SummaryThe aim of this study was to compare different concentrations of soy lecithin (LEC0.01%, LEC0.05% and LEC0.1%) with egg yolk (Control) in cooling extenders during the storage of semen at 5ºC for 5 days. Twelve dogs (n = 12) were selected, and semen was cooled and assessed after 2, 24, 48, 72, 96 or 120 h. At each time point, sperm were analyzed for kinetic patterns (using computer-assisted sperm analysis), mitochondrial activity (3′3- diaminobenzidine assay), lipid peroxidation (TBARS assay), DNA fragmentation (SCSA®) and plasma and acrosome membrane integrity (eosin/nigrosin and fast green/rose Bengal stains, respectively). The Control group (1814.4 ± 197.2) presented the highest rates of lipid peroxidation at 120 h. Conversely, progressive motility (42.8 ± 4%), linearity (45.4 ± 1%), and VAP (88 ± 3%) were higher in the Control group. In addition, there was lower mitochondrial activity in the Control group at 72 h. Therefore, our data show that lecithin used at these concentrations was not able to maintain sperm viability at as high qualities as would egg yolk. Moreover, the decrease in high mitochondrial activity and the persistence of sperm motility may indicate a compensatory mechanism in canine spermatozoa (i.e., glycolytic pathway). Furthermore, these higher lipid peroxidation indexes could indicate the necessity for future therapy using extenders and antioxidants over a long cooling time for dog sperm.

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Efeito da adição de glutationa na função e estresse oxidativo em sêmen ovino criopreservado Effect of glutathione on the function and oxidative status of ovine cryopreserved sperm

Sperm recovery from the caudae epididymides can be advantageous for preserving semen of endangered animal species. In this context, the domestic cat is a suitable model for the study of sperm physiology in endangered feline species and the research on epididymal sperm preservation combined with the use of reproductive biotechnologies including intracytoplasmic sperm injection (ICSI). The aim of the present study was to examine the sperm collected from the cauda and caput of the cat epididymis using functional tests. Testicles and epididymides from 5 adult tomcats were collected by orchiectomy and maintained at 4°C for 4 h, until semen collection. Semen samples were collected from the epididymal tail and head by careful dissection. Samples were then analysed for motility by computer assisted sperm analysis (CASA; only for the caudal sperm). The 3-3′ diaminobenzidine stain was used as an index of mitochondrial activity, the eosin nigrosin stain as an index of membrane integrity, the simple stain (fast green/Bengal rose) as an index of acrosome integrity, and the measurement of thiobarbituric acid reactive substances (TBARS) as an index of lipid peroxidation. Statistical analysis was performed using the SAS System for Windows (SAS Institute Inc., Cary, NC, USA; least significant differences test and Spearman correlation; P < 0.05). No motility was observed in samples collected from the epididymal head, whereas samples from the tail showed 50.0 ± 4.2% motile spermatozoa. Surprisingly, more spermatozoa with high mitochondrial activity were found in the epididymal head than in samples from the tail (74.0 ± 3.5 v. 50.0 ± 4.3%, respectively). Similarly, samples collected from the head showed a higher susceptibility against the attack of ROS (31.9 ± 5.5 v. 16.3 ± 7.1 ng of TBARS/106 sperm, respectively). Furthermore, epididymal head sperm showed a lower percentage of sperm with intact membrane and a higher percentage of sperm with intact acrosome (44.9 ± 3.3 and 78.4 ± 1.8 v. 66.4 ± 4.2 and 56.7 ± 4.4%, respectively). Our results demonstrate that, during maturation, feline sperm are subjected to high oxidative stress, as shown by the lipid peroxidation assay, which would lead to structural damage to biomolecules, DNA, lipids, carbohydrates and proteins, as well as other cellular components, such as mitochondria, and acrosomal impairment. Similar results were found in humans, in which higher levels of oxidative stress occurred in the post-testicular environment. The plasma membrane seems to be more resistant to damages. This may be due to the described rearrangement in the lipid profile occurring during maturation, but studies to test this hypothesis are still underway.

