Influência do método de separação dos espermatozóides viáveis (swim-up) na eficiência de seleção do sexo de bovinos por gradiente descontínuo de densidade e o impacto no melhoramento genético animal

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Transmission electron microscopy for characterization of acrosomal damage after Percoll gradient centrifugation of cryopreserved bovine spermatozoa

The objective of this study was to characterize acrosomal ultrastructure following discontinuous Percoll gradient centrifugation of cryopreserved bovine sperm. Semen was collected from six bulls of different breeds and three ejaculates per bull were evaluated. Frozen semen samples were thawed and the acrosomal region of sperm cells was evaluated by transmission electron microscopy (TEM) before (n = 18) and after (n = 18) Percoll centrifugation. The evaluation of 20 sperm heads from each of the 36 samples analyzed ensured that a large number of cells were investigated. The data were subjected to analysis of variance at a level of significance of 5%. Percoll centrifugation reduced the percentage of sperm exhibiting normal acrosomes (from 61.77 to 30.24%), reduced the percentage of sperm presenting atypical acrosome reactions (from 28.38 to 4.84%) and increased the percentage of sperm exhibiting damage in the acrosome (from 6.14 to 64.26%). The percentage of sperm with typical acrosome reactions was not significantly different before (3.70%) and after (0.67%) centrifugation. TEM distinguished four different types of acrosomal status and enabled ultrastructural characterization of acrosomal injuries. The percentage of sperm exhibiting normal acrosomes decreased and damage in the acrosome was the most frequent acrosomal injury with the Percoll gradient centrifugation protocol utilized.

Effects of semen storage and separation techniques on sperm DNA fragmentation

Objective: To determine the effect of semen storage and separation techniques on sperm DNA fragmentation. Design: Controlled clinical study. Setting: An assisted reproductive technology laboratory. Patient(s): Thirty normoozospermic semen samples obtained from patients undergoing infertility evaluation. Intervention(s): One aliquot from each sample was immediately prepared (control) for the sperm chromatin dispersion assay (SCD). Aliquots used to assess storage techniques were treated in the following ways: snap frozen by liquid nitrogen immersion, slow frozen with Tris-yolk buffer and glycerol, kept on ice for 24 hours or maintained at room temperature for 4 and 24 hours. Aliquots used to assess separation techniques were processed by the following methods: washed and centrifuged in media, swim-up from washed sperm pellet, density gradient separation, density gradient followed by swim-up. DNA integrity was then measured by SCD. Main Outcome Measure(s): DNA fragmentation as measured by SCD. Result(s): There was no significant difference in fragmentation among the snap frozen, slow frozen, and wet-ice groups. Compared to other storage methods short-term storage at room temperature did not impact DNA fragmentation yet 24 hours storage significantly increased fragmentation. Swim-up, density gradient and density gradient/swim-up had significantly reduced DNA fragmentation levels compared with washed semen. Postincubation, density gradient/swim-up showed the lowest fragmentation levels. Conclusion(s): The effect of sperm processing methods on DNA fragmentation should be considered when selecting storage or separation techniques for clinical use. (Fertil Steril (R) 2010;94:2626-30. (C) 2010 by American Society for Reproductive Medicine.)

Detection of HIV and HCV RNA in semen from Brazilian coinfected men using multiplex PCR before and after semen washing

INTRODUCTION: Prolonged survival of patients under HAART has resulted in new demands for assisted reproductive technologies. HIV serodiscordant couples wish to make use of assisted reproduction techniques in order to avoid viral transmission to the partner or to the newborn. It is therefore essential to test the effectiveness of techniques aimed at reducing HIV and HCV loads in infected semen using molecular biology tests. METHODS: After seminal analysis, semen samples from 20 coinfected patients were submitted to cell fractioning and isolation of motile spermatozoa by density gradient centrifugation and swim-up. HIV and HCV RNA detection tests were performed with RNA obtained from sperm, seminal plasma and total semen. RESULTS: In pre-washing semen, HIV RNA was detected in 100% of total semen samples, whereas HCV RNA was concomitantly amplified in only one specimen. Neither HIV nor HCV were detected either in the swim-up or in the post-washing semen fractions. CONCLUSIONS: Reduction of HIV and/or HCV shedding in semen by density gradient centrifugation followed by swim-up is an efficient method. These findings lead us to believe that, although semen is rarely found to contain HCV, semen processing is highly beneficial for HIV/HCV coinfected individuals.

