Study on Seminal Attributes of X- sperm Enriched Sahiwal Bull Semen

The present study was conducted to observe the effect of percoll density gradient centrifugation on quality of semen. Ejaculates were collected by AV method from Sahiwal bulls. X-sperm enrichment was done by percoll density gradient method i.e. 7 layers (70-10%). Centrifugation was done at 750 g (22-24°C) for 15 min. The pellets obtained were diluted in EYC medium. Semen quality was evaluated in fresh semen (Control), in pellet of normal centrifugation (Group I), supernatant of centrifugation in percoll density gradient (Group II) and pellet of centrifugation in percoll density gradient (Group III). To assess the quality of enriched semen pH, mass motility, progressive motility, live spermatozoa %, abnormal spermatozoa %, HOST % and intact acrosome % were evaluated. Number of progressively motile sperms in pellet of X- enriched semen were non-significantly increased and significantly (P<0.05) decreased in supernatant. The abnormal spermatozoa (%) were decreased in G III as compared to G II Live spermatozoa (%) were increased in enriched semen (pellet). Number of Intact sperms decreased significantly (P<0.05) in supernatant of percoll density gradient centrifuged Sahiwal semen. HOST responsive sperms number was not affected after percoll density gradient centrifugation. Thus, the semen quality of X-sperm enriched semen by percoll density gradient method (7 layer 70%) was not affected hence it can be used to increase female calves’ birth after A.I.

Correlation between adhesion molecules on the surface of red blood cell membrane and red blood cell lifespan / 中国输血杂志

【Objective】 To explore the correlation between red blood cell lifespan and adhesion molecules on the surface of red blood cell membrane, in order to establish a method to detect the duration of red blood cell storage. 【Methods】 10 samples(10 mL each) of fresh red blood cell, collectedf rom 10 healthy voluntary blood donors, were divided into 5 age groups (layers) by Percoll density gradient centrifugation. The expression of CD47, CD44 and CD147 on the surface of red blood cell membrane in each layer was detected using flow cytometry. The variance of protein expression in each layer of red blood cells was analyzed by SPSS statistical software. 【Results】 The expression levels (%) of 3 adhesion molecules on the surface of red blood cell membranes from young to old were CD47: 14.44±2.61, 9.30±1.75, 7.84±1.49, 6.54±1.32 and 5.53±1.12 (P<0.01); CD44: 25.01±1.94, 19.22±1.52, 17.10±1.28, 15.18±1.11 and 13.56±1.08 (P<0.01); CD147: 33.46±1.99, 28.31±2.95, 23.83±1.59, 20.40±1.56 and 18.03±1.65 (P<0.01). 【Conclusion】 The expression levels of CD47, CD44 and CD147 on the surface of red blood cell membranes have showed a downward trend as the storage extended. These three protein adhesion molecules have showed a correlation with red blood cells lifespan, and could be used as detection markers of cell age.

Evaluación de métodos de concentración y purificación de Giardia spp. a partir de muestras coprológicas / Evaluation of methods of concentration and purification of Giardia spp. from coprological samples

RESUMEN El objetivo del estudio fue comparar diferentes métodos de concentración para recuperar la mayor cantidad de quistes de Giardia spp. a partir de muestras coprológicas. Se analizaron 100 muestras procedentes de hospitales de referencia nacional y se aplicaron cuatro métodos parasitológicos: concentración por sedimentación espontánea en tubo (TSET), Faust, gradiente de sucrosa de una fase y gradiente de sucrosa de dos fases. Se encontró que el método de gradiente de sucrosa de dos fases alcanzó resultados significativamente mejores en concentración de quistes (121 903 quistes/ml) y cantidad de detritos (6%), en comparación con los métodos de Faust (35 355 quistes/ml), concentración por sedimentación espontánea en tubo (20,145 quistes/ml) y gradiente de sucrosa de una fase (18 702 quistes/ml). Se concluye que el método más eficaz para la concentración y purificación de quistes de Giardia spp. a partir de muestras coprológicas es el método de gradiente de sucrosa de dos fases, lo que facilitaría los cultivos in vitro de Giardia spp.

