Indução do estro em cutias (Dasyprocta leporina) utilizando-se protocolos à base de prostaglandina isolada ou em associação com análogo de GnRH / Estrus induction in agoutis (Dasyprocta leporina) using protocols based on prostaglandin isolated or in association to GnRH analogue

Comparou-se a eficiência de protocolos para indução de estro em cutias. Em cinco fêmeas, foram administradas duas doses de cloprostenol (5µg) com intervalo de nove dias, via intraperitoneal; em outras cinco, administraram-se 30µg de análogo do hormônio liberador de gonadotrofinas (GnRH), via intravulvar, seguidos de 5µg de cloprostenol, via intraperitoneal, após sete dias e, após mais dois dias, nova dose do análogo de GnRH. A cada três dias, a ciclicidade reprodutiva dos animais foi monitorada, por meio de coleta de sangue, para dosagem hormonal, ultrassonografia ovariana e citologia vaginal. Duas das fêmeas que receberam apenas prostaglandina, as quais estavam em fase luteal no início do tratamento, manifestaram o estro aos três e seis dias após a segunda administração da droga. Já nas fêmeas que receberam a prostaglandina associada ao análogo do GnRH, duas que originalmente estavam em fase luteal apresentaram estro aos quatro dias após o tratamento, e uma outra apenas após 10 dias. Não foram evidenciadas diferenças estatísticas quanto à eficiência dos tratamentos (P>0,05). Conclui-se que, de acordo com os protocolos utilizados, o uso da prostaglandina isolada ou em associação com análogo do GnRH para a indução do estro em cutias D. leporina apresenta eficiência limitada às fêmeas que estejam em fase luteal por ocasião do início do tratamento.(AU)

We compared the efficiency of protocols for estrus induction in agoutis. Five females received double intraperitoneal administration of cloprostenol (5µg) on a 2-days interval; other five females were treated with intravulvar administration of 30µg gonadotrophin release hormone analogue (GnRH associated to intraperitoneal administration of 5µg cloprostenol after seven days and a new administration of GnRH analogue after two days. Every 3 days, the agoutis' reproductive cycle was monitored by blood collection for hormonal analysis, ovarian ultrasound and vaginal cytology. Two females, originally in luteal phase, that received isolated prostaglandin presented estrous signs at 3 and 6 days after the second drug administration. From the females that received the association, two that were originally in luteal phase presented estrus at 4 days after treatment, and one other presented estrus only after 10 days. There was no significant statistical difference regarding the efficiency of treatments for estrus induction (P>0.05). We conclude that, according to the protocols tested in the study, the use of isolated prostaglandin or its association to GnRH analogue for estrus induction in D. leporine shows an efficiency limited to the females that were in luteal phase in the beginning of the treatment.(AU)

Use of Aloe vera-based extender for chilling and freezing collared peccary (Pecari tajacu) semen.

As an alternative for the conservation of collared peccary semen, this research aims at evaluating the use of Aloe vera (AV) extract as a cryoprotectant for semen chilling and freezing. Five ejaculates were divided in two aliquots that were diluted in Tris plus egg yolk (EY; 20%) or AV extract (20%) and chilled at 5 °C. In both treatments, an adequate semen conservation was achieved and values closer to 40% motile sperm with viability and osmotic response ranging from 20% to 40%, and normal morphology of 80% were found after 36 hours of storage. Moreover, 12 other ejaculates were diluted in Tris plus EY (20%) or AV extract (5, 10, or 20%) and glycerol (3%). Samples were frozen in liquid nitrogen and thawed after 1 week. After thawing, all the treatments containing EY or AV provided similar values for sperm morphology, viability, osmotic response, membrane integrity, sperm motility, amplitude of lateral head, beat cross frequency, and rapid, low, and static subpopulations, but the highest values for straightness and the lowest values for curvilinear velocity were found using 20% AV (P < 0.05). In conclusion, we found that AV extract at a 20% concentration could be used as an alternative substitute to EY in the formulation of Tris extenders for collared peccaries' semen chilling or freezing.

Morphology and vascularization of the corpus luteum of peccaries (Pecari tajacu, Linnaeus, 1758) throughout the estrous cycle / Morfologia e vascularização do corpo lúteo de catetos (Pecari tajacu, Linnaeus, 1758) ao longo do ciclo estral

