Lúcia Daniel Machado da Silva

Cryopreservation of canine semen using a coconut water extender with egg yolk and three different glycerol concentrations

Semen was collected by digital manipulation from six adult dogs. The second fraction of the ejaculate was used in this study. The semen was assessed by macroscopic and microscopic criteria 1 min after collection, first dilution, cooling, glycerol addition and thawing. Experiments were conducted to compare the effect of three different concentrations of glycerol in coconut water extender. The freezing method employed was that one described for caprine semen with slight changes. Semen was thawed at 37 degrees C for 1 min. Spermatozoal motility after thawing was 49.2+/-26, 44.2+/-18.3 and 35.8+/-26.8% for groups with 4, 6 and 8% glycerol, respectively. The vigor after thawing was 2.6+/-1.1, 2.7+/-1.0 and 2.1+/-1.2 for these groups, respectively. There was no difference among groups in motility and vigor. However, a smaller percentage of total and secondary abnormalities was observed using 6% glycerol in coconut water extender. In conclusion, the three glycerol concentrations (4, 6 or 8%) can be used successfully in cryopreservation of canine semen using a coconut water extender.

Congelação de sêmem canino com diferentes concentrações de gema de ovo e glicerol em diluidores à base de tris e água de coco

The aim of the present research was to compare the efficiency of coconut water and Tris extenders on canine semen freezing. The semen from 5 German Shepherd dogs was collected by digital manipulation. The spermatic fraction of different dogs was evaluated about its macro and microscopic parameters and mixed in a pool. It was extended in coconut water or Tris, added of egg yolk (10 or 20%) and glycerol (4, 6 or 8%). Then, it was frozen by the method of NUNES et al. (1997) and thawed at 37oC after one week. A Tris extender superiority was shown at the conservation of vigor (2.3 ± 0.8), total (14.8 ± 5.1%) and secondary (14.4 ± 5.5%) spermatic defects in the protocols with 20% of egg yolk and 6% of glycerol. In all other protocols, there were no significant differences for the observed parameters. These results suggest a superiority of Tris extender over the coconut water extender on canine semen freeze, in the use of them added of 20% of egg yolk and 6% of glycerol.

Canine preantral follicles cultured with various concentrations of follicle-stimulating hormone (FSH).

The objective was to evaluate the effects of various concentrations of exogenous FSH during in vitro culture of isolated canine preantral follicles. Preantral secondary follicles (>200 microm) were isolated by microdissection and cultured for 18 d in supplemented alpha-Minimum Essential Medium (alpha-MEM). There were three treatment groups: 1) absence of FSH (control medium); 2) FSH100 (fixed concentration of 100 ng/mL throughout the entire culture period); and 3) sequential FSH (FSHSeq - 100, 500, and 1,000 ng/mL were added sequentially). Following culture, all follicles from all treatments were still viable (marked green by calcein-AM). The initial (D0) average follicle diameter for the control, FSH100, and FSHSeq was (mean +/- SEM) 298.96 +/- 7.02, 286.00 +/- 5.87, and 275.39 +/- 174 6.55 microm, respectively (P > 0.05). Mean diameter of follicles treated with FSHSeq on Day 18 (D18-439.80 +/- 14.08 microm) was greater than those of the other treatments (P < 0.05). Daily follicular growth rate (microm/d) of follicles in the FSHSeq treatment (6.47 +/- 0.55) was significantly faster than for both the control (3.67 +/- 0.32) and FSH100 (4.47 +/- 0.38) treatments. Furthermore, FSH100 and FSHSeq treatments had a significantly higher rate of antrum formation than the control group on D12 of culture, whereas after D12, FSH100 had a significantly higher rate of extrusion compared to the control (P < 0.05). In conclusion, the sequential addition of FSH to the culture medium maintained the survival of isolated canine preantral follicles and promoted an increased rate of follicular growth and antrum formation.

High insulin concentrations promote the in vitro growth and viability of canine preantral follicles

To determine whether the effects of different concentrations of insulin on the development of canine preantral follicles in vitro were associated or not with FSH, secondary follicles were isolated and cultured. In Experiment 1, follicles were cultured in the following media: modified minimum essential medium (CtrlMEM) alone; CtrlMEM plus 5 ng mL⁻¹ insulin (Ins5ng); CtrlMEM plus 10 ng mL⁻¹ insulin (Ins10ng); and CtrlMEM plus 10 μg mL⁻¹ insulin. In Experiment 2, follicles were cultured in the same media but in the presence of sequential FSH (i.e. CtrlFSH, Ins5ngF, Ins10ngF and 10μgF, respectively). Increasing concentrations of FSH (100, 500 and 1000 ng mL⁻¹) were added sequentially to the culture medium on Days 0, 6 and 12 of culture. Viability were assessed at the end of culture and follicular diameter and the antrum formation rate at four time points (Days 0, 6, 12 and 18). In Experiment 1, the high insulin concentration significantly increased follicular viability (P<0.05). In contrast, in Experiment 2, viability was not affected by the inclusion of insulin. In addition, viability was significantly better in follicles cultured in CtrlFSH (P<0.05). The diameter of follicles in the high-insulin group in Experiment 1 and high-insulin plus FSH group in Experiment 2 was superior to other groups tested. In experiment 2, the Ins10μg and Ins10μgF groups exhibited significantly higher antrum formation rates than the other groups. In conclusion, in the absence of FSH, high concentrations of insulin have beneficial effects on follicular viability. However, to promote the growth of canine preantral follicles in vitro, it is recommended that a combination of insulin and FSH be added to the medium.