The seasonal and ovarian status effects on in vitro production of domestic cat embryos between Equator and Tropic of Capricorn

From the Tropic of Capricorn to Equator, the seasonality of domestic cat is known to be absent, i.e., these animals are considered non-seasonal breeders at these regions. We hypothesized that this particularity might have some influence on in vitro embryo production. The aim of this experiment was to determine the percentage of cleavage and morulae and blastocyst formation produced from oocytes recovered from queen ovaries of three distinct status - follicular, luteal or inactive - during two different reproductive seasons experienced by cats in southeast of Brazil (22°53'09" S and 48°26'42" W) - non breeding season (NBS), comprehending January to March; and breeding season (BS), August to October. Thirty queens were neutered. Ovaries were classified according to their status and were sliced in PBS for cumulus oocyte complex (COC) releasing. Grade I COC were washed three times in H-MEM supplemented with BSA, glutamine, sodium pyruvate, cysteine, streptomycin and penicillin. Oocytes were incubated in groups of 20-30 in 400µL of DMEM supplemented with FSH, LH, estradiol, IGF-I and basic fibroblast growth factor under mineral oil for 30 or 36 hours at 38°C in humidified environment of 5% de O2, 5% CO2 and 90% N2. COC were fertilized in Ham's F-10 medium supplemented with BSA, cysteine, pyruvate and streptomycin/penicillin (culture medium) with fresh semen selected through swim up technique. Eighteen hours later, the presumptive zygotes were denuded, the percentage of cleavage was determined and every 10 zygotes were transferred to 100mL drops of culture medium for culture during three days. After 72 hours of culture the percentage of morulae formation was evaluated and these structures were transferred to drops of the same culture medium. At the eighth day of culture blastocyst formation was analyzed. During NBS, from a total of 272 (inactive), 162 (luteal) and 134 (follicular) fertilized oocytes, the percentage of cleaved zygotes, morulae and blastocysts derived from inactive ovaries were 24.63, 16.54 and 8.09 respectively; for those derived from luteal ovaries, the percentage was 21.6, 12.96 and 8.64, and for those from follicular ovaries, they were 24.62, 16.41 and 8.21. Considering BS, from a total of 102 (inactive), 198 (luteal) and 86 (follicular) fertilized oocytes, the relative frequency (%) of cleaved zygotes, morulae and blastocysts derived from inactive ovaries were 64.7, 41.17 and 23.53 respectively; for those derived from luteal ovaries, the percentage was 64.14, 40.41 and 23.73, and for those from follicular ovaries, they were 63.95, 39.54 and 24.41. The results of this experiment demonstrate that no statistically significant difference (P<0.05) was verified in the frequency of cleaved embryos and morulae and blastocyst formation when comparing the three ovarian conditions in the same season. However the breeding season presented better results considering cleavage and morulae and blastocyst formation.

Chronic exposure to sub-lethal concentration of a glyphosate-based herbicide alters hormone profiles and affects reproduction of female Jundia (Rhamdia quelen)

This work was carried out to verify the effect of a glyphosate-based herbicide on Jundia hormones (cortisol, 17 beta-estradiol and testosterone), oocyte and swim-up fry production. Earthen ponds containing Jundia females were contaminated with glyphosate (3.6 mg/L); blood samples were collected from eight females from each treatment immediately before, or at 1, 10, 20 30 and 40 days following contamination. A typical post-stress rise in cortisol levels was observed at the 20th and 40th days following exposure to glyphosate. At the 40th day, 17 beta-estradiol was decreased in the exposed females. A similar number of oocytes were stripped out from females from both groups, however, a lower number of viable swim-up fry were obtained from the herbicide exposed females, which also had a higher liver-somatic index (LSI). The results indicate that the presence of glyphosate in water was deleterious to Rhamdia quelen reproduction, altering steroid profiles and egg viability. (c) 2006 Elsevier B.V. All rights reserved.

Penetration of zona-free hamster, bovine and equine oocytes by stallion and bull spermatozoa pretreated with equine follicular fluid, dilauroylphosphatidylcholine or calcium ionophore A23187