The current paper characterizes the changes in morphology and vascularization of the corpus luteum of collared peccaries during the estrous cycle and correlates progesterone synthesis (P4). Twenty females were subjected to a treatment for estrus synchronization; an ear implant containing 1.5 mg of norgestomet was implanted on D0, whereas on D9 the peccaries received an IM injection of eCG 200UI and 50g of PGF2a. The animals were divided into four groups (G1, G2, G3 and G4) and euthanized on post-ovulation days 3, 12, 18 and 22. The ovaries were collected and the corpora lutea were measured and processed for histological and vascular density (Dv). Blood was collected for dosage of P4 serum. The morphology of the ovaries, the corpora lutea and P4 varied significantly during the estrous cycle (P<0.001). There was a significant co-relationship between weight and length of the ovaries and CL (r = 0.66, r = 0.52, P<0.05, respectively) and between weight, length and width of the CL and P4 (r = 0.51, r = 0.54 and r = 0.68, P<0.05, respectively). The luteal Dv was highly influenced by the estrous cycle phase (P<0.0001). The P4 and luteal Dv concentrations were higher in G2 and evidenced maximum secretory activity, with a highly significant correlation (P<0.0001). Assessed lutein parameters may estimate the phase of the estrous cycle in peccaries and the functional activity of the corpus luteum.

Objetivou-se caracterizar as variações na morfologia e vascularização do corpo lúteo (CL) de catetos durante ciclo estral (CE) e correlacioná-las com a concentração de progesterona (P4). Vinte fêmeas de cateto foram submetidas a tratamento de sincronização do estro; no D0 receberam implante auricular contendo 1,5mg de norgestomet, no D9 injeção via IM de 200UI de eCG e 50µg de PGF2α. Os animais foram divididos em quatro grupos (G1, G2, G3 e G4) e eutanasiados nos dias três, 12, 18 e 22 pós-ovulação. Os ovários foram coletados e os CL foram mensurados e processados para avaliação histológica e da densidade vascular (Dv). O sangue foi coletado para dosagem da P4 sérica. A morfologia dos ovários, CL e a concentração de P4 variaram significativamente durante o CE (P<0,001). Houve correlação significativa entre peso e comprimento dos ovários e CL (r = 0,66, r = 0,52, P<0,05, respectivamente) e entre peso, comprimento e largura do CL e a concentração de P4 (r=0,51, r=0,54 e r=0,68; P<0,05, respectivamente). A Dv do CL se mostrou muito influenciada pela fase do CE (P<0,001) e apresentou alta correlação significativa (P< 0,001). No G2 os maiores valores de P4 e Dv confirmaram máxima atividade secretória do CL nesse estádio. Os parâmetros luteínicos avaliados podem ser usados para estimar a fase do ciclo estral em catetos e a atividade funcional do CL.

Use of Non-invasive Methods for Evaluating the Testicular Biometry in Collared Peccaries (Pecari tajacu Linnaeus, 1758).

The aim of this study was to compare the accuracy of two methods used to estimate testicular volume in the collared peccary. Calliper and ultrasonographic measurements of testicular dimensions (length, width and height) of both testes were taken on five adult collared peccaries. The testicular volume was calculated by Lambert's empiric formula: length (L) × width (W) × height (H) × 0.71, the formula of an ellipsoid L × W × H × 0.52, and Hansen's formula: L × W(2)  × 0.52. The calculated volumes were then compared with the actual ones, which were estimated by water displacement. The mean of true testicular volume was 22.65 ± 1.52 ml. Lambert's formula estimated testicular volume more accurately when ultrasound measurements were taken. However, when the calliper was the methodology used, the results were closest to the true volume, especially when Ellipsoid formula and Hansen's formula were applied, and underestimated the true volumes by 1.53 ± 1.75 ml and 1.53 ± 1.65 ml, respectively. This specific application of technologies in wild animals has the potential to revolutionize the selection process for the collared peccary entering artificial insemination or natural breeding programmes.

Interactions Among Different Devices and Electrical Stimulus on the Electroejaculation of Captive Agoutis (Dasyprocta leporina).

The interactions among different electroejaculation devices associated with serial or continuous stimuli were investigated to improve the efficiency of the electroejaculation for semen collection in agoutis. Ten sexually matured male Dasyprocta leporina were restrained by the intramuscular administration of xylazine-ketamine association. Each individual was randomly subjected to four electroejaculation protocols, by combining two devices (one presenting longitudinal electrodes emitting square waves and other presenting ring electrodes emitting sine waves) and two electrical stimuli protocols (serial or continuous). A total of 40 attempts for electroejaculation were conducted in agoutis, being 10 per treatment. The most efficient treatment in providing ejaculates containing sperm (p < 0.05) was that using and electroejaculator connected to a probe with ring electrodes and associated with serial stimuli (4/7; 57%). In spite of semen parameters obtained by sine waves were adequate for using the samples for assisted reproduction, higher values for sperm motility and functional membrane integrity were obtained in the use of the square wave, independently of the electric stimulation protocol used (p < 0.05). In conclusion, we verified that the use of a device presenting a probe with ring electrodes and emitting sine waves, associated with a serial stimuli protocol, improves the efficiency for semen obtaining by electroejaculation in adults D. leporina.

