E.A.A. Santos

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.

Use of two anesthetic combinations for semen collection by electroejaculation from captive coatis (Nasua nasua)

The objective was to compare the effects of ketamine-xylazine or tiletamine-zolazepam combinations as anesthetic protocols for captive coatis (Nasua nasua) for semen collection by electroejaculation. Five mature male coatis were physically restrained and then anesthetized by im injections of ketamine (10mg/kg) plus xylazine (1mg/kg) or a tiletamine-zolazepam combination (8 mg/kg). For the two combinations, additional quarter-doses of ketamine or the tiletamine-zolazepam combination were administered when necessary. Semen was collected by electroejaculation and immediately evaluated for color, volume, pH, sperm motility, vigor, morphology, acrosomal integrity, and percentage of live cells. Overall, collection of nine ejaculates was attempted from five animals for each treatment. Regardless of the anesthetic combination, all animals developed an erection during each attempt to collect semen. Ejaculates were obtained in all (9 of 9) attempts that used ketamine-xylazine for anesthesia, but in only 3 of 9 attempts (P<0.05) when tiletamine-zolazepam was used. All ejaculates contained sperm, with no significant differences in semen characteristics between the two anesthetic combinations. Recovery was smooth in all animals. In conclusion, semen collection by electroejaculation in coatis was significantly more successful with the use of a ketamine-xylazine combination than with tiletamine-zolazepam.

Protein profile of the seminal plasma of collared peccaries (Pecari tajacu Linnaeus, 1758)

This study was conducted to characterize the major proteins of the peccary seminal plasma, based on the semen samples collected from nine adult and reproductively sound animals. Our approach included the use of two-dimensional electrophoresis followed by Coomassie blue staining and analysis of polypeptide maps with PDQuest Software (Bio-Rad). Proteins were identified by tandem mass spectrometry (LC-MS/MS). We detected 179 protein spots per gel and 98 spots were identified by mass spectrometry, corresponding to 23 different proteins. The combined intensity of those spots accounted for 56.2±6% of the intensities of all spots and 60.9% of the intensities of spots presented in every protein map. Protein spots identified as clusterin represented 19.7±8.3% of the integrated optical densities of all spots detected in the seminal plasma maps. There was a negative association (r=-0.87; P<0.05) between the intensity of a clusterin spot and the percentage of sperm with functional membrane. Spermadhesin porcine seminal plasma protein 1 and bodhesin 2 comprised 5.4±1.9 and 8.8±3.9% of the total intensity of all spots respectively. Many proteins appeared in a polymorphic pattern, such as clusterin (27 spots), epididymal secretory glutathione peroxidase (ten spots), inter-α-trypsin inhibitor (12 spots), and IgG-binding protein (ten spots), among others. In conclusion, we presently describe the major seminal plasma proteome of the peccary, which exhibits a distinct high expression of clusterin isoforms. Knowledge of wild species reproductive biology is crucial for an understanding of their survival strategies and adaptation in a changing environment.

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.

Sperm morphological and morphometric evaluation in captive collared peccaries (Pecari tajacu)

ABSTRACT.- Sousa P.C., Santos E.A.A., Souza A.L.P., Lima G.L., Barros F.F.P.C., Oliveira M.F. & Silva A.R. 2013. Sperm morphological and morphometric evaluation in captive collared peccaries ( Pecari tajacu ). Pesquisa Veterinaria Brasileira 33(7):924-930. Laboratorio de Con -servacao de Germoplasma Animal, Universidade Federal Rural do Semi-Arido, BR 110 Km 47, Presidente Costa e Silva, Mossoro, RN 59625-900, Brazil. E-mail: legio2000@yahoo.comThe aim of this study was to compare different staining methods for the evaluation of sperm morphology by light microscopy and also to describe the morphometry of the entire sperm in collared peccaries ( Pecari tajacu ). Semen from 10 males was obtained by electro -ejaculation and evaluated for sperm motility, vigor, and concentration. Semen smears were prepared through three different staining methods: Bengal rose, brome-phenol blue, and eosin-nigrosin. Smears were evaluated under light microscopy and sperm morphologic al-terations were determined in percentage. In addition, sperm morphometric analysis was conducted by light microscopy coupled to image analyzer software. The smears stained with Bengal Rose provide the best results for the visualization of the sperm tail, midpiece, and head. The use of eosin-nigrosin stain did not allow an adequate impregnation, and some sperm presented a few contrasts with the background. A higher incidence of bent coiled tails was verified in the use of brome-phenol blue staining (P<0.05). Through mor-phometric evaluation, it was observed that the tail occupies the greatest proportion (89%) of the sperm which presents a discretely elongated head. According to the results, the use of the Bengal Rose stain is recommended for the morphologic evaluation of the collared peccary sperm.