Experiments evaluated the ability of follicular fluid (FF), dilauroylphosphatidylcholine (PC12) and the calcium ionophore A23187 (A23187) to induce capacitation in stallion and bull spermatozoa, determined by the ability of the spermatozoa to penetrate zona-free hamster, bovine and equine oocytes. Spermatozoa suspensions were incubated at 37 degreesC in one of the following treatments: 1) a modified Tyrode's medium (BGM3) alone, 2) BGM3 + FF; 3) BGM3 + PC12; 4) BGM3 + FF + PC12; 5) BGM3 + A23187; and 6) BGM3 + FF + A23187. Treated spermatozoa were incubated with zona-free hamster, bovine and equine oocytes for 3 h, after which oocytes were stained to assess spermatozoa penetration. The number of hamster oocytes penetrated by spermatozoa incubated in BGM3 alone (1/30) or in presence of FF (2/31) was significantly lower (P < 0.05) than by spermatozoa treated with PC12 or A23187 (16/30 and 17/30, respectively). Processing stallion spermatozoa either by a swim-up procedure or by centrifugation through a Percoll gradient increased the percentages of motile spermatozoa in the final sample, and spermatozoa collected by both processes penetrated similar numbers of zona-free hamster oocytes (P > 0.05). Although treating spermatozoa with PC12 or A23187 enabled both stallion and bull spermatozoa to penetrate oocytes, higher numbers of bovine oocytes were penetrated by bull spermatozoa (25/30) than by stallion spermatozoa (4/30) regardless of spermatozoal treatment. However, the number of zona-free hamster and equine oocytes penetrated by bull spermatozoa (25/30 and 12/18 respectively) and stallion spermatozoa (17/30 and 15/21 respectively) were similar (P > 0.05). We conclude that both PC12 and A23187 capacitate stallion and bull spermatozoa sufficiently to permit the acrosome reaction to occur, enabling spermatozoa to penetrate homologous and heterologous zona-free oocytes. (C) 2001 by Elsevier B.V.

The effects of stressful broodstock handling on hormonal profiles and reproductive performance of rhamdia quelen (Quoy & Gaimard) females

A stressful environment induces cortisol that might affect fish breeding and reproduction. In the present work, which aimed to mimic aquacultural conditions of the jundia (Rhamdia quelen) hatcheries in southern South America, females were submitted to normal or stressful handling and the effects of cortisol on serum levels of 17β-estradiol (E2) and testosterone (T) were determined. In addition, the effect of stress on reproductive parameters such as eggs and swim-up fry production was also measured. Eight females from a group submitted to stressful handling (SH) conditions and eight females from a group with normal handling (NH) conditions were captured for blood sampling at D 0 and at D 1, 10, 20, 30, and 40 of the experiment. A typical cortisol response was observed in the SH females group in that they presented higher cortisol level in contrast to the NH female group, in all days sampled, except at D 0. In the 10th and 20th d, the E2 levels were lower in SH females, but cortisol levels were higher, suggesting an effect of cortisol on E2 production and/or release. Stressful handling appeared to affect both the number and the quality of the gametes because a lower number of oocytes was stripped from SH females, and from SH fertilized eggs, a lower number of viable swim-up fry was obtained to be transferred to earthen larviculture ponds. Taken together, the results indicated that stressful handling of broodstock impairs R. quelen reproduction. © Copyright by the World Aquaculture Society 2008.

Efeito do meio diluidor e da dose inseminante sobre a congebilidade e fertilidade do sêmen bovino utilizado em programas de inseminação artificial em tempo-fixo(LATF)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The seasonal and ovarian status effects on in vitro production of domestic cat embryos between Equator and Tropic of Capricorn

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Relationship between thirty post-thaw spermatozoal characteristics and the field fertility of 11 high-use Australian dairy AI sires

This study determined the relationship between two measures of field fertility of I I high-use Australian artificial insemination (AI) dairy bulls and thirty standard laboratory assessments of spermatozoal post-thaw viability. The two measures of field fertility used, conception rates (cCR) and non-return rates (cNRR), were both corrected for all major non-bull variables. Sperm viability assessments were conducted on semen collected within the same season as that used to derive the field fertility estimates. These assessments measured sperm concentration, motility, morphology and membrane integrity at thawing, after 2 h incubation and after the swim-up sperm selection procedure. Derivations of these measures and in vitro embryo fertilizing and developmental capacity were also determined. The Genstat Statistical Package [Genstat 5 Release 4.2 Reference Manual, VSN International, Oxford, 20001 was used to conduct an analysis of variance on the viability parameters across semen straws and bulls, and to calculate the strength of correlation between each semen parameter, cNRR and cCR in a correlation matrix. Step forward multiple regression identified the combination of semen parameters that were most highly correlated with cCR and with cNRR. The sperm parameters identified as being most predictive of cCR were the percentage of morphologically normal sperm immediately post-thaw (zeroNorm), the number of morphologically normal sperm after the swim-up procedure (nSuNorm), and the rate of zygote cleavage in vitro (Clv); the predictive equation formed by these parameters accounted for 70% of variance. The predictive equation produced for cNRR contained the variables zeroNorm, the proportion of membrane intact sperm after 2 h incubation at 37 degreesC (twoMem) and Clv and accounted for 76.5% of the variation. ZeroNorm was found to be consistent across straws and semen batches within-bull and the sperm parameter with the strongest individual predictive capacity for both cCR (P = 0.1) and cNRR (P = 0.001). Post-thaw sperm parameters can be used to predict field fertility of Australian dairy sires; the calculated predictive equations are particularly useful for identifying and monitoring bulls of very high and very low potential fertility within a group. (C) 2003 Elsevier B.V. All rights reserved.

