Effect of quercetin or butylated hydroxytoluene on cooled or frozen-thawed ram sperm quality

Cooling and freezing processes cause physical and chemical damage to sperm by cold shock and oxidative stress. This study aimed to evaluate the effect of two antioxidants on sperm parameters of cooled and frozen-thawed ram semen diluted in an egg yolk-based extender. Semen was collected from 30 rams and processed in two consecutive experiments to test the inclusion of different concentrations of quercetin and butylated hydroxytoluene (BHT) in an egg yolk-based semen extender. Dimethyl sulfoxide (DMSO) was added as a solvent to the semen extender in a ratio of 1 mL DMSO for 90 mg of quercetin and 1 mL DMSO for 880 mg of BHT. After collection, semen was diluted at 200 × 106 motile sperm/mL (control) and split into different groups in each experiment. In experiment 1, semen was diluted with the extender containing quercetin (Q5, 5 μg/mL; Q10, 10 μg/mL; Q15, 15 μg/mL) or DMSO alone (DMSO1, 0.055 μL DMSO per mL; DMSO2, 0.165 μL DMSO per mL). In experiment 2, semen was diluted with the extender with BHT (BHT1, 0.5 μg/mL; BHT2, 1 μg/mL; BHT3, 1.5 μg/mL) or DMSO alone (DMSO3, 0.375 μL DMSO per mL; DMSO4, 1.125 μL DMSO per mL). After dilution, the semen was divided into two aliquots. Treated ram sperm samples were also subjected to different storage methods. The first set of samples was cooled at 5 °C for 24 h, whereas the second set of samples was frozen-thawed. Sperm motility parameters and plasma membrane integrity (PMI) were evaluated immediately after dilution (0h) and 24 h after cooling and in the frozen-thawed samples via computer-assisted sperm analysis and epifluorescence microscopy, respectively. The inclusion of quercetin or BHT did not affect sperm motility parameters or PMI of fresh, cooled, or frozen-thawed sperm in this study (P < 0.05). However, further studies are needed to test the effects of these antioxidants on the fertility of cryopreserved ram semen.

Chlorogenic Acid Improves Quality of Chilled Ram Sperm by Mitigating Oxidative Stress

The purpose of this study was to investigate whether the addition of chlorogenic acid (CGA) to a sheep semen extender could improve the quality of chilled sheep sperm. Ejaculates (n = 80) were collected from five Hu rams with an artificial vagina. The ejaculates were mixed and divided into five equal parts, diluted with a CGA-free Tris–egg yolk extender (control), or supplemented with 0.2, 0.4, 0.8, and 1.2 mg/mL. The sperm kinematic parameters (viability, progressive motility), functional integrity of plasma membrane and acrosome, adenosine triphosphate (ATP) concentration and antioxidant parameters (Catalase (CAT), Superoxide dismutase (SOD) activity, total antioxidant capacity (T-AOC), ROS level and Malondialdehyde (MDA) content) were evaluated during storage of the semen. The results indicated that: PM, plasmatic membrane integrity and acrosomal integrity in 0.8 mg/mL CGA were higher (p < 0.05) from day 1 to 5. The ROS level in CGA groups was lower than the control (p < 0.05). CAT, SOD, ATP, and T-AOC were highest at 0.8 mg/mL concentration within 1 to 5 days. The above results indicated that the right concentration of CGA improved the quality of Hu ram sperm during chilling storage.

Centrifugal force assessment in ram sperm: identifying species-specific impact

Background Centrifugation is routinely employed in handling the ejaculates of some species, but it is not part of the commonly used protocols in ram. However, the development and implementation of new assisted reproductive technologies, alternative preservation models based on washing sperm from a cellular ageing-accelerating substance such as the seminal plasma, and basic studies in spermatology is associated with the use of centrifugation. This requires a specific evaluation of the centrifugation protocols considering the species-specific relationship with the potential damage produced by this procedure. No previous studies have determined the effect of different centrifugation forces on ram sperm. Therefore, we aimed to assess the performance of three centrifugal forces (600×g, 3000×g, and 6000×g for 10 min at room temperature) and their effects on ram sperm motility and functionality. Results Sperm motility and functionality parameters were assessed at 0 h and after 2 h of incubation at 37 °C. As expected, a higher cell packaging degree was obtained at high centrifugation forces (P ≤ 0.0001). Cell packaging was unstable at all centrifugal forces. Thus, there was a high cell resuspension rate after less than 2 min. Regarding sperm quality, there was a change in movement pattern of 3000×g and 6000×g centrifuged sperm after 2 h of incubation at 37 °C, characterized by an increase in rapid progressive motility, linearity, straightness, and beat frequency, and a decrease in medium progressive motility, curvilinear velocity, path velocity, and head lateral amplitude. Non-significant differences were obtained among the different treatments concerning the total viability. However, we observed a significant increase (P ≤ 0.05) in the percentage of viable apoptotic sperm in the samples centrifuged at 6000×g at 0 h. Conclusions Centrifugal forces equal to or greater than 3000×g induced some deleterious effects in ram sperm quality, and lower forces did not provide a successful cell packaging degree.