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.

Plasma profile of pregnancy associated glycoprotein (PAG) in pregnant Alpine goats using two radioimmunoassay (RIA) systems

Abstract This paper evaluates the profiles obtained for caprine pregnancy associated glycoprotein (cPAG) in pregnant multiparous and nulliparous goats using the heterologous-radioimmunoassay (htPAG-RIA) and the homologous-RIA (hmPAG-RIA) systems during the pregnancy and postpartum periods. The results show that the cPAG concentrations detected through the hmPAG-RIA procedure were lower than those found through the htPAG-RIA method. No statistical differences were observed due to the maternal reproductive status (nulliparous or multiparous goats) during pregnancy using either one of the methods. However, pregnant and cyclic goats were distinguished from each other by cPAG concentrations verified through the hmPAG-RIA procedure. The lower cPAG concentrations detected through this method suggest that this system is more specific than the htPAG-RIA procedure to measure cPAG during pregnancy and postpartum period in goats. The homologous system was also an efficient method to predict pregnancies of only 35 days in both nulliparous and multiparous goats.

Doppler velocimetric parameters of the testicular artery in healthy dogs.

This study aimed to examine the Doppler velocimetric pattern of the testicular artery of small dogs in two different locations. Testes of 21 dogs were evaluated by two-dimensional ultrasonography to measure testicular volume and by Doppler ultrasonography to record the velocimetric patterns of the testicular artery in the spermatic cord and marginal location. The volume of left testes (4.70 ± 1.22 cm(3)) was significantly higher than the volume of the right testes (4.45 ± 1.17 cm(3)). Peak systolic velocity (PSV) of the left spermatic cord was significantly higher than the right side. End-diastolic velocity was significantly higher in the marginal artery than the spermatic cord on both sides; however, resistance and pulsatility indexes were significantly lower in the marginal artery. Results demonstrate the viability of Doppler ultrasonography for characterization of the testicular artery in small dogs and Doppler velocimetric values vary according to the location of measurement along the artery.

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.

Relationship between maternal concentrations of caprine pregnancy-associated glycoprotein in Alpine goats and the number of fetuses using a homologous radioimmunoassay

Abstract The relationship between maternal caprine pregnancy-associated glycoprotein (cPAG) concentrations and fetal number was investigated in 21 nulliparous and 17 multiparous goats. Using a homologous radioimmunoassay (hm-RIA) cPAG was detected in plasma of pregnant goats from the 21st day of pregnancy. Statistical differences between goats bearing single and multiple fetuses were observed after 63 days to the end of pregnancy, except on day 133. After 126 days (3rd week pre-partum) cPAG levels began to decrease slowly in both the groups. The hm-RIA was an efficient method to describe the relationship between maternal PAG concentrations and number of fetuses in goats.

Impact of growth hormone (GH) and follicle stimulating hormone (FSH) on in vitro canine preantral follicle development and estradiol production

OBJECTIVE Evaluate the effect of different concentrations of growth hormone (GH) on the in vitro development of domestic dog (Canis lupus familiaris) preantral follicles in the presence or absence of follicle stimulating hormone (FSH). METHODS Secondary preantral follicles, isolated by microdissection, were cultured in a medium composed of αMEM with bovine serum albumin (BSA), glutamine, hypoxanthine, insulin, transferrin, selenium and ascorbic acid (αMEM(+)-control) added at different concentrations of GH (GH10 ng/ml or GH50 ng/ml) and FSH (GH10+FSH, GH50+FSH). Follicle development was evaluated based on the percentage of intact follicles, antrum formation, follicular diameter, follicular viability using fluorescent markers and estradiol production. RESULTS GH50 was the only treatment that maintained the same percentage of normal morphologically follicles from day 0 to day 18 of culture (P<0.05). For all treatments, except the control, follicles were viable throughout the 18 days of culture (P<0.05). GH50 supplemented with FSH (GH50+FSH) resulted in the highest average follicular diameter (P<0.05) from day 12 to 18. Follicles from both the control and the GH50+FSH treatment groups actively and increasingly secreted estradiol from day 6 to 18 of culture (P<0.05). CONCLUSIONS Our study demonstrates that GH benefits the maintenance of follicular morphology in a dose-dependent manner and, in association with FSH, stimulates in vitro follicular growth and estradiol production.