Short-term storage sperm of coho salmon (Oncorhynchus kisutch) at 4 °C: Effect of sperm: Extender dilution ratios and antioxidant butyl-hydroxytoluene (BHT) on sperm function.

Short-term storage of semen is a necessary key procedure in fish; it allows maximizing the use of gametes. Nevertheless, sperm quality decreases during storage has been associated with oxidative stress damage due to an increase in reactive oxygen species (ROS) during storage. This study was designed to optimize a short-term storage protocol for Coho salmon (Oncorhynchus kisutch) spermatozoa, evaluating the effect of extender dilution and the addition of butyl-hydroxytoluene (BHT) antioxidant on sperm function parameters. In the first experiment, fresh semen was diluted in Storfish®: extender dilution (1:2 and 1:3) and a control sample undiluted and stored at 4 °C for 7-days. In both experiments motility (MO), viability and integrity of plasma membrane, mitochondrial membrane potential (MMP) and superoxide anion level (O2-) were evaluated at 0, 3 and 7 days. Result shows that, 1:3 dilution maintained a higher sperm function for a longer period time. In the second experiment, spermatozoa were suspended in Storfish® (1:3) supplemented with two different concentrations of BHT (1.0 mM and 2.0 mM) and a control sample without antioxidant and stored at 4 °C for 7 days. The results demonstrated that, antioxidant-supplemented samples greater MO than control samples (P < 0.05). The viability remained >75% during storage in all groups. MMP was higher in 2.0 mM BHT compared to 1.0 mM and control (P < 0.05), in addition, this concentration reduced O2- level (P < 0.05). In conclusion, sperm: extender dilution 1:3 and adding of 2.0 mM BHT in sperm storage extender may enhance protection sperm function in Oncorhynchus kisutch against effects harmful of the oxidative stress during the in vitro storage.

Positive effect of butylated hydroxytoluene (BHT) on the quality of cryopreserved cat spermatozoa.

The semen cryopreservation processes are associated with state of oxidative stress induced by high levels of reactive oxygen species (ROS), causing damage to functional spermatozoa. Whereby, antioxidants have been utilized to scavenge or neutralize the elevated levels of ROS. The aim of at the present study was to evaluate the effect of adding BHT to the freezing extenders on post-thaw characteristics of domestic cat spermatozoa. Semen samples were frozen in Tris-fructose-citric acid-based extender, supplemented with different concentrations of BHT (0.5 mM, 1.0 mM and 2.0 mM) and a control sample without antioxidant. After thawing, sperm samples were assessed for motility by computer-assisted sperm analysis and viability, acrosome integrity, superoxide anion production and membrane lipid peroxidation status by flow cytometry. In the study, the parameters of sperm motility and acrosome integrity were significantly higher in 2.0 mM BHT compared to sperm frozen in the extender with other concentrations and control (P < 0.05), in addition, this concentration reduced significantly the superoxide anion production and lipid peroxidation of the sperm. The results demonstrated that the supplementation of BHT to the freezing extender could protect the function and cellular structure of domestic cat sperm from cryoinjuries.

Change in the swimming pattern of Salmo salar spermatozoa caused by the high temperature of the sperm motility activation medium.

Fish are ectotherms and many have an external reproductive mode. An environmental factor which triggers fish reproductive activity in fish is water temperature. However, climate change is causing increasingly frequent events in which the water temperature varies rapidly; as a result, both in hatchery and in natural conditions, fish sperm are exposed to varying environmental temperatures during their journey toward the egg. This study was based on two experiments: The first experiment was designed to determine how storage at 4 °C for four days affected the sperm functions of Atlantic salmon (Salmo salar) sperm collected by either abdominal massage (stripping/Pure) or testicular dissection (testicular macerate/Macerated). Further, computer-assisted semen analysis (CASA) was used to compare sperm velocity parameters (VCL, VSL, and VAP) and progressivity (STR, LIN, and WOB) after motility activation at different temperatures (8 and 16 °C) of sperm collected by both methods (Pure vs Macerated). The results show that spermatozoa from Macerated samples maintained a higher sperm function when stored at 4 °C for 4 days compared to Pure sperm samples. In the second experiment, CASA determined that all parameters for sperm velocity (VCL, VSL, and VAP) and progressivity (STR (50%/55%), LIN (25%-32%), and WOB (51%-57%) were affected by activation temperature (P < 0.05) and that the motility patterns after activation at 16 °C (P < 0.05), specifically the LIN or STR swimming trajectories of the sperm differed between the two groups. In conclusion, the sperm quality of testicular Macerate was superior to that of Pure sperm abdominal mass, based on the higher quality of various sperm functions during short-term storage. Moreover, there was a significant effect of the temperature of the activation medium on sperm speed and progressivity (motility pattern) in the collected samples of testicular macerate. The sensitivity of Salmo salar spermatozoa to elevated temperature varies markedly between collection methods (Pure and Macerated).

