C A Baptista Sobrinho

University of São Paulo, San Paulo, São Paulo, Brazil

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Publications (3)7.25 Total impact

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    ABSTRACT: 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 4h, 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/10(6) 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.
    Reproduction Fertility and Development 01/2011; 23(1):218-219. · 2.58 Impact Factor
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    ABSTRACT: One of the main causes of poor quality of frozen-thawed dog sperm is oxidative stress (i.e. higher production of reactive oxygen species not compensated by improved antioxidant protection). This event is known to impair sperm functionality by attacking plasma membrane, acrosome, mitochondria, and DNA. Spermatozoa are particularly susceptible the oxidative stress, mainly due to the reduced cytoplasm and the high content of polyunsaturated fatty acids (PUFA) in the membrane, which allows the spermatozoa to be motile and confers a higher resistance against the damages caused by cryopreservation, but makes the sperm more susceptible to the attack of the reactive oxygen species (ROS). The present study aimed to evaluate the effects of antioxidant supplementation on semen extender (Tris-egg yolk-citrate-glicerol) with glutathione (GSH) and vitamin E on the quality of cryopreserved dog sperm. Ejaculates of 12 dogs were divided in pools of 3 ejaculates with at least 70% of motility. Each pool was diluted with 7 different extenders for treatment groups as follows: control, vitamin E (1, 5, and 10mM), and reduced glutathione (GSH; 1, 5, and 10mM) and submitted to cryopreservation. Samples were thawed (37°C/30') and evaluated for motility, vigor, percentage of sperm showing intact membrane (eosin/nigrosin), and acrosome (simple stain fast-green and bengal rose), mitochondrial activity (3-3'-diaminobenzidine-DAB), and sperm susceptibility to oxidative stress (TBARS). Statistical analyses were performed using the SAS system for Windows (SAS Institute Inc., Cary, NC, USA; least significant differences test and Spearman correlation; P<0.05). Samples treated with 1mM of GSH showed a higher percentage of sperm with intact membrane when compared with the control (11.21±2.84 and 6.21±1.16%, respectively; P<0.05). On the other hand, treatment with 5mM of GSH showed better results regarding mitochondrial activity. Vitamin E supplementation also played a protective role on mitochondrial activity; samples treated with 1mM showed a lower percentage of DAB III sperm (cells with severely compromised mitochondrial activity) when compared with the control group (5.61±0.7 and 8.62±1.05%, respectively; P<0.05). Both vitamin E and GSH are important non-enzymatic antioxidants responsible for the destruction of the hydroxyl radical. Despite the positive influence of these antioxidants on mitochondrial status, no effect was found on the other variables studied. These results indicate that the action of both antioxidants in dog sperm would be mainly intracellular. Furthermore, other ROS could be responsible for the other damages caused by cryopreservation on the other sperm functionalities (i.e. membrane, acrosome, DNA, oxidative status). Therefore, the use of a combination of enzymatic and non-enzymatic antioxidants could be an alternative to overcome the deleterious influence of oxidative stress in cryopreserved semen of dogs.
    Reproduction Fertility and Development 01/2011; 23(1):152. · 2.58 Impact Factor
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    ABSTRACT: The objective was to determine if treatment with dexamethasone (to mimic stress) has a deleterious effect on the spermiogram and on the composition of seminal plasma in the dog and whether adverse effects were reduced by oral supplementation with Vitamin E. Eighteen adult male Rottweiler dogs were randomly allocated in a 2 x 2 factorial treatment design (with or without dexamethasone treatment versus with or without Vitamin E supplementation). Dogs in the supplemented group received 500 mg of alpha-tocopherol (Vitamin E)/dog/day per os for 10 weeks. Dexamethasone (0.01 mg/kg/day i.m.) was given once daily for 7 days, starting 7 days after the onset of Vitamin E supplementation. Food intake, body condition score and body weight were assessed daily. Semen collections (digital manipulation) were performed twice weekly for 14 weeks and blood samples (for plasma concentrations of cortisol and testosterone) were collected once a week. Dexamethasone treatment significantly reduced ejaculate volume and increased thiobarbituric acid-reactive substances (TBARS) in the seminal plasma. In contrast, supplementation with Vitamin E increased sperm motility, vigor and concentration and decreased the percentage of major sperm defects. In conclusion, dexamethasone treatment (to mimic stress) had a deleterious effect on the spermiogram and on the seminal plasma lipid peroxidation in dogs; however, some of these effects were prevented by oral supplementation with Vitamin E.
    Theriogenology 11/2006; 66(6-7):1610-4. · 2.08 Impact Factor