ABSTRACT The aim of this study was to compare different methods of concentration to recover the largest number of Giardia spp. cysts from coprological samples. One hundred (100) samples from national reference hospitals were analyzed and four parasitological methods were applied: spontaneous tube sedimentation concentration (TSET), Faust, single-phase sucrose gradient, and two-phase sucrose gradient. The two-phase sucrose gradient method was found to achieve significantly better results in cyst concentration (121,903 cysts/ml) and amount of debris (6%), compared to Faust methods (35,355 cysts/ml), spontaneous tube sedimentation concentration (20,145 cysts/ml), and single-phase sucrose gradient (18,702 cysts/ml). It is concluded that the most effective method for the concentration and purification of Giardia spp. cysts from coprological samples is the two-phase sucrose gradient method, which would facilitate in vitro culture of Giardia spp.

Evaluation of normal morphology, DNA fragmentation, and hyaluronic acid binding ability of human spermatozoa after using four different commercial media for density gradient centrifugation / 대한생식의학회지

OBJECTIVE: Density gradient centrifugation (DGC) is frequently used to isolate high-motility fractions of spermatozoa. We compared the efficacy of four DGC media in terms of the percentage of morphologically normal spermatozoa, DNA fragmentation level, and hyaluronic acid (HA) binding ability. METHODS: Thirty men with a total motile spermatozoa count >80 million participated. Semen samples were divided into four aliquots, which were processed using PureSperm, PureCeption, Sidney, and SpermGrad media, respectively. The DNA fragmentation level was measured using the Halosperm assay kit and HA binding ability was measured using the HBA assay kit. RESULTS: The mean percentage of morphologically normal spermatozoa was significantly enhanced after DGC using all four media (10.3%, 9.9%, 9.8%, and 10.7%, respectively; p0.05). HA binding ability did not change after DGC using any of the four media. CONCLUSION: The four media were equally effective for obtaining a sperm fraction with highly motile, morphologically normal sperm. PureSperm, PureCeption, and SpermGrad media were equally effective for acquiring a sperm fraction with less DNA fragmentation.

A new method for separation of human pancreatic stellate cells / 中华肝胆外科杂志

Objective To explore a new method for the separation of human pancreatic stellate cells.Methods Single-cell suspension of normal pancreatic tissue and pancreatic cancer tissue was prepared by gentle MACSTM tissue processor-constant temperature shaking digestion.Human pancreatic stellate cells of quiescent and activated state were isolated by density gradient centrifugation.Results A new type of isolation method could obtain about (2.6 ± 0.7) × 106 quiescent pancreatic stellate cells in 1 g of human normal pancreatic tissue,with a viability of about 90.0％.The morphology of the cells were conformed to the representative for the quiescent state characteristics and transient blue-green autofluorescence was observed at the 328 nm excitation wavelength;1 g of human pancreatic cancer was able to obtain approximately (4.1 ± 1.1) × 106 activated PSCs with a viability of 92.0％,and all of the activated cells expressed α-SMA vimentin,FSP-1 and other characteristic markers.Conclusions The new separation method of this experiment is suitable for both human resting and activated human pancreatic stellate cells.At the same time,the purity is high and the separation time is greatly shortened,which is worth promoting.

Sperm DNA fragmentation and sex chromosome aneuploidy after swim-up versus density gradient centrifugation / 대한생식의학회지

OBJECTIVE: The aim of this study was to compare the efficacy of swim-up and density gradient centrifugation (DGC) for reducing the amount of sperm with fragmented DNA, sex chromosome aneuploidy, and abnormal chromatin structure. METHODS: Semen samples were obtained from 18 healthy male partners who attended infertility clinics for infertility investigations and were processed with swim-up and DGC. The percentages of sperm cells with fragmented DNA measured by the sperm chromatin dispersion test, normal sex chromosomes assessed by fluorescence in situ hybridization, and abnormal chromatin structure identified by toluidine blue staining were examined. RESULTS: The percentage of sperm cells with fragmented DNA was significantly lower in the swim-up fraction (9.7%, p=0.001) than in the unprocessed fraction (27.0%), but not in the DGC fraction (27.8%, p=0.098). The percentage of sperm cells with normal X or Y chromosomes was comparable in the three fractions. The percentage of sperm cells with abnormal chromatin structure significantly decreased after DGC (from 15.7% to 10.3%, p=0.002). The swim-up method also tended to reduce the percentage of sperm cells with abnormal chromatin structure, but the difference was not significant (from 15.7% to 11.6%, p=0.316). CONCLUSION: The swim-up method is superior for enriching genetically competent sperm.

