Effect of ultracentrifuge and ultrasonic treatment of egg yolk before extender supplementation on ram semen quality and fertility outcome after storage at 4°C

Introduction: Egg yolk is an economical option for farm animal semen extenders. However, there are some potential disadvantages and concerns associated with whole egg yolk use as a component in extender media. Preparation of clarified egg yolk by high-speed centrifugation or probe ultrasonic treatment of egg yolk are novel, low-cost, fast, safe, and effective method in providing high-quality extenders for semen preservation. Ultracentrifuge and ultrasonic technology are mechanical methods used for separating and dispersing low-density lipoproteins (LDL), the primary protective component of egg yolk. Each method has its advantages and disadvantages, and as with any novel technique, it is essential to thoroughly evaluate and optimize the ultrasonic technology before using it for extender treatment. Therefore, the purpose of this study was to investigate the effect of ultracentrifuge and ultrasonic treatment of egg yolk before extender supplementation on the ram semen quality and fertility outcome after storage at 4°C for 72 hours.
Materials and methods: This study was conducted in a completely randomized block design with three treatments and six replicates. The treatments included: 1) Whole egg yolk (WEY as control), 2) Clarified egg yolk (CEY) prepared by high-speed centrifuge at 25000 g for 60 minutes, and 3) Ultrasonicated egg yolk (SEY) with 50% power (500 W) for 10 minutes. In the breeding season, semen samples of three fertility-proven Mehraban rams (live body weight of 76.37 kg, age of 3-4 years, and BCS of 3.5-4) were collected by artificial vagina once a week, over six consecutive weeks (as an experimental block). Collected ejaculates were passed the minimum quality requirements before dilution. Ejaculates of rams in each week were mixed and divided equally into three parts and diluted up to the concentration of 200×10⁶ spermatozoa/mL and stored at 4°C for 72 hours. Semen qualitative parameters were evaluated every 24 hours. Sperm viability, membrane integrity, and motility parameters were determined by eosin-nigrosin staining, the hypo-osmotic swelling test (HOST), and computer-assisted sperm analysis (CASA), respectively. In addition, a total of 42 non-pregnant Mehraban ewes, aged 2-4 years, with almost the same body weight and score, were used for the detection of fertility performance. The ewes' estrous cycle was synchronized by flurogestone acetate-loaded sponges and eCG (500 IU), and they were subjected to laparoscopic artificial insemination (LAI) 54 h after sponge removal, and the semen sample of each treatment was deposited into the uterine horns bilaterally. The pregnancy rate was detected by ultrasound scanning on d-45 after LAI.
Results and discussion: Semen storage for 72 hours in extenders containing ultrasonic and ultracentrifuged egg yolk (CEY and SEY treatments) significantly showed higher sperm membrane integrity compared to the WEY (P<0.05). Total motility in the CEY treatment (P=0.0807) and progressively motile spermatozoa in the SEY treatment (P=0.0897) tended to increase. Stored semen in the SEY extender showed significantly higher progressively motile spermatozoa compared to the WEY (control), 24 hours after storage (P<0.05). In addition, the amplitude of lateral head displacement (P=0.07) and linearity (P=0.09) tended to increase 24 hours after storage. The highest level of sperm membrane integrity, 48 hours after storage, and the highest viability and total motility, 72 hours after storage, were related to the SEY treatment (P<0.05). The progressive motility (P=0.10) and linear velocity (P=0.07) tended to increase 72 hours after sperm storage in the CEY treatment. Other parameters, as well as the fertility outcome after laparoscopic artificial insemination, were not affected by the treatments. Very few studies have been conducted on the storage of ram semen with a diluent containing ultrasonicated or ultracentrifuged egg yolk at 4°C. Most of the studies reported the effect of a diluent containing ultrasonicated or ultracentrifuged egg yolk after the freeze-thaw process. Consistent with the findings of this study, a study showed that total motility, progressive motility, and membrane integrity of sperm after freeze-thawing of dog sperm in a diluent containing ultrasonicated egg yolk were better preserved than in a diluent containing ultracentrifuged egg yolk. In contrast, another study showed that sperm motility parameters and membrane integrity of pig sperm after freeze-thawing were improved only in a diluent containing centrifuged egg yolk, which is contrary to this finding of this study. Consistent with the findings of this study, it has been shown that freezing of donkey semen in diluents containing ultrasonicated egg yolk with different ultrasonic intensities and durations increased total and progressive motility, curvilinear line velocity, average path velocity, and straight-line velocity after thawing. It could be deduced from our results that during the centrifugation process, some constituents of egg yolk that could be detrimental to sperm cells were pelleted, leaving only LDL molecules in the extender, which had good results in the kinematic parameters. Ultrasonic treatment of egg yolk improves the dispersion of egg yolk components in the extender media, and can improve the extender's ability to protect sperm during chilled semen storage, resulting in increased sperm survival and viability. The sperm plasma membrane is the structure that is mainly injured during semen preservation. The storage temperature at 4°C causes the membrane to move into a gel phase, increasing the loss of lipids that are part of the cell structure, causing a loss in membrane integrity. The results of the present study revealed that ultrasonic and ultracentrifuge treatment of egg yolk were more efficient in maintaining the sperm plasma membrane integrity than the whole egg yolk, probably due to higher surface reactivity and a probable increase in the interaction capacity of egg yolk components due to ultrasonic and ultracentrifuge treatment. In this study, the fertility outcome was not affected by treatments. The reason for this could be related to the artificial insemination method and the time of storage of the inseminated semen. The quality of stored semen decreases over time of storage, but the insemination technique used in this study could compensate for this decline in sperm quality.
Conclusions: Ultrasonic and ultracentrifugation processing of egg yolk with a change in its functional characteristics exert a positive effect on membrane integrity and some sperm motility characteristics, and can be used as a suitable approach to improve sperm preservation for artificial insemination.