Sheyla Farhayldes Souza Domingues

Conceptus ecobiometry and triplex Doppler ultrasonography of uterine and umbilical arteries for assessment of fetal viability in dogs

The objectives were to: 1) evaluate blood flow in the uterine (UA) and umbilical (Uma) arteries in the pregnant bitch, by measuring the resistive index (RI) and pulsatility index (PI); 2) to note the presence or absence of the early diastolic notch and diastolic flow in the UA and Uma flow waveforms, respectively; and 3) perform conceptus ecobiometry for fetal growth assessment during pregnancy. Six healthy bitches were examined on approximately Days -44, -42, -36, -31, -28, -25, -21, -18, -14, -8, -4, and -2 of pregnancy (whelping = Day 0). Triplex Doppler and B-mode ultrasonography were used to assess blood flow and conceptus ecobiometry. All pregnancies ended with a normal whelping and birth of live puppies. Prior to whelping, all conceptus dimensions increased significantly, whereas RI and PI of both the Uma and UA decreased significantly. For the UA, RI and PI were (mean +/- SEM) 0.95 +/- 0.02 and 2.75 +/- 0.41, respectively, on Day -44, and were 0.60 +/- 0.01 and 0.99 +/- 0.03 on Day -4. For the Uma, RI and PI were 0.99 +/- 0.01 and 2.42 +/- 0.03 on Day -31, and were 0.62 +/- 0.01 and 1.15 +/- 0.02 on Day -4. The complete disappearance of the early diastolic notch in the UA, and the appearance of diastolic flow in the Uma occurred on Days -16 +/- 5 and -21 +/- 1. The authors concluded that UA and Uma perfusion were important end points to assess fetal vitality in bitches. Furthermore, the current reference values provided a baseline for monitoring normal and abnormal pregnancies in bitches.

Semen coagulum liquefaction, sperm activation and cryopreservation of capuchin monkey (Cebus apella) semen in coconut water solution (CWS) and TES-TRIS

The objectives of the present study were to test the effect of coconut water solution and TES-TRIS on the seminal coagulum liquefaction, sperm activation in fresh diluted semen, and on the cryopreservation of semen from capuchin monkeys (Cebus apella). Semen was collected from six males by electro-ejaculation, diluted in TES-TRIS or coconut water solution (CWS), and incubated at 35°C until the coagulated fraction of the semen was completely liquefied. In the experiment I, after liquefaction, samples were diluted in TES-TRIS or CWS, plus 6 and 10mM/mL of caffeine. Sperm motility and vigor were evaluated during 5h. For experiment II, after liquefaction, semen samples were extended in TES-TRIS (3.5% glycerol in the final solution) or CWS (2.5% glycerol in the final solution), cryopreserved and stored in liquid nitrogen for 1 week. The seminal coagulum was liquefied in (mean±SDM) 4.5±1.7 and 2.8±1.1h in TES-TRIS and CWS, respectively. Sperm were motile in TES-TRIS and CWS for 5.0±1.4 and 1.0±0.5h, respectively. The mean motility in this period was 38±22% (TES-TRIS) and 22.0±16.0 (CWS). Motility increased after caffeine addition only in samples diluted in CWS containing 6mM (22.5±16.0) or 10mM (28.0±19.0) caffeine. Post-thaw live sperm percentage was 26.2% in TES-TRIS and 13.2% in CWS. For cryopreservation of semen from C. apella TES-TRIS (3.5% glycerol) was more appropriate than CWS (2.5% glycerol). CWS+caffeine potentially increase sperm motility and may be useful in artificial insemination of fresh diluted semen.