Mangalarga, due to its marching abilities, is the mostly widespread and numerous equine breed in Brazil. Furthermore, previous studies indicate that the semen of these horses is particularly susceptible to cryo-injuries. Therefore, the use of chilled semen is crucial when employing reproductive biotechnologies. However, previous studies indicate that chilled semen is highly impaired by the oxidative stress, which is caused by reactive oxygen species (ROS). An alternative to overcome the injuries caused by oxidative stress is antioxidant treatment, which requires the identification of those ROS that are the most deleterious. The aim of this study was to identify the most harmful ROS to Mangalarga sperm. Semen samples from 4 horses were collected, mixed with chilling media (Equimix®, Nutricell) and transported to the laboratory at 15°C. Samples were then incubated (1 h, 37°C) with 4 ROS inducing mechanisms: xanthine/xanthine oxidase (produces superoxide anion), hydrogen peroxide (4 mM), ascorbate and ferrous sulfate (4 mM; produces hydroxyl radical). Samples were analysed for motility using computer assisted sperm analysis (CASA). The 3-3′ diaminobenzidine stain was used as an index of mitochondrial activity, the eosin nigrosin stain as an index of membrane integrity, the simple stain (fast green/Bengal rose) as an index of acrosome integrity, sperm chromatin structure assay (SCSA) as an index of DNA fragmentation, and the measurement of thiobarbituric acid reactive substances (TBARS) an index of lipid peroxidation. Statistical analysis was performed using the SAS System for Windows (SAS Institute Inc., Cary, NC, USA; least significant differences test and Spearman correlation; P < 0.05). Results showed that Mangalarga sperm is highly susceptible to the hydroxyl radical. Samples treated with this ROS showed a lower percentage of sperm with high mitochondrial activity then samples treated with hydrogen peroxide (24.6 ± 5.9 v. 43.7 ± 6.8%, respectively). Similarly, lipid peroxidation (TBARS) was higher in samples treated with hydroxyl radical when compared with those treated with both superoxide anion and hydrogen peroxide (2037.7 ± 154.8, 681.2 ± 170.1, and 789.4 ± 124.5 ng/106 sperm). In addition, for all variables analysed using CASA except for ALH and BCF, samples treated with hydroxyl radical showed decreased quality when compared with the other samples. A positive correlation was found between TBARS and mitochondrial activity, indicating that the higher the sperm susceptibility of sperm against oxidative stress, the lower the mitochondrial activity. Level of TBARS also correlated negatively with most of the variables evaluated by CASA. The present results suggest that Mangalarga sperm is highly susceptible to the hydroxyl radical, a mechanism apparently related to the mitochondrial activity. Therefore, an alternative to overcome the deleterious influence of oxidative stress in semen of Mangalarga stallions would be the treatment with hydroxyl radical scavengers such as vitamins C and E, reduced glutathione, and other nonenzymatic antioxidants. The authors acknowledge Nutricell for the media used and CAPES for financial support.

Cryopreservation of equine semen has been widely studied by several research groups because of the large breed and individual variation in sperm freezability. A key factor in sperm cryopreservation is the high incidence of oxidative stress, an imbalance between reactive oxygen species (ROS) and antioxidant protection, which impairs sperm functionality by attacking plasma membrane, acrosome, mitochondria, and DNA. In order to study the resistance of equine spermatozoa to different reactive oxygen species (ROS), sperm samples from 4 Mangalarga stallions were collected using an artificial vagina. Samples were cryopreserved in extenders containing dimethylformamide (DMF) or methylformamide (MF). After thawing and washing, sperm samples were then incubated (1 h, 37°C) with 4 ROS inducer mechanisms: xanthine/xanthine oxidase (produces superoxide anion), hydrogen peroxide (4 mM), ascorbate/ferrous sulfate (4 mM; produced hidroxyl radical), and malondialdehyde (MDA, lipid peroxidation product). Samples were evaluated using the 3-3′ diamino benzidine (DAB) stain, as an indicator of mitochondrial activity; the eosin nigrosin staining, to evaluate plasma membrane integrity; the simple stain (fast green/Bengal rose), to assess acrosome integrity; and the measurement of thiobarbituric acid reactive substances (TBARS), a lipid peroxidation product. Statistical analysis was performed using the Student t-test and LSD test. Results showed that sperm mitochondrial potential of frozen-thawed samples in MF was highly susceptible to the attack of hydroxyl radical and hydrogen peroxide. No effect of ROS was observed on membrane and acrosome integrity. On the other hand, samples cryopreserved in DMF showed no differences in susceptibility to ROS. When evaluating the main effects of different extenders, results showed a higher protective effect of the MF extender on acrosome integrity and mitochondrial potential (MF: 12.1 ± 2.2 and 7.8 ± 2.3% v. DMF: 3.4 ± 0.7 and 1.1 ± 0.7%, respectively, P < 0.05). However, a negative effect of MF extender was observed regarding the percentage of sperm showing intact membrane and TBARS content (MF: 2.0 ± 0.8% and 517 ± 115 ng/106 sperm v. DMF: 20.6 ± 1.7% and 118 ± 44 ng/106 sperm, respectively, P < 0.05). A strong negative correlation was found between TBARS and plasma membrane integrity (r = -0.88; P = 0.004) for samples cryopreserved in DMF, whereas a positive correlation was found between TBARS and sperm with full mitochondrial potential (r = 0.73; P = 0.04). Results of the present study indicate that DMF may play a role in the protection of sperm against the attack of ROS. However, such action is apparently limited to the plasma membrane. On the other hand, the MF-supplemented extender exerts an intracellular protection. Therefore, the antioxidant therapy, especially hydrogen peroxide and hydroxyl radical scavengers, may be an alternative to improve the post-thaw quality of MF-supplemented cryopreserved semen in stallions, by increasing extracellular antioxidant capacity. The authors thank Nutricell for financial support and the media used in the present experiment.

GURGEL, J. R. C. Effect of the seminal plasma on stallion sperm susceptibility to distinct reactive oxygen species. [Efeito do plasma seminal sobre a susceptibilidade dos espermatozóides equinos às diferentes espécies reativas de oxigênio]. 2014. 72 f. Dissertação (Mestrado