Valquiria Hyppolito Barnabe

Influence of bovine sperm DNA fragmentation and oxidative stress on early embryo in vitro development outcome.

Sperm chromatin fragmentation may be caused by a number of factors, the most significant of which is reactive oxygen species. However, little is known about the effect of sperm oxidative stress (OS) on DNA integrity, fertilization, and embryonic development in cattle. Therefore, the goal of this study was to evaluate the influence of sperm OS susceptibility on the DNA fragmentation rate and in vitro embryo production (IVP) in a population of bulls. Groups of cryopreserved sperm samples were divided into four groups, based on their susceptibility to OS (G1, low OS; G2, average OS; G3, high OS; and G4, highest OS). Our results demonstrated that the sperm DNA integrity was compromised in response to increased OS susceptibility. Furthermore, semen samples with lower susceptibility to OS were also less susceptible to DNA damage (G1, 4.06%; G2, 6.09%; G3, 6.19%; and G4, 6.20%). In addition, embryo IVP provided evidence that the embryo cleavage rate decreased as the OS increased (G1, 70.18%; G2, 62.24%; G3, 55.85%; and G4, 50.93%), but no significant difference in the blastocyst rate or the number of blastomeres was observed among the groups. The groups with greater sensitivity to OS were also associated with a greater percentage of apoptotic cells (G1, 2.6%; G2, 2.76%; G3, 5.59%; and G4, 4.49%). In conclusion, we demonstrated that an increased susceptibility to OS compromises sperm DNA integrity and consequently reduces embryo quality.

Avaliaçäo da capacidade de penetraçäo de sêmen congelado de onça pintada (Panthera onca) em oócitos heterólogos

Assisted reproductive technologies can be viewed as one potential approach for safeguarding wild species. In this study were evaluated the fertility of captivity male jaguar (Panthera onca) and different capacitation media using the golden hamster zona free oocyte penetration assay. We used frozen/thawed semen from 3 animals housed at Bosque dos Jequitibas, Campinas/SP, to test the Percoll gradient, Swim-up and Swim-up + 1 h incubation (5% CO2 / 38°C), considering as penetration the spermatozoa head descondensation visualized within the oocyte. The results rate was greater for Percoll (26.5%) as compared to Swim-up (8.1%) (X2 = 19.93; p < 0.05). It was not observed penetration with Swim-up + 1 h incubation (5% CO2 / 38°C). It is concluded that Percoll and Swim-up are efficient methods to perform the golden hamster zona free oocyte penetration assay using frozen/thawed jaguar (Panthera onca) spermatozoa. The low rate of penetration could be related to the high rate of morphological abnormal spermatozoa observed in the samples examined.

The Stimulated Glycolytic Pathway Is Able to Maintain ATP Levels and Kinetic Patterns of Bovine Epididymal Sperm Subjected to Mitochondrial Uncoupling

Studies have reported the importance of mitochondria in sperm functionality. However, for some species, the glycolytic pathway appears to be as important as oxidative phosphorylation in ATP synthesis and sperm kinetics. These mechanisms have not been fully elucidated for bovine spermatozoa. Therefore, the aim of this study was to evaluate the role of mitochondria and the glycolytic pathway in ATP synthesis, sperm movement patterns, and oxidative homeostasis of epididymal spermatozoa in bovine specimens. We observed that mitochondrial uncoupling with protonophores significantly reduced ATP levels. However, these levels were reestablished after stimulation of the glycolytic pathway. We verified the same pattern of results for sperm kinetic variables and the production of reactive oxygen species (ROS). Thus, we suggest that, after its appropriate stimulation, the glycolytic pathway is capable of maintaining ATP levels, sperm kinetic patterns, and oxidative balance of bovine epididymal spermatozoa submitted to mitochondrial uncoupling.

Influence of Osteopontin in Bovine Uterine Tube Fluid on Sperm Binding and Fertilization in RCA-1 Lectin-treated Oocytes