The effect of semen optimization on the rate of sperm DNA fragmentation and its effect on pregnancy out-come of IVF-ET / 实用医学杂志

Objective To investigate the method of effectively density gradient centrifugation combined with swim-up improves sperm nuclear integrity and determine whether the sperm chromatin dispersion test of sperm DNA fragmentation in raw or DGC-swim-up treated semen can influence the outcome of IVF. Method The DNA integrity of spermatozoa from 120 patients underwent IVF were analyzed by SCD before and after DGC and swim-up. The predictive value of the SDFI for IVF outcomes were assessed in a cohort of 100 patients who were underwent new embryo transfer. Result In male infertility group,DGC combined with swim-up decreased the SDFI from 22.75(14.44,30.25)to 11.50(5.60,22.79),while the control group decreased the SDFI from 20.86(15.00,26.81) to 7.50(3.63,15.44),respectively(P<0.05);SDFI after optimization in clinical pregnancy group was significantly lower than that of non-pregnant group. The area under the receiver operating characteristic curve was 0.667. The patients with low sperm DFI had a higher implantation rate and pregnancy rate compared with patients with high sperm DFI. Conclusions DGC and swim-up treated Sperm DNA fragmentation can predict the outcome of IVF. The effect of semen optimization on the rate of sperm DNA fragmentation is limited,once exceed,pregnancy rate and birth rate are decreased although fertilization is normal.

The effect of semen optimization on the rate of sperm DNA fragmentation and its effect on pregnancy out-come of IVF-ET / 实用医学杂志

Objective To investigate the method of effectively density gradient centrifugation combined with swim-up improves sperm nuclear integrity and determine whether the sperm chromatin dispersion test of sperm DNA fragmentation in raw or DGC-swim-up treated semen can influence the outcome of IVF. Method The DNA integrity of spermatozoa from 120 patients underwent IVF were analyzed by SCD before and after DGC and swim-up. The predictive value of the SDFI for IVF outcomes were assessed in a cohort of 100 patients who were underwent new embryo transfer. Result In male infertility group,DGC combined with swim-up decreased the SDFI from 22.75(14.44,30.25)to 11.50(5.60,22.79),while the control group decreased the SDFI from 20.86(15.00,26.81) to 7.50(3.63,15.44),respectively(P<0.05);SDFI after optimization in clinical pregnancy group was significantly lower than that of non-pregnant group. The area under the receiver operating characteristic curve was 0.667. The patients with low sperm DFI had a higher implantation rate and pregnancy rate compared with patients with high sperm DFI. Conclusions DGC and swim-up treated Sperm DNA fragmentation can predict the outcome of IVF. The effect of semen optimization on the rate of sperm DNA fragmentation is limited,once exceed,pregnancy rate and birth rate are decreased although fertilization is normal.

Efficient isolation of sperm with high DNA integrity and stable chromatin packaging by a combination of density-gradient centrifugation and magnetic-activated cell sorting / 대한생식의학회지

OBJECTIVE: This study was carried out to investigate the correlations of the sperm DNA fragmentation index (DFI) with semen parameters and apoptosis, and to investigate the effects of density-gradient centrifugation (DGC) and magnetic-activated cell sorting (MACS) on reducing the proportion of sperm with DNA fragmentation and protamine deficiency. METHODS: Semen analysis and a sperm DNA fragmentation assay were performed to assess the correlations between semen parameters and the DFI in 458 semen samples. Sperm with progressive motility or non-apoptosis were isolated by DGC or MACS, respectively, in 29 normozoospermic semen samples. The effects of DGC or MACS alone and of DGC and MACS combined on reducing the amount of sperm in the sample with DNA fragmentation and protamine deficiency were investigated. RESULTS: The sperm DFI showed a significant correlation (r=–0.347, p<0.001) with sperm motility and morphology (r=–0.114, p<0.05) but not with other semen parameters. The DFI (11.5%±2.0%) of semen samples was significantly reduced by DGC (8.1%±4.1%) or MACS alone (7.4%±3.9%) (p<0.05). The DFI was significantly further reduced by a combination of DGC and MACS (4.1%±1.3%, p<0.05). Moreover, the combination of DGC and MACS (1.6%±1.1%, p<0.05) significantly reduced the protamine deficiency rate of semen samples compared to DGC (4.4%±3.2%) or MACS alone (3.4%±2.2%). CONCLUSION: The combination of DGC and MACS may be an effective method to isolate high-quality sperm with progressive motility, non-apoptosis, high DNA integrity, and low protamine deficiency in clinical use.

