Gislayne Christianne Xavier Peixoto

Recovery and cryopreservation of epididymal sperm from agouti (Dasiprocta aguti) using powdered coconut water (ACP-109c) and Tris extenders

The objective was to compare the use of powdered coconut water (ACP-109c; ACP Biotecnologia, Fortaleza, CE, Brazil) and Tris extenders for recovery and cryopreservation of epididymal sperm from agouti. The caudae epididymus and proximal ductus deferens from 10 sexually mature agoutis were subjected to retrograde washing using ACP-109c (ACP Biotecnologia) or Tris. Epididymal sperm were evaluated for motility, vigor, sperm viability, membrane integrity, and morphology. Samples were centrifuged, and extended in the same diluents plus egg yolk (20%) and glycerol (6%), frozen in liquid nitrogen, and subsequently thawed at 37°C for 1 min, followed by re-evaluation of sperm characteristics. The two extenders were similarly efficient for epididymal recovery, with regard to the number and quality of sperm recovered. However, for both extenders, sperm quality decreased (P < 0.05) after centrifugation and dilution. After sperm cryopreservation and thawing, there were (mean ± SEM) 26.5 ± 2.6% motile sperm with 2.6 ± 0.2 vigor in the ACP-109c (ACP Biotecnologia) group, which was significantly better than 9.7 ± 2.6% motile sperm with 1.2 ± 0.3 vigor in Tris. In conclusion, agouti epididymal sperm were successfully recovered using either ACP-109c (ACP Biotecnologia) or Tris extenders; however, ACP-109c (ACP Biotecnologia) was a significantly better extender for processing and cryopreserving these sperm.

Effect of centrifugation and sugar supplementation on the semen cryopreservation of captive collared peccaries (Tayassu tajacu)

The present study is aimed at evaluating the effect of centrifugation for seminal plasma removal and the supplementation of fructose or glucose to the Tris-based extender on the kinematic patterns of the motility parameters of frozen-thawed semen obtained from captive collared peccaries (Tayassu tajacu). Semen samples (n = 14) were collected from 10 sexually mature male collared peccaries by electroejaculation. These samples were further evaluated for parameters such as motility, vigor, sperm viability, membrane integrity, and sperm morphology. The samples were divided into four aliquots, and only two of these aliquots were centrifuged. The semen aliquots (centrifuged and raw semen samples) were diluted in Tris-based extenders supplemented with fructose or glucose. Egg yolk (20%) and glycerol (3%) were added to all the samples which were cryopreserved in liquid nitrogen and thawed at 37 °C/1 min. The frozen-thawed semen was evaluated for the same parameters described for the fresh semen. On the other hand, the kinematic motility patterns were evaluated by a computer-aided system. After thawing, it was observed that the values for the total sperm motility were around 30% for all the samples. A negative effect of centrifugation was verified for parameters such as sperm morphology, linearity, straightness, and beat cross frequency (P < 0.05). However, no differences between fructose and glucose were verified for any semen end point (P > 0.05). In conclusion, it is not recommended to centrifuge the ejaculates from collared peccaries prior to conducting the cryopreservative procedures using a Tris-based extender supplemented with fructose or glucose.

Cryopreservation of collared peccaries (Tayassu tajacu) semen using a powdered coconut water (ACP-116c) based extender plus various concentrations of egg yolk and glycerol.

The objective was to determine the effectiveness of a powdered coconut water-based extender (ACP-116c), plus various concentrations of egg-yolk and glycerol, as an alternative for cryopreservation of collared peccary semen. Twelve ejaculates were obtained from captive adult males by electroejaculation, and evaluated for sperm motility, kinetic rating, viability, morphology, and functional membrane integrity. The ejaculates were apportioned into aliquots that were diluted in Tris plus 10% egg yolk and 3% glycerol, or in ACP-116c plus 10 or 20% egg yolk and 1.5 or 3% glycerol. Samples were frozen in liquid nitrogen and, after 1 mo, thawed at 37 °C for 1 min. After thawing, samples were evaluated as reported for fresh semen, and also for sperm membrane integrity (fluorescent probes) and kinematic parameters (computerized analysis). Results were presented as means ± SEM. Freezing and thawing decreased sperm characteristics relative to fresh semen. Overall, ACP-116c plus 20% egg yolk and 3% glycerol provided better (P < 0.05) sperm motility and kinetic rating (48 ± 6.1% and 2.8 ± 0.2, respectively) after thawing than Tris extender (30.4 ± 5.7% and 2.4 ± 0.2). However, there were no differences (P > 0.05) among treatments with regard to the other sperm characteristics. Based on computerized motion analysis, total (26.5 ± 5.9%) and progressive (8.1 ± 2.2%) motility were best preserved (P < 0.05) with the above-mentioned treatment. In conclusion, a coconut water-based extender, ACP-116c, plus 20% egg yolk and 3% glycerol, was effective for cryopreservation of semen from collared peccaries.

