M. A. Torres

An Efficient Technique to Detect Sperm Reactive Oxygen Species: The CellRox Deep Red ® Fluorescent Probe

Heat stress and testicular traumas increase sperm Reactive Oxygen Species (ROS) resulting in Oxidative Stress (OS) and injury of sperm. The fluorescent probe CellROX Deep Red® is not already used to detect ROS in spermatozoa. On this way, this study aims to evaluate its efficiency in detecting ROS in ram sperm. For that, it was used ejaculates from rams performing the in vitro induction of sperm OS in T0, T50 and T100. T0 was semen sample that was not submitted to OS induction, T50 was 50% induced to OS induction and T100 was entire submitted to OS induction. The production of ROS was detected by CellROX Deep Red®. Data polynomial regression analysis was performed and the result of determination coefficient was 0.728. Besides, sixteen rams were submitted to scrotal insulation during 72 hours performing the in vivo induction of OS sperm. Analyses of sperm motility, morphology, DNA fragmentation and ROS (detected by CellROX Deep Red®) were done two times before the scrotal insulation and two times afterwards. Data analysis of variance was performed from the periods (before x after) and it is observed that after scrotal insulation the total and progressive motility decrease (p<0.05) and total and major sperm defects and sperm DNA fragmentation increase (p<0.05). Moreover, after insulation, it was observed increase (p<0.05) in sperm showing moderate and intense OS. Thus, it is possible to conclude that CellROX® fluorescent probe is able to detect ROS production in ram sperm by in vitro and in vivo induction being a new technique to detect sperm OS.

Determination of fatty acid profile in ram spermatozoa and seminal plasma

Fatty acids are important in male reproductive function because they are associated with membrane fluidity, acrosome reaction, sperm motility and viability, but limited information exists about the fatty acid profile of ram semen. Our aim was to determine the fatty acid composition in ram spermatozoa and seminal plasma. Sixty ejaculates were obtained from three ram (20 ejaculates/ram) using artificial vagina. Ram spermatozoa (RS) and seminal plasma (SP) were separated using centrifugation, and the fatty acids were analysed by gas chromatography. Total lipids obtained in ram spermatozoa were 1.8% and 1.6% in seminal plasma. Saturated fatty acid (SFA) was proportionally major in SP (66.6%) that RS (49.9%). The highest proportions of SFA corresponded to C4:0 (RS = 16.3% and SP = 28.8%) and C16:0 (RS = 16.3% and PS = 20%). The most important unsaturated fatty acid (UFA) was docosahexaenoic acid (DHA), 44.9% in RS and 31.5% in SP. The profile of fatty acid and their proportions showed differences between spermatozoa and seminal plasma.

Novel Flow Cytometry Analyses of Boar Sperm Viability: Can the Addition of Whole Sperm-Rich Fraction Seminal Plasma to Frozen-Thawed Boar Sperm Affect It?

Boar semen cryopreservation remains a challenge due to the extension of cold shock damage. Thus, many alternatives have emerged to improve the quality of frozen-thawed boar sperm. Although the use of seminal plasma arising from boar sperm-rich fraction (SP-SRF) has shown good efficacy; however, the majority of actual sperm evaluation techniques include a single or dual sperm parameter analysis, which overrates the real sperm viability. Within this context, this work was performed to introduce a sperm flow cytometry fourfold stain technique for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential. We then used the sperm flow cytometry fourfold stain technique to study the effect of SP-SRF on frozen-thawed boar sperm and further evaluated the effect of this treatment on sperm movement, tyrosine phosphorylation and fertility rate (FR). The sperm fourfold stain technique is accurate (R2 = 0.9356, p > 0.01) for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential (IPIAH cells). Centrifugation pre-cryopreservation was not deleterious (p > 0.05) for any analyzed variables. Addition of SP-SRF after cryopreservation was able to improve total and progressive motility (p < 0.05) when boar semen was cryopreserved without SP-SRF; however, it was not able to decrease tyrosine phosphorylation (p > 0.05) or improve IPIAH cells (p > 0.05). FR was not (p > 0.05) statistically increased by the addition of seminal plasma, though females inseminated with frozen-thawed boar semen plus SP-SRF did perform better than those inseminated with sperm lacking seminal plasma. Thus, we conclude that sperm fourfold stain can be used to simultaneously evaluate plasma and acrosomal membrane integrity and mitochondrial membrane potential, and the addition of SP-SRF at thawed boar semen cryopreserved in absence of SP-SRF improve its total and progressive motility.

Seminal plasma arising from the whole boar sperm-rich fraction increases the stability of sperm membrane after thawing.

Boar spermatozoa arising from the sperm-rich ejaculate fraction are reported to have a more stable plasma membrane and are more resistant to cold shock and premature acrosome reaction than spermatozoa from the whole ejaculate. Furthermore, seminal plasma (SP) can increase the cryotolerance of boar spermatozoa, and in other domestic species, it has the ability to reverse cryopreservation damage. This study aimed to evaluate the effects of boar SP arising from the whole sperm-rich ejaculate fraction (SP-SRF) on the integrity, stability, and peroxidation of sperm membranes after thawing. Each ejaculate ( = 24) was divided among 4 treatments: control (CT), centrifuged and suspended in autologous SP-SRF (CS), centrifuged with withdrawn SP-SRF (CW), and post-thawed SP arising from the whole sperm-rich fraction addition to CW (CWSP). After thawing, all treatments were incubated for 5, 60, and 120 min and were analyzed for membrane integrity, fluidity, and peroxidation by flow cytometer. The absence of SP-SRF increased the lipid disorder ( < 0.05) but had no effect on lipid peroxidation ( > 0.05) or membrane integrity ( > 0.05). However, the increase in lipid disorder by withdrawal of SP-SRF was reversed by SP-SRF addition ( < 0.05) to the post-thawing medium, whereas plasma and acrosomal membrane integrity ( > 0.05) and lipid peroxidation ( > 0.05) were unchanged. In conclusion, despite the centrifugation effects, the addition of SP arising from the whole sperm-rich fraction to post-thawed boar semen decreased sperm lipid disorder without an influence of the sperm membrane integrity and peroxidation.