Assessment of feline fetal viability by conceptus echobiometry and triplex Doppler ultrasonography of uterine and umbilical arteries

The aims of the present study were to: (1) evaluate blood flow in the uterine (UA) and umbilical arteries (Uma) in the pregnant queen, by measuring the resistive index (RI) and pulsatility index (PI); (2) to note the presence or absence of the early diastolic notch and diastolic flow in the UA and Uma flow waveforms, respectively; and (3) perform conceptus echobiometry for fetal growth assessment during pregnancy. Eight healthy pregnant domestic Brazilian Shorthair queens were examined from Days 10 to 50 after mating (mating=Day 0). Triplex Doppler and B-mode ultrasonography were used to assess blood flow and conceptus echobiometry. All pregnancies ended with a normal parturition and birth of live kittens. Prior to parturition, all conceptus dimensions increased significantly, whereas RI and PI peaked between Days 33 and 43 followed by a decrease (P<0.05). The PI least on Day 50. The RI and PI of Uma decreased (P<0.05) during two periods in the fetal development, i.e. from Days 22 to 40 (0.79 ± 0.01 and 1.64 ± 0.04), and from Days 41 to 50 (0.75 ± 0.01 and 1.39 ± 0.05), representing the increased Uma perfusion. Both the complete disappearance of the early diastolic notch in the UA, and the appearance of diastolic flow in the Uma occurred on Day 42 ± 1. It was concluded fetal echobiometry, UA and Uma perfusion, were important end points to assess fetal viability in queens. Furthermore, the current reference values provided a baseline for monitoring normal and abnormal pregnancies in queens.

Seminal characteristics and cryopreservation of sperm from the squirrel monkey, Saimiri collinsi

The Neotropical nonhuman primate squirrel monkey (Saimiri sp.) is one of the most commonly used species in research in several areas of knowledge. However, little progress has been reported in respect to techniques for preservation of their gametes. Thus, the main objectives of this study were (1) to describe testicular and seminal aspects of a new species, Saimiri collinsi, (2) to preserve semen of this species by cooling or freezing using ACP-118 (powdered coconut water), and (3) to test two glycerol (GLY) concentrations (1.5% or 3%) for semen freezing in the presence of ACP-118. The experimental group started with 14 captive males, but only 11 were suitable to collect ejaculates containing sperm. After anesthesia, both testes were evaluated: length, width, height, and testicular circumference. Semen was collected by electroejaculation and evaluated, followed by dilution, cooling, and freezing. Seminal parameters and sperm motility, vigor, plasma membrane integrity, and normal morphology were evaluated after each step; functionality was also checked in fresh and frozen-thawed sperm. Sperm motility, plasma membrane integrity, and normal sperm in cooled semen (n = 11) were 44.1 ± 34.0, 63.1 ± 15.6, and 73.8 ± 19.8, respectively, with vigor ranging of 2 to 3. Sperm motility, plasma membrane integrity, normal and functional sperm in frozen semen (n = 5) were 0.6 ± 1.3 (1.5% and 3% GLY); 4.4 ± 4.9 (1.5% GLY) and 6.6 ± 7.2 (3% GLY); 86.8 ± 3.0 (1.5% GLY) and 88.8 ± 5.1 (3% GLY); 13.3 ± 11.9 (1.5% GLY) and 14.3 ± 13.5 (3% GLY), respectively, and vigor 0 for both 1.5% and 3% GLY. No significant difference between GLY concentrations was observed. We concluded that electroejaculation was efficient for semen collection of S collinsi and tested the cooling protocol that allowed to recover a satisfactory percentage (63%) of membrane intact sperm. However, the freezing protocol was not appropriate to sperm preservation.

Histological study of capuchin monkey (Cebus apella) ovarian follicles

O objetivo do presente trabalho foi obter dados quantitativos e qualitativos da populacao folicular ovariana de femeas de Cebus apella. Foram obtidos 7 ovarios de 4 femeas adultas de C. apella atraves de ovariectomia. Os ovarios foram submetidos a preparacao para histologia otica de rotina. O numero de foliculos pre-antrais e antrais por ovario foi estimado utilizando o Metodo Fracionador. Os foliculos pre-antrais foram classificados em primordial, transicao, primario e secundario. Foram considerados foliculos antrais todos aqueles que apresentavam uma cavidade antral. Todos os foliculos contados foram classificados em normais ou degenerados. Com o intuito de acompanhar o desenvolvimento folicular, os diâmetros medios folicular, oocitario e do nucleo do oocito foram determinados. Todos os resultados foram apresentados em Media ± Erro Padrao. A populacao media de foliculos pre-antrais foi de 56.938 ± 21.888 e 49.133 ± 26.896 para os ovarios direito e esquerdo, respectivamente. A percentagem de foliculos pre-antrais estimados normais foi de 80,00 ± 4,95 %. O diâmetro medio folicular variou de 22,0 ± 0,5 µm a 61,2 ± 4,0 µm. No tocante aos foliculos antrais, a populacao media de foliculos normais e degenerados por ovario foi de 60,0 ± 19,0 e 3 ± 1,8 foliculos, respectivamente. O diâmetro medio folicular foi de 514,4 ± 56,6 µm. Para concluir, as informacoes obtidas neste trabalho poderao servir como parâmetro para posteriores estudos in vivo ou in vitro da foliculogenese de primatas nao-humanos neotropicais da especie C. apella.