Morphology, morphometry and ultrastructure of captive six-banded armadillo (Euphractus sexcinctus) sperm.

We analyzed the sperm characteristics of captive six-banded armadillos (Euphractus sexcinctus), by the assessment of sperm morphology, morphometry, and ultrastructure. In general, armadillos ejaculates present more than 80% of sperm within the range considered normal for sperm morphology currently accepted for other mammals. Coiled tails (3.9%) and detached heads (2.8%) were the defects most frequently verified. The morphometric analysis revealed that the total length of six-banded armadillo sperm is 77.6±1.2μm, and the length of the tail is 64.7±1.1μm on average. They also present a big head that corresponds to 16.6% of the entire sperm. Through transmission electron microscopy, we identified the presence of electron lucent points into the nucleus and the presence of about 45 mitochondria spirals in the mitochondrial sheath midpiece as a peculiarity of the six-banded armadillo sperm.

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.

Morphological Characterization of the Ovarian Preantral Follicle Population of Collared Peccaries (Tayassu tajacu Linnaeus, 1758)

The aim of this research was to characterize the preantral ovarian follicular population in collared peccaries (Tayassu tajacu) using light and electron microscopy. Ovaries from six mature females were collected and further fixed for histological and ultrastructural analysis. A total of 33273.45 ± 5789.99 preantral follicles (PFs) were estimated for the population in each ovary. Most preantral follicles were primordial (91.56%), followed by primary (6.29%) and secondary (2.15%) ones. Most PFs were morphologically normal (94.4%), and only a few were atretic (5.6%). At histology assessment, amounts of lipid droplets were observed into the oocyte cytoplasm, which was confirmed through ultrastructural analysis. This work characterizes for the first time the ovarian population of preantral follicles, total and per category, in collared peccaries (Tayassu tajacu). The general follicles featured at primordial, primary and secondary categories are very similar to those described for other species.

Identification of ultrastructural and functional damages in sperm from six-banded armadillos (Euphractus sexcinctus) due to cryopreservation

O objetivo deste estudo foi criopreservar o semen de tatus-peba (Euphractus sexcinctus) em diluente Tris-gema e glicerol, e determinar os danos causados pelo processo de congelacao-descongelacao, utilizando marcadores fluorescentes e analise ultraestrutural. As amostras de semen (n=11) coletadas de 4 tatus-peba adultos por eletroejaculacao foram criopreservadas em diluente Tris acrescido de 20% de gema de ovo e 3% de glicerol, em curva rapida de congelacao. A analise classica das amostras foi realizada apos a diluicao, refrigeracao e descongelacao, seguida por analise de fluorescencia, utilizando uma combinacao de sondas fluorescentes para avaliar a integridade da membrana (Iodeto de Propidio - PI e Hoechst - H342), e a atividade mitocondrial (CMXRos - Mito Tracker RED®). Foi tambem utilizada a analise ultraestrutural para verificar possiveis alteracoes morfologicas causadas pela crioinjuria. Quando comparadas com as amostras a fresco, verificou-se uma queda significativa em todos os parâmetros seminais dos tatus apos a descongelacao, em que apenas 6,1% de espermatozoides moveis foram encontrados. No entanto, o percentual de espermatozoides que permaneceu com membrana viavel (13%) e funcional (24,7%) apos a descongelacao sugere que algumas celulas podem estar vivas, mas imoveis. Analises utilizando marcadores fluorescentes revelaram que as mitocondrias dos espermatozoides de tatus sao altamente sensiveis ao protocolo de congelacao e os achados atraves da analise ultraestrutural comprovaram esta afirmacao. Alem disso, as imagens obtidas por microscopia eletronica de transmissao revelaram que espermatozoides congelados-descongelados apresentaram membranas plasmaticas danificadas, modificacoes nucleares como alteracoes na cromatina, e alteracoes acrossomais relativas a capacitacao espermatica. Em conclusao, este estudo e a primeira tentativa de criopreservacao de semen em uma especie de tatu, e nos auxiliou a identificar pontos criticos no processo de congelacao-descongelacao, a fim de melhorar o protocolo.