Isolation,culture and identification of bi-directional differentiation potential liver stem cells in fetal mice / 重庆医学

Objective To optimize the method of isolating ,culturing and screening fetal mouse liver stem cells in vitro ,and to identify the potential of bi‐directional differentiation .Methods The fetal liver stem cells of mouse were isolated by the density gra‐dient centrifugation and cell difference adherence method ,the proliferation of stem cells was determined by cell plate cloning tech‐nique and MTT method;stem cells were induced for differentiation by adding DMSO and HGF .Results The isolated stem cells showed adherence within 24 h ,which were orbicular‐ovate ,closely packed ,activated within 1~2 weeks ;the positive rates of CD133 , CD49f and EPCAM were (97 .95 ± 1 .21)% ,(92 .71 ± 3 .49)% and (50 .73 ± 3 .45)% respectively ;AFP and CK19 proteins were expressed;red glycogen granules were seen by PAS after induced differentiation;ALB and HNF‐4αwere expressed .Conclusion Fe‐tal hepatic stem cells are successfully isolated by the density gradient centrifugation combined with difference adherence method ,and the isolated cells have strong stemness and proliferation ability ,as well as the ability of bi‐directional differentiation towards hepato‐cytes and bile duct epithelial cells .

Comparison of the effect of different media on the clinical outcomes of the density-gradient centrifugation/swim-up and swim-up methods / 대한생식의학회지

OBJECTIVE: Sperm must be properly prepared in in vitro fertilization (IVF)-embryo transfer (ET) programs in order to control the fertilization rate and ensure that embryos are of high quality and have appropriate developmental abilities. The objective of this study was to determine the most optimal sperm preparation method for IVF. METHODS: Patients less than 40 years of age who participated in a fresh IVF-ET cycle from November 2012 to March 2013 were included in this study. Poor responders with less than three mature oocytes were excluded. Ham's F-10 medium or sperm-washing medium (SWM) was used in combination with the density-gradient centrifugation/swim-up (DGC-SUP) or SUP methods for sperm preparation. A total of 429 fresh IVF-ET cycles were grouped according to the media and methods used for sperm preparation and retrospectively analyzed (DGC-SUP/Ham's F-10, n=82; DGC-SUP/SWM, n=43; SUP/Ham's F-10, n=181; SUP/SWM, n=123). RESULTS: There were no significant differences among these four groups with respect to the mean age of the female partners, duration of infertility, number of previous IVF cycles, and retrieved oocytes. We determined that both the DGC-SUP and SUP methods for sperm preparation from whole semen, using either Ham's F-10 or SWM media, result in comparable clinical outcomes, including fertilization and pregnancy rates. CONCLUSION: We suggest that both media and both methods for sperm preparation can be used for selecting high-quality sperm for assistive reproductive technology programs.

Progress in the isolation and purification methods of adipose-derived stem cells / 中国比较医学杂志

Adipose-derived stem cells ( ASCs ) as potential seeded cells have been widely used in tissue engineering.Thus to obtain enough, high activity, high purity adipose-derived stem cells is the particular important premise of the application in tissue engineering.In this paper, the isolation and purification methods of ASCs were reviewed and the merit and demerit of different methods were compared in order to provide theoretical basis for safe and high-effective isolation and purification of ASCs.

A comparative study of tumor-associated macrophages isolation in colorectal carcinoma by Ficoll-Hy-paque density gradient centrifugation and Percoll density gradient centrifugation / 实用医学杂志

Objective Percoll density gradient centrifugation and Ficoll-Hypaque density gradient cen-trifugation, which are frequently-used methods for separation of tumor-associated macrophages (TAMs) from solid carcinoma were compared, in order to find an effective way to separate TAMs from colorectal carcinoma (CRC). Furthermore, we studied the best adherence time of separating macrophage among mononuclear cells. Methods specimens were collected from CRC patients , after digesting into single cells , TAMs were separated from the same specimen by 100% Ficoll, 35% percoll and 25% combined with 65% percoll respectively. After these pre-liminary separation, the collected cells were purified a second time by adherence separation. The purity of TAMs were detected by immunofluorescence. Results TAMs purity from Ficoll-Hypaque density gradient centrifugation was 80.18%, statistically higher than that from Percoll density gradient centrifugations (54.33% and 10.93% re-spectively). Conclusion Compared to Percoll density gradient centrifugation, Ficoll-Hypaque density gradient centrifugation is a more effective and simple way to isolate TAMs from colorectal carcinoma , suggesting it can be wildly used in clinical and basic medical research. 2-4 hours is the best adherence time for isolating macrophage.