Effects of insulin on sperm cell quality in ram semen cooled at 5°C

Insulin is present in the seminal plasma and is involved in sperm activities like motility and capacitation. However, the effects of insulin on the viability of cooled ram sperm are not fully understood. Therefore, the objective of the current study was to evaluate the effect of insulin addition on ram sperm maintained at 5ºC. Sperm samples were collected from six healthy, mature Santa Inês rams. The ejaculates were divided into two aliquots with (insulin group) or without (control group) insulin (3 IU mL-1) in the semen extender, and then cooled at 5°C for 48 hours. Subsequently, the sperm cells were evaluated for motility and kinetics using computer-assisted semen analysis. The samples were evaluated for acrosomal integrity by fluorescein using isothiocyanate combined with peanut agglutinin (FITC-PNA) and membrane functionality by the hypoosmotic swelling test. The semen analysis was performed after 24 or 48 hours of cooling. There was an increased percentage of progressive sperm motility (%), straightness (%), linearity (%) and beat caudal frequency (Hz) in the insulin group after 24 and 48 hours of cooling (p < 0.05). However, insulin did not affect total sperm motility, sperm velocities (VSL, VAP and VCL) (μm seg-1), acrosomal integrity and membrane functionality during cooling (p > 0.05). In conclusion, the addition of 3 IU mL-1 insulin to ram semen extender improved the quality of sperm motility after cooling.

Effect of anisosmotic stress on the structural and functional integrity of the plasma and acrosomal membrane of ram sperm

The integrity of the plasma membrane (MP) and the acrosome (MA) have been two of the most studied seminal evaluation parameters due to their role as a cell boundary and because they are responsible for interactions between cells effective. To assessing more objectively the effects of osmotic stress on the integrity of the PM and MA, as well as the rate of change that occurred during seminal cryopreservation, five freshly collected ejaculates were evaluated, refrigerated at 5 ºC and thawed per ram/session during 5 consecutive weeks. Using eosin-nigrosin (EN) staining, vitality (VIT), morpho abnormalities and cellular response were evaluated after performing osmotic resistance (ORT) and endosmosis (HOST) tests. The direct effect of anysosmosis and cryopreservation on the dependent variables were analyzed using the GLM procedure (SAS®) and when differences were observed, the effects were quantified using the LSMEANS. All the sperm quality values studied were significantly affected (P <0.001) by cryopreservation (VIT, ORT, HOST). The ORT demonstrated how the acrosome was one of the structures most affected by cryopreservation (P <0.001). In conclusion, the present study confirms that anysosmotic stress affects the sperm cell in an important way, compromising the reference values that quantify semen quality, especially MA and MP.

Las subpoblaciones de espermatozoides y su calidad en fracciones producidas por la centrifugación de una sola capa en muestras frescas y normospérmicas de esperma de cordero

Single layer centrifugation (SLC) technique has been developed to select the best sperm population in the ejaculate in order to increase the fertilization rates by artificial insemination or in vitro fertilization. Normospermic ram semen samples containing 800 and 3,000 × 106 sperms/ml (C800 and C3000, respectively) were processed by SLC. Three sperm fractions were separated in each sample following silica-coloidal sperm centrifugation and sperm yield, quality and subpopulations were analyzed in each one. In C800 group, the sperm recovery rate did not vary in any studied fraction, but when samples were highly concentrated (C3000) the top fraction (F1) contained significantly higher spermatozoa than bottom fraction (F3). Also, it was observed that F1 in C3000 had got a significantly higher percentage of spermatozoa (53.2 %) than in C800, while the quantity of spermatozoa recovered in fraction 2 was lower (25.2 % vs 45.4 %). Based on the sperm motility parameters, three sperm subpopulations were identified: SP1, low velocity spermatozoa showing no progressive movement (19.1 %); SP2, rapid and progressive spermatozoa (43.7 %); and SP3, rapid spermatozoa but non-linear movement (37.2 %). While SLC has been implemented for sperm separation in suboptimal and/or low concentrated sperm samples, this trial demonstrates that SLC is not efficient to separate different sperm populations in normospermic ram sperm samples containing high concentrations of spermatozoa.

THE PRODUCTION OF FREEZE-DRIED EGG YOLK POWDER AND ITS EFFECT ON THE QUALITY OF GARUT RAM LIQUID SEMEN

This research aims to evaluate the protective potency of freeze-dried egg yolk powder on Garut ram sperm during the liquid semen preservation process. Semen with good sperm quality was divided into three groups and diluted using the following Tris diluents: fresh egg yolk-Tris (FEY-Tris), commercial egg yolk powder-Tris (CEY-Tris), and freeze-dried egg yolk powder-Tris (DEY-Tris). Semen that had been diluted was observed every 12 hours until sperm progressive motility was 50%. Sperm quality was tested for progressive motility percentages, viability, intact plasma membrane (IPM), and intact acrosome (IA). Fresh egg yolk had a particle size of 14460.00±330.76 nm, with polydispersity index value (PI) and zeta potential (ZP) being 1.00±0.11 and -9.22±0.30 mV, respectively. The particle size of commercial egg yolk powder was 877.90±168.86 nm with PI value 0.34±0.04 and ZP-28.7±1.24 mV. The particle size of freeze-dried egg yolk powder was 1296.00±86.73 nm, with PI value 0.70±0.04 and ZP -34.5±0.64 mV. Progressive motility percentage showed that DEY-Tris diluent managed to survive to 51.11±6.06% for 168 hours, compared to FEY-Tris (51.25±6.74%) which survived for 156 hours, although no longer than CEY-Tris diluent (53.47±6.33%) which survive for 180 hours (P0.05). Higher sperm motility was also supported by viability percentage, IPM, and IA, which were all higher (P0.05). In conclusion, freeze dried egg yolk powder mixed with Tris buffer was able to maintain sperm quality during the liquid semen preservation process.