Luana Azevedo de Freitas

Cryopreservation of canine epididymal sperm using ACP-106c and TRIS.

The objective was to cryopreserve sperm recovered from the canine epididymal cauda immediately after an orchiectomy. The sperm was stored for 12h at 4 °C using ACP-106c and TRIS as extenders. Sixty adult male dogs were used. The testis-epididymis complex (TEC) was removed, immersed in 0.9% saline and transported to the laboratory. The 60 TEC were divided into groups according to the 4 °C cooling time (0 h or 12 h) and according to the extender used for sperm recovery (ACP-106c or TRIS), forming 4 experimental groups: G0h-ACP, G12h-ACP, G0h-TRIS and G12h-TRIS. The sperm were recovered from the epididymal cauda using the retrograde flow technique. Next, 1.0 mL of ACP-106c or 1.0 mL of TRIS (preheated to 37 °C for 5 min) was added to the sperm of each epididymis. One week later, the sperm was thawed at 37 °C for 1 min, and its morphology, functionality and total and progressive sperm motilities were analyzed. Other parameters were obtained by Computer Assisted Semen Analysis (CASA). The data were submitted to multivariate analysis of variance (MANOVA) (P<0.05). The total motility values were 52.17 ± 1.78 and 49.8 ± 1.93 for groups G0h-ACP and G12h-ACP and 50.7 ± 2.06 and 43.90 ± 2.51 for groups G0h-TRIS and G12h-TRIS, respectively. A decrease in total sperm motility was observed after 12h of cooling for both extenders (P<0.05). ACP-106c can be used as an extender for freezing canine epididymal sperm, and the freezing procedure must be performed immediately after sperm recovery.

Two-dimensional and Doppler sonographic prostatic appearance of sexually intact French Bulldogs.

The aim of this study was to determine the relationship between age and prostate size and measure and characterize the prostates of French Bulldogs through two-dimensional and Doppler ultrasound. Thirty-three healthy French Bulldogs were used in this study. The dogs were divided into three groups of 11 animals according to age: 8 to 19 months (group 1), 24 to 36 months (group 2), and 48 to 72 months (group 3). The animals were evaluated once with ultrasound to obtain the following: the two-dimensional sonographic appearance of the prostate, prostatic dimensions, and characteristics from colored Doppler (perfusion characteristics, diameter, and number of pixels of the vessel) and spectral Doppler (wave morphology, resistive index, and pulsatility). There was a high positive correlation between age and prostate volume (r = 0.906). The prostatic volume gradually increased with age (P < 0.05). The characteristics in color and spectral Doppler differed according to the location of the prostatic artery. In cranial and subcapsular location: prostatic artery diameter was 0.22 ± 0.02 and 0.15 ± 0.02; number of pixels: 15,431.09 ± 1753 and 10,095.18 ± 1079.85; resistance index: 0.86 ± 0.06 and 0.64 ± 0.05; pulsatility index: 2.45 ± 0.32 and 1.14 ± 0.11, respectively. All parameters evaluated increased between groups 1 and 3 (P < 0.05), except at the parenchymal location. The resistance and pulsatility indices were significantly lower in group 1 compared with group 3 in all locations studied. In addition, the indices were significantly reduced among the locations of the prostatic artery, with the exception of the cranial and caudal locations. Groups 1 and 3 differed in all evaluations, suggesting that there is a relationship between age and the Doppler parameters evaluated in this study. It can be concluded that two-dimensional ultrasound combined with Doppler ultrasound plays an important role in the evaluation of the canine prostate. Furthermore, the location of the prostatic artery may influence Doppler parameters, and age is an important factor that should be considered when evaluating the canine prostate.

Assessment of uterine involution in bitches using B-mode and Doppler ultrasonography

The aim of this study was to measure the uterine diameter by B-mode and Doppler velocimetry patterns of uterine arteries in postpartum female dogs after normal delivery or cesarean section (c-section). Ten female dogs were assessed on weeks 0, 1, 2, 3 and 4 postpartum. Only at week 0, bitches submitted to c-section presented higher body diameters and uterine horns when compared to normal delivery. It was observed a reduction in uterine diameters over the weeks in both groups. In general, bitches submitted to c-section presented lower uterine perfusion. Each group presented distinct Doppler velocimetric characteristics. It was concluded that the B-mode ultrasound and Doppler are important tools for assessing puerperal uterine with distinct characteristics influenced by the type of delivery.

Can maternal-fetal hemodynamics influence prenatal development in dogs?

