بررسی اثر مکمل‌سازی جیره با منابع خوراکی حاوی اسیدچرب امگا-3 و امگا-6 برکیفیت و باروری اسپرم منجمد خروس‌های گله مادرگوشتی

Introduction
One of the major costs in commercial poultry production is the purchase and upkeep of roosters, which are the sires and key contributors to flock fertility. Although roosters make up only about 10 to 15 percent of the flock, their genetic quality accounts for over 50 percent of the flock’s fertility. Finding cost-effective solutions, such as artificial insemination, to reduce the expenses associated with acquiring and maintaining breeding roosters is a major concern for producers. Unlike in mammals, artificial insemination is not used in birds because sperm quality and fertility decline after the freezing and thawing processes. Cryopreserving bird sperm would allow us to use roosters with excellent genetics without them needing to be present. This would reduce genetic defects, lower costs, and help with the global distribution of sperm. It would also decrease disease transmission risks, prevent extinction, and create gene banks for better breeds. Despite extensive efforts, developing an effective cryopreservation method for avian sperm remains a challenge. The lower fertility rates observed with cryopreserve poultry sperm, compared to other animals are a significant hurdle for commercial applications. This difficulty may be related to the unique physiological features of bird sperm. Many hypotheses have been proposed about avian sperm, including the idea that poultry sperm contains a distinctive fatty acid profile, rich in omega-6 fatty acids. Supplementing sperm with essential fatty acids like omega-3 through dietary means has been shown to improve its reproductive performance. The success of cryopreservation heavily relies on maintaining sperm’s biological functions, which are influenced by factors such as extenders, cooling rates, and thawing protocols. Research has focused on optimizing parameters for sperm survival after thawing, including motility, viability, membrane integrity, and structural stability. Despite these efforts, fertility rates above 60% using cryopreserved sperm have not been reached. Studies indicate that providing diets rich in omega-3 fatty acids can enhance various sperm quality parameters and boost fertility. Dietary fats’ type and source affect sperm performance. This is because of their involvement in intricate molecular processes and their role as hormone and signaling pathway precursors, which are crucial for sperm function. Research on fatty acids’ impact on sperm uses fresh samples. Data is lacking on whether dietary supplements containing essential fatty acids can improve cryopreserved and thawed sperm quality. In this study, besides assessing motility and viability, parameters such as plasma membrane integrity, sperm morphology, membrane stability, lipid peroxidation, and DNA fragmentation will be evaluated.
Materials and Methods
Thirty-six 42-week-old Ross 308 roosters were randomly allocated to four groups (n=6/group) and housed in individual cages under standard conditions (21-24°C, 75% humidity, 15L:9D photoperiod). Experimental diets included: 1) Control (no oil), 2) 2% flaxseed oil, 3) 2% sesame oil, and 4) 1% flaxseed + 1% sesame oil. All diets were isoenergetic and isonitrogenous, formulated using WUFFDA software. Semen was gathered using abdominal massage on days 1, 20, 40, and 60. It was immediately diluted (1:20) in Lake’s extender, which had 5% glycerol, and held at 5°C for 2 hours. Afterward, it was frozen in 0.25 mL straws with liquid nitrogen vapor before being stored at -196°C. Thawing was performed in 4°C water for 3 minutes. Motility was assessed using computer-assisted sperm analysis (CASA). Viability was determined by eosin-nigrosine staining. Membrane integrity was measured with the hypo-osmotic swelling test (HOST). Lipid peroxidation (MDA) was measured using the thiobarbituric acid assay. Morphology was evaluated by phase-contrast microscopy.
For fertility trials, pooled semen from each group was used to inseminate 20 hens/group (250 μL containing 300×10⁶ sperm). They gathered the eggs for five days after insemination, placed them in the incubator, and candled them on day 10 to determine their fertility. The data obtained from this experiment were statistically analyzed using the GLM procedure in SAS software. The comparison of means was conducted using the Tukey test at P<0.05.
Results
Flaxseed oil (2% of diet) or a mix of flaxseed and sesame oil (1:1 ratio) improved total motility, progressive motility, sperm lipid peroxidation, membrane integrity, sperm viability, and fertility. These results were superior to control and sesame oil treatments (P < 0.05). Fertility rates were significantly higher in the flaxseed (80%) and mixed oil (79%) groups versus control (60%, P<0.05), though hatchability did not differ significantly. The findings show that dietary flaxseed oil or its combination with sesame oil significantly enhances frozen-thaw sperm quality in broiler breeders. The improvements in motility, membrane integrity, and oxidative stability likely result from increased incorporation of omega-3 fatty acids (particularly DHA) into sperm membranes, improving fluidity and cryoresistance. The antioxidant properties of omega-3s may also mitigate freeze-thaw induced lipid peroxidation.
Conclusion
The study suggests that adding 2% flaxseed oil or a 1:1 flaxseed-sesame mixture to rooster diets can improve frozen-thawed semen quality and fertility in commercial poultry. Future research should optimize fatty acid ratios and evaluate long-term reproductive performance. The protocol provides a practical approach to reducing male maintenance costs while enabling global semen distribution in breeding programs.