Immunolocalization of Growth, Inhibitory, and Proliferative Factors Involved in Initial Ovarian Folliculogenesis From Adult Common Squirrel Monkey (Saimiri collinsi)

We performed an immunohistochemical (IHC) study to determine the follicular expression of growth differentiation factor 9 (GDF-9), anti-Müllerian hormone (AMH), Kit Ligand (KL), and c-Kit in squirrel monkey ovary. Ovarian tissue fragments from 4 squirrel monkeys were collected by laparotomy and processed for classical histology and IHC. Additionally, follicle development was assessed by Ki67 immunostaining to evaluate proliferative status of granulosa cells. A total of 4025 follicles were examined (1475 for classical histology and 2550 for immunohistochemistry). More than 80% of the evaluated follicles were morphologically normal. The GDF-9 protein was detectable in oocyte cytoplasm from primordial (100%), primary (99.1%), and secondary (100%) follicles. The AMH was not expressed in primordial follicles but just in few primary follicles (13.8%). On the other hand, it was highly expressed in granulosa cells from secondary follicles (67.9%). c-Kit, KL receptor, was found in the oolemma of primordial (100%), primary (100%), and secondary (100%) follicles. The KL expression was observed in oocytes and granulosa cells from primordial (94.9%), primary (91.6%) and secondary follicles (100%). Ki67 immunostaining was observed in granulosa cells from primary (5.7%) and secondary (54.8%) follicles but not in primordial follicles. In conclusion, we described the localization of GDF-9, KL, c-Kit, and Ki67 proteins and confirmed the presence of AMH protein in preantral follicles from squirrel monkey. Our results offer contribution for understanding of folliculogenesis in neotropical nonhuman primates. Moreover, these markers can be used to assess follicular viability and functionality after cryopreservation, transplantation, or in vitro culture of ovarian tissue.

Morphological and morphometrical characterization, and estimation of population of preantral ovarian follicles from senile common squirrel monkey (Saimiri sciureus)

The experiment described the morphological and morphometrical characteristics as well as estimate the population of primordial, primary and secondary ovarian follicles from common squirrel monkey (Saimiri sciureus). Ovaries (n=10) from five senile squirrel monkeys were collected after natural death and processed for classical histology. The mean ovarian population was estimated as 915.04 ± 78.83, 230.46 ± 20.82 and 115.88 ± 15.72 primordial, primary and secondary follicles per ovary, respectively. 73.30% were classified as primordial, 18.62% as primary, and 8.09% as secondary follicles. From all these developmental stages, the mean diameters of follicles, oocytes, oocytes nuclei and the mean number of granulosa cells were described. The number of granulosa cells surrounding normal primordial follicles (5.65 ± 0.001) was lower (P<0.05) than the number of granulosa cells (13.17 ± 0.02) surrounding the primary follicles. Secondary follicles presented the highest (P<0.001) number of granulosa cells surrounding each oocyte (273.73 ± 20.80). We have estimated the follicular population, as well as described the morphometric and morphological characteristics of preantral follicles from senile squirrel monkeys, which may be a valuable animal model for female ovarian aging studies.