Oxidative Stress Induces Changes in Molecular Markers Associated with Ferroptosis in Human Spermatozoa.

PURPOSE: Ferroptosis is a type of iron-dependent regulated cell death characterized by increased bioavailability of redox-active iron, loss of GPX4 antioxidant capacity, and oxidation of polyunsaturated fatty acid-containing phospholipids mediated by reactive oxygen species (ROS). The aim of this study was to evaluate the effect of oxidative stress induced by arachidonic acid (AA) on ferroptotic cell death in human spermatozoa. MATERIALS AND METHODS: Spermatozoa from normozoospermic donors were exposed to AA (5, 25, and 50 µM) for 1 hour at 37 ℃, including an untreated control. Oxidative stress was confirmed by evaluation of cytosolic and mitochondrial ROS production, viability, mitochondrial membrane potential (ΔΨm) and motility. Subsequently, molecular markers of ferroptosis including iron content, levels of GPX4, SLC7A11, ACSL4, IREB2 and lipid peroxidation were evaluated. The analyses were carried out using either flow cytometry, a microplate reader or confocal laser microscopy. RESULTS: AA-induced oxidative stress showed increased cytosolic and mitochondrial ROS production accompanied by impairedΔΨm, viability and motility in human spermatozoa. These results were associated with biochemical and molecular markers related to ferroptotic cell death including an increase in iron content in the form of ferrous (Fe2+) ions, SLC7A11, ACSL4, IREB2, a decrease in the level of GPX4, and an increase in the level of lipid peroxidation compared to the untreated control. CONCLUSIONS: This study revealed that AA-induced oxidative stress induces cell death with biochemical characteristics of ferroptosis in human spermatozoa, demonstrating another mechanism of alteration of sperm function induced by oxidative stress and could establish new therapeutic objectives to prevent the decrease in sperm quality mediated by oxidative stress.

Effect of exogenous sperm capacitation inducers on stallion sperm.

Although under appropriate laboratory conditions, sperm from different mammalian species can be capacitated in vitro, the optimal conditions for sperm capacitation in the stallion have been elusive. This study evaluated the effect of different capacitating inducers in Whitten and Tyrode media and assessed their impact on capacitation-related factors. Stallion sperm were incubated with different combinations of capacitating inducers at 38.5 °C in an air atmosphere. Sperm quality variables such as motility, mitochondrial membrane potential, and lipid peroxidation were assessed. Membrane fluidity and intracellular calcium levels were evaluated as early markers of capacitation, while tyrosine phosphorylation events and the sperm's ability to perform acrosomal exocytosis were used as late capacitation markers. Finally, these sperm were evaluated using a heterologous zona pellucida binding assay. The findings confirm that capacitating conditions evaluated increase intracellular calcium levels and membrane fluidity in both media. Similarly, including 2 or 3 inducers in both media increased tyrosine phosphorylation levels and acrosomal exocytosis after exposure to progesterone, confirming that stallion sperm incubated in these conditions shows cellular and molecular changes consistent with sperm capacitation. Furthermore, the zona pellucida binding assay confirmed the binding capacity of sperm incubated in capacitation conditions, a key step for stallion in vitro fertilization success. Further studies are needed to evaluate the effect of these conditions on in vitro fertilization in the horse.

Importance of banana flour and its effect on growth performance of broiler.

The main objective of this work was to study the effects of banana flour as energy sources on broiler performance. Seventy-five broilers were randomly distributed into five groups each with 15 broilers (n = 15 broilers/group). The broilers were grouped to maize-soybean meal diet as control, T1 : (5% of banana flour), T2 : (10% of banana flour), T3 : (15% of banana flour), and T4 : (20% of banana flour). The parameters analyzed in this research were body weight, daily weight gain, and daily feed intake at days 0, 10, 20, 30, and 40. The results showed no significant effects on body weight during the time of assessment, showing healthy values (>1,400 g) in all treatments (p > .05). Daily Weight gain was affected significantly during the days of assessment (p < .05). In all treatments and at different days of assessment, T3 showed the highest daily weight gain at day 10 (37.56 ± 4.52 g) compared to the other experimental treatments. Regarding daily feed intake, significant differences were observed at day 10 in the control and treatments T1 , T2 , T3 , and T4 compared to days 20, 30, and 40 (p < .05), being the highest value for T1 (35.14 ± 2.77).