Establishing the hypoosmotic swelling test for sperm analysis in collared peccaries (Pecari tajacu) / Estabelecimento do teste hiposmótico para a análise de sêmen de catetos (Pecari tajacu)

Soluções hiposmóticas com diferentes concentrações (0, 50, 100, 150, 200mOsm/L) foram testadas para a avaliação funcional da membrana espermática de catetos (n=13). Foi verificado que o número de espermatozoides reagidos diminuía (P<0,05) de acordo com o aumento da osmolaridade do meio. A maior porcentagem (71,8%) de espermatozoides reagidos, bem como a menor variação nas respostas osmóticas, foi detectada com o uso de água destilada (0mOsm/L) (P<0,05), a qual também apresentou a menor variação nos resultados, de acordo com os erros padrão verificados. Em conclusão, a água destilada aparenta ser uma solução adequada para o uso no teste hiposmótico para sêmen de catetos.

Cryopreservation of collared peccary (Pecari tajacu) semen using different freezing curves, straw sizes, and thawing rates.

The objective of this study was to verify the effect of different freezing curves, straw sizes, and thawing rates on the 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 diluted in a coconut water extender (ACP-116c) with egg yolk and glycerol, packaged into 0.25 mL or 0.50 mL plastic straws and cryopreserved in liquid nitrogen following a slow (-10 °C/min) or a fast (-40 °C/min) freezing curve. After one week, samples were thawed at 37 °C/1 min or 70 °C/8s and evaluated as reported for fresh semen, and also for kinematic parameters (computerized analysis). A significant decrease in sperm motility and kinetic rating was observed after glycerol addition at 5 °C and also after thawing for all the treatments (P<0.05). Regarding post-thaw semen variables, no differences were verified between freezing curves when the same straw size and thawing rate were taken as reference (P>0.05). In general, values for sperm characteristics found after thawing at 37 °C were better preserved than at 70 °C (P<0.05), both in the use of 0.25 mL or 0.50 mL straws, which were similar for semen packaging (P>0.05). The evaluation of the kinematic parameters of sperm motility confirmed these results at values varying from 20% to 30% motile sperm for the samples tha wed at 37 °C, and values fewer than 12% motile sperm for samples thawed at 70 °C (P<0.05). In conclusion, we recommend the use of a fast freezing curve that reduces the time spent on the cryopreservation of collared peccary semen, which could be packaged both in 0.25 mL or 0.50 mL straws, but the thawing should be conducted at 37 °C/1 min.

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 Lambert's 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.

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.

Abdominal exploration in captive collared peccaries (Tayassu tajacu) by ultrasonography.

This study determines the morphology and ultrasound features of the abdominal organs in male, nestling and healthy collared peccaries. The bladder wall is hyperechogenic, with a thickness of 0.2 ± 0.08 cm. The kidneys present a well-defined cortex, medulla and pelvis, and the dimensions are 2.56 ± 0.3 × 4.6 ± 0.8 cm for the left and 2.51 ± 0.4 × 4.86 ± 1.1 cm for the right kidney. The spleen has a uniform echotexture over its entire surface. The largest dimensions of the liver are 2.0 ± 0.57 cm for the left lobe and 1.42 ± 0.66 cm for the caudate lobe. The liver presents a homogeneous echotexture in the majority of cases, but sometimes some hyperechoic spots are present. The stomach wall has a thickness of 0.42 ± 0.28 cm. The bowel loops show alternate hyperechoic and hypoechoic layers with a uniform diameter and a wall thickness of 0.19 ± 0.07 cm.

Interactions between straw size and thawing rates on the cryopreservation of agouti (Dasyprocta aguti) epididymal sperm.

This study verifies the interactions between straw size and thawing rates and their impact on the epididymal sperm from this species. Caudae epididymidum from 10 agoutis were subjected to retrograde washing using a coconut water extender (ACP-109c(®) ). Epididymal sperm were evaluated and extended in ACP-109c(®) plus egg yolk (20%) and glycerol (6%). The samples were packaged in 0.25- or 0.50-ml straws, frozen in liquid nitrogen and thawed at 37°C/1 min or 70°C/8 s, followed by a re-evaluation. The use of 0.25-ml straws thawed at 37°C/1 min provided a value of 26.6% for sperm motility. No interactions between straw size and thawing rates were verified on agouti sperm (p > 0.05), but when 0.5-ml straws were thawed at 70°C/8 s, sperm vigour decreased significantly (p < 0.05). It is recommended that the agouti epididymal sperm cryopreserved in ACP-109c(®) extender should be packaged in 0.25- or 0.50-ml straws and thawed at 37°C/60 s.

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.

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.

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.