Triplex Doppler da artéria renal e a relação entre a ecobiometria dos rins com distância atlanto-coccígea e altura em Canis familiaris

The aim of this paper was to establish the relation between the kidney ecobiometry with atlanto-coccyges distance (ACD) and height (H) in adult healthy dogs, to obtain normality parameters for assessment of renal size and volume, as well as establish reference values to evaluate kidney blood perfusion by the resistivity index (RI) and pulsatility index (PI) of main renal artery. The study was applied at 22 adult dogs, 11 males and 11 females. Previously, the DAC and H of all animals were measured. For ultrasonographic examination, the ultra-sound system HDI 4000 PHILIPS equipped with a multi-frequency microconvex transducer, Color Doppler and Spectral Doppler devices was used. The animals were placed into right or left lateral decubitus position, in agreement with the kidney to be assessed. The longitudinal (LD) and dorsoventral diameters (DVD) of kidney were measured in longitudinal plane, and the transverse diameter (TD) was determined in transversal section. The renal volume (V) was automatically calculated by the ultrasound software. With Triplex Doppler, the RI and PI of right and left main renal arteries were obtained. All data were represented in mean ± SEM. Linear regression analyses were performed with renal LD, DVD, TD and V as dependent variable, and ACD and H as independent variable. RI and PI were compared between right and left renal arteries with Students t-test. The LD, DVD, TD (cm) and V (ml) mean measurements for the left and right kidneys were: 5.24±0.27, 3.07±0.15, 3.07±0.9, 28.01±3.4 and 4.50±0.19, 2.88±0.14, 2.71±0.15, 21.27±2.6. All regression analyses were significant for the intercept and regression coefficient (P<0.01). There were statistical differences on RI and PI means between right and left renal arteries (P=0.001). The RI and PI means of left and right renal artery obtained were: 0.62±0.08 and 1.34±0.18; 0.70±0.06 and 1.62±0.13. The data obtained in the present paper can be used as parameters for evaluation of the renal size, volume and perfusion in adult dogs.

Efficacious long-term cooling and freezing of Sapajus apella semen in ACP-118®

The objectives of the present study were to test the effect of coconut water solution (CWS), TES-TRIS and ACP-118(®) on the seminal cooling and cryopreservation of semen from capuchin monkeys (Sapajus apella). Semen was collected from six males by electro-ejaculation, diluted in TES-TRIS, CWS or ACP-118(®), and maintained at 4°C for 28h. Semen was subsequently evaluated (Experiment I) or cryopreserved in the presence of different glycerol concentrations (3%, 5% or 7%) (Experiment II). ACP-118(®) was the preferred extender to preserve sperm motility and viability after 28h incubation at 4°C. Cooled sperm were successfully frozen-thawed in a medium containing 3% glycerol. After thawing, sperm retained the capacity to fertilize oocytes and zygotes were obtained. In conclusion, ACP-118(®) can be effectively and efficiently used as extender for the cooling of S. apella semen. Furthermore, cryopreservation using ACP-118(®) by adding 3% glycerol is suitable to maintain sperm morphology and the capacity of these cells to fertilize in vitro.

Study of the development of uteroplacental and fetal feline circulation by triplex Doppler

The objective was to evaluate blood flow in fetal and maternal vessels by Triplex Doppler and its association with development of blood vessels during gestation in the domestic cat. Ten queens were examined weekly from 14 to 63 d after mating. Peak systolic velocity (PSV), end diastolic velocity (EDV), resistance index (RI) and pulsatility index (PI) of uteroplacental, aorta and umbilical fetal arteries and caudal vena cava of the fetus were evaluated. Throughout pregnancy, there was an increase in PSV and EDV in the aorta and umbilical arteries. In the caudal vena cava, there was an increase in PSV, whereas the EDV was constant, with a significant increase on Day 63. Peak systolic velocity and EDV of the uteroplacental artery reduced significantly on Day 63. Resistance index of the umbilical artery progressively decreased. In the aorta, this reduction was detected only on Day 42, with no defined pattern in the caudal vena cava and uteroplacental artery. Pulsatility index of the aorta varied. Although pulsatility increased in the caudal vena cava on Day 35 and remained elevated, pulsatility was significantly reduced in the umbilical artery by Day 63. The pulsatility index of the uteroplacental artery was constant (increased only on Day 63). Triplex Doppler evaluation could be a useful adjunct for prenatal care of pregnant queens, including assessment of vascular gestational development and prediction of gestational age.