Objective assessment of the cryoprotective effects of dimethylformamide for freezing goat semen

The aim of this work was to assess the cryoprotective effects of dimethylformamide (DMF) for freezing goat semen, using an objective analysis by computer-assisted sperm analysis (CASA). Twenty-one ejaculates (seven per animal) were collected from three stud bucks with the aid of an artificial vagina and immediately evaluated for gross and microscopic characteristics. The semen was diluted in two steps with a Tris-egg yolk extender containing 6% glycerol or 6% DMF, frozen in 0.50-mL straws, and stored in liquid nitrogen. Samples were accessed for sperm morphology, sperm membrane structural and functional integrity, and by CASA, immediately after thawing. There were differences (P<0.05) between glycerol and DMF with regard to subjective progressive motility (23.9±2.2% vs. 16.6±2.0%), objective progressive motility (3.5±0.4% vs. 1.8±0.3%), linearity (53.9±1.6% vs. 48.1±1.4%) and amplitude of lateral head (2.3±0.1 vs. 2.9±0.1 mm), which confirmed the efficiency of glycerol. In conclusion, dimethylformamide could be used as an alternative cryoprotectant for goat semen freezing. However it was showed that no benefits were derived by using dimethylformamide to replace glycerol at an equal 6% concentration.

Monitoring of Lutzomyia longipalpis Lutz & Neiva, 1912 in an area of intense transmission of visceral leishmaniasis in Rio Grande do Norte, Northeast Brazil

Urban increase of visceral leishmaniasis (VL) in Brazil is associated with the adaptation of its vector, Lutzomyia longipalpis, to environments modified by humans. The present study reports the results of an entomological monitoring of L. longipalpis and the effect of environmental variables on its population density. Sandflies were captured in the municipality of Mossoró, State of Rio Grande do Norte, Northeastern Brazil, from January 2005 to December 2006. Two CDC light traps were placed monthly for four consecutive nights in the peridomicile of selected households. Data analysis was based on the chi-square test and linear regression. A total of 2,087 sandflies were captured, 99.86% of which were L. longipalpis. A higher proportion of females were captured (p < 0.05). Monthly analysis of the variables temperature, relative humidity and rainfall did not show a significant influence on population density. However, there were seasonal differences: approximately 70% of sand flies were captured during the rainy season (p < 0.05). The predominant species, L. longipalpis, is present in substantial number, representing a public health risk. Therefore, because of higher prevalence during the rainy season, we recommend intensified VL control measures before and during this season to reduce the risk of disease transmission.

Sandflies (Psychodidae: Phlebotominae) survey in an urban transmission area of visceral leishmaniasis, Northeastern Brazil

Visceral leishmaniasis (VL) is a major public health challenge in Brazil, especially in states where it is endemic. The aim of this study was to investigate the relationship of sand fly population density with environmental variables (temperature, rainfall and relative humidity) in urban areas of the city of Mossoró, Rio Grande do Norte, Northeastern Brazil. Sand flies were captured with Center Disease Control (CDC) traps installed monthly in the intra and peridomicile of three houses. Data analysis was based on the chi-square test and linear regression. A total of 7,347 sand flies were captured, being 93.85% Lutzomyia longipalpis and 6.15% Lutzomyia evandroi. Sand flies were more commonly found in the peridomicile and there was no difference between the number of males and females. The variables rainy season as well as relative humidity and rainfall, alone or together, did not have an effect on sand fly population density. However, high temperatures had a negative effect. The study of the behavior of sand flies in specific units of endemic areas can provide input to public health authorities for planning appropriate VL vector control measures.

Establishing the hypoosmotic swelling test for sperm analysis in collared peccaries (Pecari tajacu)

Solucoes hiposmoticas com diferentes concentracoes (0, 50, 100, 150, 200mOsm/L) foram testadas para a avaliacao funcional da membrana espermatica de catetos (n=13). Foi verificado que o numero de espermatozoides reagidos diminuia (P<0,05) de acordo com o aumento da osmolaridade do meio. A maior porcentagem (71,8%) de espermatozoides reagidos, bem como a menor variacao nas respostas osmoticas, foi detectada com o uso de agua destilada (0mOsm/L) (P<0,05), a qual tambem apresentou a menor variacao nos resultados, de acordo com os erros padrao verificados. Em conclusao, a agua destilada aparenta ser uma solucao adequada para o uso no teste hiposmotico para semen de catetos.