Absence of seminal plasma from sperm-rich fraction decreases boar sperm quality characteristics during the course of liquid storage

Seminal plasma (SP), the fluid that surrounds the sperm cells, is known to exert substantial influence on sperm physiology. The SP has a pivotal role in sperm function in vivo, and due to its components, it functions in an ambiguous manner in vitro, simultaneously possessing deleterious and beneficial effects. This experiment aimed to describe the differences between the presence or absence of SP from the sperm-rich fraction on some spermatozoa characteristics (kinetics, plasma and acrosome membrane integrity, lipid peroxidation and capacitation-like changes). Furthermore, this experiment focused on distinguishing the effects of SP on the variables evaluated from the effects of centrifugation during SP removal. Total and progressive sperm motility, as well as integrity of plasma and acrosome membranes, were less (P <  0.05) in the absence of SP. Membrane lipid peroxidation (P <  0.05) and sperm membrane stability (P < 0.05) did not differ among treatments. The SP from the sperm-rich fraction is important for the maintenance of adequate structural and functional characteristics of extended liquid boar semen and should be present in seminal doses throughout storage. Furthermore, the detrimental effect on the variables evaluated was caused solely by the absence of SP and not by the process of removal through centrifugation at 500 x g for 10 min.

Nitric oxide in frozen-thawed equine sperm: Effects on motility, membrane integrity and sperm capacitation

Nitric oxide (NO) is a reactive nitrogen species (RSN) that, over the years, has been shown to be integrated with biological and physiological events, including reproductive processes. NO can affect the functionality of spermatozoa through free radical scavenging, deactivating and inhibiting the production of superoxide anions (O2.-). However, the role of NO in mammalian spermatozoa physiology seems paradoxical. The aim of this study was to investigate the effects of NO on motility, hyperactivation, membrane integrity, peroxidation, and capacitation in cryopreserved equine sperm. Ejaculates were collected and cryopreserved. After thawing, samples were centrifuged, suspended in an in vitro fertilization (IVF) medium and incubated with the following treatments: 1) C = control (IVF); 2) A = l-arginine (10 mM - In); 3) L = L-NAME (1 mM - Ih); 4) M = methylene blue (100 mM - Re); 5) AL = L-arginine + L-NAME (In + Ih); 6) AM = L-arginine + methylene blue (In + Re). The samples were evaluated for spermatic kinetics by CASA and other analyses [plasma and acrosomal membranes used the propidium iodide (PI) and Pisum sativum agglutinin (PSA), detection of tyrosine residues phosphorylation in the membrane (F0426), nitric oxide (DAF-2/DA), lipid peroxidation (C11-BODIPY581/591)] by flow cytometry. The l-arginine treatments reduced MOT, PROG, RAP and LIN only at time 0 min compared to the control and L-NAME. These treatments (MT and MP, VAP, VSL, LIN, RAP) also reduced the sperm movement characteristics but only at the beginning of the incubation period. After this period of incubation, motility recovered. NO removal by methylene blue almost completely inhibited sperm motility, but these treatments had the highest percentages of intact membranes. l-arginine treatments improved acrosome reactions and differed from M and AM. NO production, tyrosine phosphorylation and lipid peroxidation did not differ among treatments, except for M and AM, where a reduction in these variables was detected. Therefore, equine sperm capacitation and the acrosome reaction are part of an oxidative process that involves the participation of ROS, and NO plays an important role in the maintenance and regulation of motility, hyperactivation, induction of acrosome reaction and possibly in capacitation, which are indispensable processes for the fertility of equine sperm.

Dietary inclusion of fish oil changes the semen lipid composition but does not improve the post-thaw semen quality of ram spermatozoa

The aim of this study was to investigate the effects of dietary fish oil (FO) time-response on the fatty acid profile, cholesterol levels and sperm cryosurvival in ram semen. Criollo Araucano rams were randomly assigned to two groups (n=4) according to the type of supplementation: a control group without FO and a supplemented group fed a diet with 3% FO for 8 weeks. The semen lipid profile and post-thaw sperm quality were analyzed at weeks 0 (pre-supplementation), 4, 8, 12 and 16 (post-supplementation) to evaluate the effects of FO supplementation by time interaction. Post-thaw sperm quality was determined by CASA and flow cytometry. In spermatozoa, the supplemented group increased the linoleic acid (C18:2n6c) and docosahexaenoic acid (DHA; C22:6n3) with levels higher at week 16 (P<0.05). The effect of FO on cholesterol concentration in sperm was significant at the end of the experiment (week 16). In seminal plasma, statistical differences of butyric acid (C4:0), palmitic acid (C16:0), stearic acid (C18:0), eicosatrienoic acid (C20:3n3) and DHA were observed at week 12. The cholesterol concentration was not affected by dietary treatments (P>0.05). However, the post-thaw sperm quality of the FO treatment group decreased. Motility percentage decreased 50% and spermatozoa with permeable plasma membrane and reacted acrosome were higher (63%) at week 16 than the control group. These results showed that DHA was effectively incorporated into semen through dietary supplementation with FO, but evaluations of post-thaw sperm quality confirm alteration specificity related to the structure of the lipid bilayer.