10 µl fennel nanoemulsion per ml of base extender.
Treatment 2: 20 µl fennel nanoemulsion per ml of base extender.
Treatment 3: 30 µl fennel nanoemulsion per ml of base extender
This study aimed to reduce sperm damage and extend its lifespan through cooling. A tris-based extender enriched with fennel extract nanoemulsion was employed, and its effect on the quantitative and qualitative traits of ram sperm was evaluated during the breeding season. The rams were conditioned to semen collection via an artificial vagina for 2 to 3 weeks, and a total of 20 ejaculates were collected from four rams twice weekly. Then the samples were evaluated for volume, wave motion, total motility, progressive motility, percentage of live sperm, and concentration. The samples were divided into four groups: a control group without any additives, and three experimental groups in which 10, 20, and 30 μl of fennel nanoemulsion were added per ml of tris-based extender. All samples were stored at 4°c for fifteen days. On days 1, 5, 10, and 15 of storage, sperm viability and kinematic parameters were assessed. Adding fennel nanoemulsion to the tris-based extender significantly improved sperm quality traits, including progressive motility, in-place motility, total motility, viability, and the hypo-osmotic swelling test (HOST). Furthermore, this additive reduced abnormalities, acrosomal membrane damage, and malondialdehyde (MDA) levels, extending the storage longevity of sperm compared to the control group. The treatment significantly increased viability, progressive motility, and HOST (p<0.001), while reducing MDA levels (p<0.001). Progressive motility in the 20 and 30 μl treatments was higher than in other groups, and viability and HOST in the 20 μl treatment were superior to other treatments. The control group exhibited the highest percentage of abnormalities. MDA levels were the lowest in the 30 μl group, followed by the 20 μl group; both significantly lower than in the 10 μl and control groups. Additionally, a significant positive correlation was observed between acrosomal abnormalities and structural defects in sperm (p<0.05). The characteristics and traits assessed in fresh sperm are presented in Table 1. The range of measured values falls within the normal limits for ram sperm, including ejaculate volume (2 to 4 milliliters), percentage of live sperm (70-95%), wave motility (4 to 5 percent), and percentage of abnormal sperm (3-10%). These values are consistent with the results obtained by Naghdi et al (2021). The observed variability in the quantitative and qualitative traits of ram sperm may be the result of a broad influence from physiological, genetic, environmental, and nutritional factors affecting sperm production. Factors influencing the biological characteristics of semen include volume, concentration, motility, percentage of live sperm, and percentage of abnormal sperm, include season, age, breed of the ram, frequency of ejaculation, and the interval between collected ejaculations, as well as environmental conditions (day length, temperature, and humidity) and management practices (Yoto et al, 2011).The findings suggest that the use of fennel extract nanoemulsion in a tris-egg yolk extender aids in maintaining the quality of ram sperm and extends its liquid storage life under cold conditions.