The goals of this study were to report embryonic and fetal ultrasound changes and compare blood flow of uteroplacental and umbilical arteries of normal and abnormal conceptus. Accordingly, from the day of mating or artificial insemination, all fetuses in 60 pregnancies were evaluated weekly. According to the ultrasound findings, the gestational age was determined and the conceptuses were divided into normal or abnormal (embryonic and fetal abnormalities). The two-dimensional ultrasound assessment consists of measuring and evaluating the echogenicity of conceptus and extra-fetal structures. Doppler velocimetry measured the resistivity index (RI) and pulsatility index (PI) of uteroplacental and umbilical arteries. Two-dimensional and Doppler measurements were expressed as mean and standard deviation. Differences between normal and abnormal groups were subject to Mann-Whitney test (P<0.05). Of 264 fetuses, 15.90% showed embryonic abnormalities (resorption) and 5.68% presented fetal abnormalities (congenital abnormalities, fetal underdevelopment and fetal death). We observed a reduced diameter and abnormalities in the contour of gestational vesicle, lack of viability, increased placental thickness, increased fluid echogenicity and increases in RI and PI of uteroplacental arteries of conceptuses with embryonic resorption between the 2nd and 4th weeks. Fetuses with abnormalities showed changes in the flow of uteroplacental and umbilical arteries prior to visualization of two-dimensional alterations and different vascular behavior according to the classification of the change. Results show that ultrasound is efficient for the detection of embryonic and fetal abnormalities. When combined with Doppler ultrasound, it allows early detection of gestational changes, as well as hemodynamic changes, in conceptuses with abnormalities, which may influence their development.

Refrigeração do epidídimo canino a 4ºC e recuperação dos espermatozoides epididimários utilizando ACP-106c

The study aimed to assess the quality of sperm recovered from the epididymal cauda after cooling the complex testis-epididymis (CTE) of dogs using ACP-106c extender. Sixty adult male dogs, weighing 10-20 kg were used. After euthanasia, CTE was removed and it was immersed in 0.9% saline and transported to the laboratory in cooler at 30oC. For cooling and recovery of epididymal spermatozoa, the 60 pairs of CTE were divided into four groups, according to the refrigeration time of the CTE and subsequent sperm recovery: G0h, G6h, G12h and G18h, wherein each pair of CTE remained zero, six, 12 or 18 hours at 4oC respectively. The recovery of sperm epididymal tail was conducted by flotation technique using ACP-106c or Tris extender. For each epididymis, it was added 1.0 mL of either extenders, preheated to 37oC for 5 minutes. They were then centrifuged at 800g/5 minutes to remove the cell debris. Morphology, functionality and total and progressive motility, and parameters obtained by CASA were evaluated. Data were analyzed by ANOVA followed by Turkey test (P 0.05). The values of total motility groups G0h, G6H, G12H, and G18h for ACP-106c were 84.4±7.7, 81.6±11.6, 88.3±6.5 and 69.5±16.9 respectively, and for Tris 85.2±8.7, 77.4±14.3, 79.0±17.8 and 65.4±17.9 respectively. A decrease in sperm quality was observed after 18 hours of cooling in both extenders. Thus it can be concluded that the ACP-106c may be used to recover the epididymal spermatozoa chilled and may be viable for up to 12h cooling.

Seminal plasma and sperm proteome of ring-tailed coatis (Nasua nasua, Linnaeus, 1766)

Ring-tailed coati is listed as a species of least concern in the International Union for Conservation of Nature (IUCN) Red List, however, there has been a sharp decline in their population. The present study was conducted to evaluate the major proteins of both seminal plasma and sperm in ring-tailed coatis. Semen sample was collected from three adult coatis and evaluated for their morphological characteristics. Further, the sample was centrifuged to separate spermatozoa from seminal plasma, and then stored in liquid nitrogen. The seminal plasma and sperm proteins were subjected to one-dimensional (1-D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and identified by mass spectrometry. Gene ontology and protein networks were analyzed using bioinformatics tools. Based on sperm concentration and average protein content of the semen, the concentration of protein/spermatozoon was found to be 104.69 ± 44.43 μg. The analysis of SDS-PAGE gels showed 20.3 ± 3.1 and 17 ± 2 protein bands/lane for seminal plasma and sperm, respectively. In-gel protein digestion and peptide analysis by mass spectrometry revealed 238 and 246 proteins in the seminal plasma and sperm, respectively. The gene ontology analysis revealed that the proteins of seminal plasma mainly participated in cellular (35%) and regulatory (21%) processes. According to their cellular localization, seminal plasma proteins were categorized as structural (18%), extracellular (17%), and nuclear (14%) proteins with molecular functions, such as catalytic activity (43%) and binding (43%). The sperm proteins were also involved in cellular (38%) and regulatory (23%) processes, and mainly categorized as extracellular (17%), nuclear (13%), and cytoplasmic (10%) proteins. The major molecular functions of the sperm proteins were catalytic activity (44%) and binding (42%). These results indicated that the seminal plasma of ring-tailed coati has an array of proteins that can potentially modulate several sperm functions, from sperm protection to oocyte binding. However, further studies are necessary to interpret the roles of these major seminal plasma proteins in coatis.