Adriel B. Brito

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.

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.

Relação entre a biometria ultra-sonográfica em modo B do bulbo ocular e os diâmetros fronto occiptal e bizigomático em Canis familiaris

In the experiment 31 healthy mongrel dogs, 8 months to 7 years of age, were used, 10 males and 21 females, weighing 1.5-28 kg. Initially, fronto-occipetal (FOD) and bizigomatic (BZD) diameters were measured using a caliper. The ophthalmologic transpalpebral B-mode ultrasonography (US) was performed to measure the ocular bulbi structures, as follows: The cornea thickness (D1), distance between cornea and anterior lens capsule (D2), distance between cornea and posterior lens capsule (D3), lens thickness (D4), lens diameter (D5), lens area (D6), distance between posterior lens capsule and retina (D7), distance between anterior lens capsule and retina (D8), and the distance between cornea and retina (D9). Except for D4, there were effect of FOD and BZD on the measures of the internal structures of BO. The Lineal Regression Analysis between the measures of the internal oculars structures and DFO and DBZ were significant for D1, D2, D3, D4, D5, D6, D7, D8 and D9.

Short-Term Culture of Ovarian Cortical Strips From Capuchin Monkeys (Sapajus apella): A Morphological, Viability, and Molecular Study of Preantral Follicular Development In Vitro

The aim of this study was to evaluate whether an in vitro culture (IVC) medium containing either or not β-mercaptoethanol (BME), bone morphogenetic protein 4 (BMP4), or pregnant mare serum gonadotrophin (PMSG) could be able to promote the development of capuchin monkeys’ preantral follicles enclosed in ovarian cortical strips. Follicular viability after IVC was similar to control (89.32%). Primordial follicle recruitment to primary stage was not reached with IVC, but the rate of secondary follicle formation was increased in the medium supplemented with BME, BMP4, and PMSG (44.86%) when compared to IVC control (9.20%). In the medium supplemented with BME, BMP4, and PMSG, contrary to other media, anti-müllerian hormone-messenger RNA (mRNA) expression in ovarian tissue was upregulated (3.4-fold), while that of growth differentiation factor-9 was maintained. The BMP4-mRNA expression, however, appeared downregulated in all cultured tissues. Our findings show a favorable effect of BME, BMP4, and PMSG on the in vitro development of secondary follicles from capuchin monkeys.

Validation of reference genes for ovarian tissue from capuchin monkeys (Cebus apella).

There is no tradition in studies reporting the effect of exposure to cryoprotectants or simply hypoxia and hypothermia on gene expression in the ovarian tissue and there has been only one study on reference or target genes quantification, and comparisons of normoxic with hypoxic, hypothermic and toxic conditions. Our aim in the present study was to investigate the stability of three reference genes in the ovarian tissue of capuchin monkeys (Cebus apella). To this end, fresh and cryoprotectant-exposed ovarian biopsies were used. Both fresh and exposed ovarian tissues were subjected to total RNA extraction and synthesis of cDNA. cDNA was amplified by real-time polymerase chain reaction (PCR), and GeNorm, BestKeeper and NormFinder software were used to evaluate the stability of glyceraldehyde-2-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and TATA-binding protein (TBP). Results demonstrated that, in the ovarian tissue from capuchin monkeys, HPRT1 and TBP were the most suitable reference genes and thus could be used as parameters to normalize data in future studies. In contrast, GAPDH appeared as the least stable gene among the tested reference genes. In conclusion, HPRT1 and TBP were the most stable reference genes in fresh and cryoprotectant-exposed ovarian tissue from capuchin monkeys.

Embryo production by parthenogenetic activation and fertilization of in vitro matured oocytes from Cebus apella

The efficiency of in vitro fertilization (IVF) depends on the viability of spermatozoa. For capuchin monkeys (Cebus apella), in vitro capacitation of spermatozoa is challenging because of their unique seminal coagulum. Motile spermatozoa can be obtained after liquefaction of the semen coagulum in coconut water-based solution. The objective of the present study was to establish an optimal in vitro maturation (IVM) protocol for capuchin monkeys and to observe the effect of follicle stimulating hormone (FSH) and luteinising hormone (LH) on IVF and parthenogenetic activation (PA) of oocytes collected from unstimulated females. We assessed spermatozoa quality after recovery from seminal coagulum using the solution ACP-118® as an extender. Oocytes were matured in vitro for 36 or 40 h and subjected to IVF or PA by applying ionomycin combined either with 6-dimethylaminopurine (6-DMAP) or roscovitine. In total, 87% of oocytes reached metaphase II (MII) after 40 IVM and 4-cell embryo production was obtained after IVF and parthenogenesis using ionomycin/6-DMAP. ACP-118® was used successfully to harvest viable spermatozoa from semen coagulum and in the preservation of spermatozoa, which were able to fertilize oocytes in vitro.

Seminal coagulation and sperm quality in different social contexts in captive tufted capuchin monkeys (Sapajus apella)

In the present study, we aimed to assess the influence of different social contexts on the seminal coagulation and sperm quality in captive tufted capuchin monkeys. For this, males were housed either individually, in mixed‐sex groups (with females), or in male‐only groups. Monkeys were housed in cages and each cage type (i.e., individual or group cage) was placed in a different room. Forty‐one males were subjected to semen collection by rectal electroejaculation. The degree of seminal coagulation was determined on a scale of I–IV. Seminal volume, sperm concentration, sperm motility, vigor, and plasma membrane integrity were evaluated for all ejaculate samples. All ejaculates collected showed degrees of coagulation between II and IV, where the majority presented coagulation degree IV, when collected from animals housed in groups. No statistical differences among percentages of coagula degree when samples were collected from males housed individually. Animals housed in group cages (male‐only groups and mixed‐sex groups) showed a significantly higher percentage of ejaculates at degree IV than males housed individually. Seminal volume was not affected by the coagula degree but by the housing system, where animals housed individually showed the highest volume (543 μl) when compared with those animals from male (273 μl) and mixed‐sex (318 μl) groups. No differences were observed in semen volume when comparing male‐only groups with mixed‐sex groups. Sperm motility was affected by both housing system and coagula degree. Samples with coagula degree IV from animals housed individually showed the highest (72%) sperm motility percentages. Sperm plasma membrane integrity was lower when samples were presenting coagula degree II + III and collected from male‐ (17%) or mixed‐sex (23%) groups. However, this housing system effect was not observed when sperm was obtained from coagula degree IV semen. Sperm vigor was neither affect by housing system or coagula degree.

Adaptation of a trap door technique for the recovery of ovarian cortical biopsies from Cebus apella (capuchin monkey).

There is a paucity of efficient cryopreservation protocols for primordial follicles enclosed in the ovarian tissue from non-human primates (NHP), in special New World primates. Our objective was to establish an optimal procedure for the recovery of ovarian biopsies from capuchin monkeys. To this end, we adapted a trap door biopsy method. Follicular density and quality of the biopsies were evaluated and ultrasound analysis was performed before and continuously after surgery to assess ovarian structure. Ovarian tissue biopsies recovered by the trap door technique allowed the successful harvesting of primordial follicles from capuchin monkeys, and no complication was recorded. The female cycle was not affected by surgery and no adherence was found thereafter. In conclusion, the adaptation of a trap door biopsy method is a safe procedure and allows recovery of healthy primordial follicles.