Chronic exposure to sub-lethal concentration of a glyphosate-based herbicide alters hormone profiles and affects reproduction of female Jundia (Rhamdia quelen)

This work was carried out to verify the effect of a glyphosate-based herbicide on Jundia hormones (cortisol, 17 beta-estradiol and testosterone), oocyte and swim-up fry production. Earthen ponds containing Jundia females were contaminated with glyphosate (3.6 mg/L); blood samples were collected from eight females from each treatment immediately before, or at 1, 10, 20 30 and 40 days following contamination. A typical post-stress rise in cortisol levels was observed at the 20th and 40th days following exposure to glyphosate. At the 40th day, 17 beta-estradiol was decreased in the exposed females. A similar number of oocytes were stripped out from females from both groups, however, a lower number of viable swim-up fry were obtained from the herbicide exposed females, which also had a higher liver-somatic index (LSI). The results indicate that the presence of glyphosate in water was deleterious to Rhamdia quelen reproduction, altering steroid profiles and egg viability. (c) 2006 Elsevier B.V. All rights reserved.

Evaluación cuali-cuantitativa de espermatozoides de la cola del epedídimo de cuyes cavia porcellus criollos y mejorados en dos edades reproductivas

El objetivo de esta investigación fue evaluar las características cuali-cuantitativas de espermatozoides de cuyes extraídos de la cola del epidídimo según su fenotipo y edad reproductiva. Se realizó en la granja Irquis de la U. de Cuenca en 20 reproductores identificados por sus características fenotípicas y dispuestos en cuatro grupos: 5 criollos jóvenes (CJ), 5 criollos adultos (CA), 5 mejorados jóvenes (MJ), y 5 mejorados adultos (MA). Los cuyes fueron hemicastrados y de los epidídimos fueron disectados la cola sobre una caja petri. Se recuperó los espermatozoides por Swim up, diluidos en 1ml de medio (18% rafinosa y 3% leche descremada), procesados con Triladyl®, refrigerados a 5oC/1 hora, y equilibrados por 0, 2, 24, 48, 96, 192, y 360 horass para su análisis de viabilidad espermática. Se congelaron únicamente los espermatozoides de 2 hs de equilibrio en vapores de nitrógeno. Se usó un DCA de 2x2: fenotipo y edad, y se usó un ANOVA para comprobar significancia. Se obtuvo interacción (P<0,05) entre factores con eficiencia atribuida a MJ a las 0 hs: en Concentración (C) y Anormalidades de cola (AC), a las 24 hs: en motilidad individual (MIP) y 48 hs: en Vitalidad (VE). En MIP no se encontró diferencias (P>0,05) en ningún tiempo de medición. En VE sólo encontró diferencias (P<0,05) a las 96 hs (CJ:18,0;MJ:10,2;MA:8,6;CA:6,0%). En anormalidades totales (AT) sólo se encontró diferencias (P<0,05) a las 0 hs (MJ:26,3;CJ:32,6;MA:36,2;CA:38,5%); y en AC se encontró diferencias (P<0,05) a las 0 hs (MJ:4,6; CJ:9,5; CA:11,5; MA:16,4%), y a las 48 hs (CA:5,7;CJ:7,3;MJ:16,0;MA:18,1%). En Integridad de la membrana (HOS-Test) se obtuvo (P<0,05) diferencias a las 2 hs (MJ:20,0; MA:13,1;CA:10,7;CJ:9,0%) y a las 96 hs (CA:25,4;CJ:15,3;MJ:9,7; MA:8,8%). A la congelabilidad no se obtuvo sobrevivencia de espermatozoides en ninguno de los tratamientos. En conclusión, la cantidad y calidad de espermatozoides epididimarios de cuyes identificados fenotípicamente varía según su edad; sin embargo, no se pudo comprobar su variación en la congelabilidad mostrándose absolutamente inviables a la crío conservación

Influence of gender and estrous cycle in the forced swim test in rats

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)