Individual variation related to testicular biometry and semen characteristics in collared peccaries (Tayassu Tajacu Linnaeus, 1758)

The aim of this research was to study the individual variation with regard to the morphometry of the testes evaluated by ultrasonography and semen characteristics and to verify the existence of relationship between these variables in collared peccaries. In addition, the testes of the animals were evaluated by histology in order to determine the proportion occupied by the seminiferous tubules. A total of 52 ejaculates were obtained from ten adult specimens that had been restrained by anesthesia. The testicular measurements (length, height, and width) were performed by ultrasonography, and the testicular volume was calculated according to Lamberts formula. The scrotal circumference was measured by encircling the thickest portion of the testicle with a graduated nylon tape. The semen was collected by electroejaculation. Testicular fragments were analyzed through classic histology for the determination of the area occupied by the seminiferous tubules. The results show a great amount of individual variation with regard to testicular morphometry and semen characteristics. No significant correlations were obtained between testicular measurements and semen characteristics. The histometric analysis revealed that 67.8% of the testes are occupied by seminiferous tubules. Results show that the measurement of testicular dimensions does not serve as an indicator of the quality of semen obtained by electroejaculation in collared peccaries, as there is no correlation between testicular morphometry and semen characteristics in this species that presents large variations among individuals.

Assessment of sperm survival and functional membrane integrity of the six-banded armadillo (Euphractus sexcinctus)

The objective was to evaluate sperm survival in the six-banded armadillo, using a thermoresistance test, and to compare sugar solutions with varying osmolarities to analyze the integrity of the functional sperm plasma membrane in this species. Twelve ejaculates were obtained from four mature males by electroejaculation and evaluated for sperm motility, vigor, live sperm, and morphology. Sperm survival was evaluated during a thermoresistance test at 34 °C (the body temperature of this species). The functional integrity of the plasma membrane was evaluated by means of the hypo-osmotic swelling test (HOST), using solutions of varying osmolarities (0, 50, 100, and 150 mOsm/L). During the thermoresistance test, at each evaluation, there was a reduction (P < 0.05) in mean values for sperm motility, sperm vigor, and percentage of live sperm (no movement was observed at 360 min). Sperm survival varied among individual armadillos (P < 0.05). In two individuals, sperm vigor was significantly enhanced when semen was diluted in Tris extender. The response of armadillo sperm to the HOST varied among individuals (P < 0.05). On average, maximal values (P < 0.05) of reactive sperm (59%) were detected with 50 mOsm/L solution; furthermore, this concentration had the largest significant positive correlation (r = 0.84) to live sperm percentage. In conclusion, six-banded armadillos had significant individual variation with regard to sperm survival in a thermoresistance test at 34 °C; in some individuals, sperm survived until 360 min. The use of a 50 mOsm/L fructose solution was recommended for conducting a HOST in this species.

Monitoramento de ciclo estral em catetos (Pecari tajacu) criados em cativeiro em condições semiáridas

Collared peccaries (Peccary tajacu) are among the most hunted species in Latin America due the appreciation of their pelt and meat. In order to optimize breeding management of captive born collared peccaries in semiarid conditions, the objective was to describe and correlate the changes in the ovarian ultrasonographic pattern, hormonal profile, vulvar appearance, and vaginal cytology during the estrus cycle in this species. During 45 days, females (n=4) were subjected each three days to blood collection destined to hormonal dosage by enzyme immunoassay (EIA). In the same occasions, evaluation of external genitalia, ovarian ultrasonography and vaginal cytology were conducted. Results are presented as means and standard deviations. According to hormonal dosage, six estrous cycles were identified as lasting 21.0 ± 5.7 days, being on average 6 days for the estrogenic phase and 15 days for the progesterone phase. Estrogen presented mean peak values of 55.6 ± 20.5 pg/mL. During the luteal phase, the high values for progesterone were 35.3 ± 4.4 ng/mL. The presence of vaginal mucus, a reddish vaginal mucosa and the separation of the vulvar lips were verified in all animals during the estrogenic peak. Through ultrasonography, ovarian follicles measuring 0.2±0.1 cm were visualized during the estrogen peak. Corpora lutea presented hyperechoic regions measuring 0.4±0.2 cm identified during luteal phase. No significant differences (P>0.05) between proportions of vaginal epithelial cells were identified when comparing estrogenic and progesterone phases. In conclusion, female collared peccaries, captive born in semiarid conditions, have an estral cycle that lasts 21.0±5.7 days, with estrous signs characterized by vulvar lips edema and hyperemic vaginal mucosa, coinciding with developed follicles and high estrogen levels.