This study was conducted to determine the effect of pre-exposure of oocytes to Ricinus communis (RCA-1) lectin and osteopontin (OPN) in uterine tube fluid (UTF) on in vitro sperm-egg binding and fertilization. In vitro-matured bovine oocytes were incubated (39 degrees C, 5% CO(2) in air) for 2 h in the following treatments: (i) 500 microl of fertilization medium (FM); (ii) 250 microl of FM with 0.25 ml of non-luteal ampullary uterine tube fluid (NLAUTF); (iii) 250 microl of FM with 250 microl of NLAUTF and 4 microl of RCA-1 lectin; (iv) 250 microl of FM with 250 microl of NLAUTF, a rabbit polyclonal antibody (1:200) against purified bovine milk OPN, and RCA-1 lectin; (v) 500 microl of FM and RCA-1 lectin. Following incubation, oocytes were washed, placed in FM with 2 microg heparin, and incubated with 1 x 10(5) frozen-thawed spermatozoa per 10 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zona pellucida counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate-orcein and observed to determine the presence of pronuclei. More sperm bound to the zona pellucida (mean +/- SEM) when oocytes were incubated in treatment 3 (59.0 +/- 5.5) than in treatments 2 (46.4 +/- 5.6), 4 (18.1 +/- 5.4), 5 (33.4 +/- 5.6) or 1 (32.5 +/- 5.6). More oocytes were fertilized when incubated in treatment 3 (91% +/- 3.0) than in 2 (84% +/- 3.0), 4 (40% +/- 3.0), 5 (77% +/- 3.0) or 1 (76% +/- 3.0). As in previous studies, this study suggests that RCA-1 lectin enhances binding of UTF-derived OPN to bovine oocytes, resulting in increased sperm-egg binding and fertilization in vitro and a possible role in fertilization.

Effect of two ram sperm capacitating media on acrosome reaction and zona-free hamster oocyte penetration test

An in vitro zona-free hamster oocyte penetration test was utilized in 24 trials in order to evaluate the capacitation media used for ram sperm. A pool of fresh semen was collected from three crossed breed rams. Two semen drops were washed by centrifugation and incubated in high ionic strength treatment (HIS) or in a defined medium with HEPES, on heat ewe serum and heparin. After the incubation to promote capacitation, simplified triple-stain technique was used to evaluate the spontaneous acrosome reaction of the capacitated sperm. Superovulation in 96 golden hamsters was induced by PMSG and hCG. The oocytes were treated with hyaluronidase and trypsin to remove, respectively, the cumulus cells and the zona pellucida. Oocytes and capacitated sperm were incubated during 3 hours for further penetration. Then, oocytes were fixed and stained, being evaluated under phase contrast microscope. No significant statistical difference (p >; 0.05) was found between media, concerning the penetration rate of the capacitated sperm and between number of sperm viable with acrosome reaction after the capacitation treatment using two different media. It was concluded that both media utilized were effective in capacitating ram sperm.

Susceptibility of Stallion Spermatozoa to Different Oxidative Challenges: Role of Seminal Plasma

Abstract Seminal cryopreservation provides several advantages in horse breeding. However, improvement on post‐thaw sperm survival is still necessary. One of the main factors known to impair post‐thaw quality of stallion’s sperm is the oxidative damage caused by reactive oxygen species (ROS). In this context, the aim of this study was evaluated the effects of ROS on stallion sperm and the possible influence of seminal plasma (SP). Toward this aim, 13 ejaculates from adult stallions (n = 13) were divided into two aliquots which were centrifuged (600g/10 minutes), and the SP was removed and reserved. Pellets were suspended in physiological saline solution (A) or SP (B). Each of these solutions was divided into four aliquots and subjected to challenge with different ROS (superoxide anion, hydrogen peroxide, and hydroxyl radical [OH−]) and the toxic by‐product of lipid peroxidation (malondialdehyde [MDA]). Samples were then evaluated for the susceptibility to lipid peroxidation (thiobarbituric acid reactive substances) plasma membrane integrity (eosin–nigrosin), acrosome integrity (Fast Green/rose bengal), mitochondrial activity (3′3 diaminobenzidine), and DNA integrity. Samples incubated in the presence of SP were highly impaired by OH− with regard to motility, plasma membrane integrity, mitochondrial activity, and DNA integrity. On the other hand, in the absence of SP, MDA was highly deleterious, especially with regard to motility, plasma membrane integrity, and mitochondrial activity. Thus, these results indicate that SP may have an important role on the protection of stallion sperm against the damages caused by MDA, an important product of lipid peroxidation. HighlightsSeminal plasma (SP) has a protective effect against oxidative damages.Hydroxyl radical in the presence of SP is the most deleterious reactive oxygen species (ROS) to equine spermatozoa.Malondialdehyde is more deleterious than ROS to equine spermatozoa in the absence of SP.Hydroxyl radical is extremely deleterious to equine sperm DNA.Malondialdehyde impaired membrane integrity and mitochondrial activity.

Efeito de dois tipos de diluidores e dois métodos de congelação na qualidade do sêmen criopreservado de caprinos

Foram colhidos 85 ejaculados de 4 reprodutores caprinos das racas Alpina, Saanen, Toggemburg e Anglonubiana, que foram divididos em 193 amostras de semen com o objetivo de proceder a congelacao nos diluidores a base de TRIS e a base de leite desnatado, usando 2 metodos de congelacao: com presenca e ausencia do plasma seminal. Analisaram-se as caracteristicas fisicas e morfologicas do semen logo apos a colheita. Foi observada variacao individual no volume, motilidade retilinea e progressiva e patologia espermatica dos ejaculados dos animais estudados. Apos a congelacao, foi feita avaliacao da motilidade retilinea e progressiva, do teste de termorresistencia e da porcentagem de acrossomos intactos dos espermatozoides. O diluidor a base de TRIS com 2,6% de gema de ovo mostrou-se mais eficiente na congelacao do semen de caprinos em relacao ao diluidor a base de leite desnatado. A retirada do plasma seminal nao alterou os resultados de congelacao do semen de caprinos. Apos 3 meses de estocagem em nitrogenio liquido, realizou-se uma nova analise da motilidade retilinea e progressiva dos espermatozoides, quando os ejaculados submetidos a retirada do plasma seminal e congelados em diluidor a base de TRIS apresentaram perda da motilidade retilinea e progressiva.

Epididymal contraction and sperm parameters are affected by clonidine.

The use of clonidine, a selective agonist of α2‐adrenoceptors, is related to the fertility impairment. Thus, it has been described that changes in the epididymal function are related to the loss of fertility. Therefore, this study was sought to further evaluate the effects of clonidine in the rat distal cauda epididymis contractions and its consequence in the sperm parameters. The in vitro effects of clonidine in the isolated distal cauda epididymis were evaluated by pharmacological experiments. The consecutive contractile responses for clonidine in distal cauda epididymis showed desensitization. The noradrenaline‐induced contractions were desensitized after in vitro clonidine pre‐treatment (10−5 m for 10 min). Clonidine was unable to alter the noradrenaline contractions if the in vitro pre‐treatment was made in the presence of idazoxan (α2‐adrenoceptor antagonist), whereas prazosin (α1‐adrenoceptor antagonist) was ineffective. Moreover, the in vitro clonidine pre‐treatment increased frequency and amplitude of spontaneous contraction of distal cauda epididymis. In addition, to induce in vivo desensitization of α2‐adrenoceptors, male Wistar rats were treated with crescent doses of clonidine and distal cauda of epididymis contraction and sperm parameters were analyzed. The in vivo treatment with clonidine diminished the potency of the contractions induced by adrenergic agonists and augmented the frequency and amplitude of spontaneous contraction of distal cauda epididymis. This treatment also altered the sperm transit time in epididymis, epididymal sperm reserves, sperm lipid peroxidation, and antioxidant enzymes activity. The results suggest that clonidine was able to affect the sperm quantity and quality by decreasing the transit time related to the increase in the frequency and amplitude of spontaneous contractions in epididymis, although the contractions induced by adrenergic agonists were desensitized.

Detecção de estro em jaguatirica (Leopardus pardalis) utilizando citologia vaginal

Utilizou-se a citologia vaginal por meio de diferentes metodos de coloracao para deteccao de cio em jaguatirica, Leopardus pardalis, pela estimulacao hormonal exogena e pela avaliacao das estruturas ovarianas por videolaparoscopia. Cinco femeas foram tratadas com eCG/hCG e FSH/LH a cada quatro meses pelo periodo de dois anos. Videolaparoscopia foi realizada apos cada tratamento utilizando-se cetamina-xilazina e isoflurano. Esfregacos vaginais foram obtidos 15 dias antes e apos a videolaparoscopia. As lâminas foram analisadas ao microscopio de luz quanto aos tipos celulares predominantes. Todos os animais apresentaram foliculos maduros (>2mm) e corpos luteos recentes em todas as intervencoes. Nao houve diferenca significativa entre os resultados obtidos na mesma coloracao de acordo com os tratamentos utilizados. Todas as tecnicas mostraram-se eficientes na deteccao de celulas superficiais queratinizadas anucleadas e nucleadas, intermediarias, parabasais e basais. Foi possivel determinar a fase de estro em Leopardus pardalis por meio da citologia vaginal

Inseminação artificial em cabras com sêmen congelado: resultados preliminares

Aiming to evaluate frozen goat semen in TRIS diluent with low egg yolk addition, progressive motility was evaluated soon after freezing and again 4 years later, resulting in 38.2 ± 7.4% and 31.4 ± 13.5%, respectively. Sixteen goats were oestrus synchronized through progesterone impregnated intravaginal sponges followed by PMSG injection on day 9. Vaginal sponges were withdrawn on day 11, with goats being artificially inseminated 46 hours later with semen frozen in straws and stocked during 4 years in liquid nitrogen. The nonreturn rate attained 31.2%. TRIS diluent with low egg yolk addition was considered as efficient in relation to frozen sperm evaluated both in vitro through progressive motility as in vivo through artificial insemination.