An optimization technique to purify the pre-ribosome and ribosome from mammalian cells using continuous sucrose density gradient ultracentrifugation / 国际生物医学工程杂志

Objective To purify pre-ribosome and ribosome of mammalian ceils using continuous sucrose density gradient ultracentrifugation.Methods Continuous sucrose density gradient was established by ultracentrifugation,and the continuous sucrose density gradient of 10％-30％ and 10％-45％ were used to extract the pre-ribosome and ribosome in mammalian cells,respectively.The mammalian cell lysis buffer was added to the established continuous sucrose density gradient.Pre-ribosome and ribosome with different sedimentation coefficients were collected and the A260 absorbance of each sample was measured.Proteins of each sample were extracted to detect the large subunit protein,RPL15 by Western Blot.Results Large subunit ribosomal protein RPL15 exists on 60S of the pre-ribosome,and also on 60S,80S and polyribosome of mature ribosome.Conclusions The continuous sucrose density gradient,which is established by the swing-out rotor,can be used to isolate the pre-ribosome and ribosome of mammalian cells rapidly.This method has the advantages of good separation effect and simple operation,which provides a good method for rapid and large amount preparation and separation of various kinds of ribosomes.

An optimized extraction protocol of prefrontal cortical and striatal synaptosomes from SHR rat / 中国比较医学杂志

Objective To introduce an improved extraction method of prefrontal cortical and striatal synaptosomes from SHR rat. Methods Synaptosomes were prepared from SHR rat brain tissue by Percoll density gradient centrifugation.Transmission electron microscopy was used to assess the morphology and structural integrity of the synaptosomes.Results The obtained synaptosomes showed oval structures surrounded by an intact membrane.Presynaptic components contained one or more mitochondria and a large number of synaptic vesicles.The synaptic clefts were clearly visible, and prominent part of the characteristic compact structure was clear, complete and with higher electron-density. The synaptosome presynaptic membrane, synaptic cleft, and postsynaptic membrane were well preserved, and the synaptosomes were densely distributed, showing typical morphological characteristics of synaptosomes.Conclusions The results of our study improved the traditional preparation method and provide a less time-consuming, highly productive protocol for preparation of structurally typical and intact synaptosomes, suitable for further research on neuroscience and neurological diseases.

Detection of the covalently closed circular DNA in peripheral blood mononuclear cells of hepatitis B patients and its clinical significance / 中华传染病杂志

Objective To analyze the correlation between covalently closed circular DNA (cccDNA) in the peripheral blood mononuclear cells (PBMC) of hepatitis B virus (HBV)-infected patients and serum HBV DNA,hepatitis B surface antigen (HBsAg),hepatitis B e antigen (HBeAg) and liver histology of hepatitis B patients,and to explore the clinical significance of HBV cccDNA detection in PBMC.Methods One hundred and eight patients with chronic HBV infection were involved in this study.PBMC were extracted using density gradient centrifugation.HBV cccDNA in PBMC and serum HBV DNA were detected by real-time fluorescence quantitative polymerase chain reaction.HBsAg and HBeAg were detected by chemiluminescence immunoassay.Liver biopsy was conducted in 59 out of the 108 patients.Chi-square test was used to compare the categorical variables.Correlation analysis was used to compare quantitative variables.Nonparametric test was used to compare the non-normal distribution parameters.Results In the overall population,HBV cccDNA in PBMC was positive in 59 patients (54.6％).Eleven of the 15 patients with liver failure were found to be HBV cccDNA positive,which was significantly higher than that in the acute hepatitis B group (only 2 of the 8 patients were HBV cccDNA positive; x2 =4.960,P＜0.05).One hundred and eight patients were categorized into three groups according to their serum HBV DNA levels,with group A:＞5 lg copy/mL,group B:3-5 lg copy/mL and group C:＜3 lg copy/mL.The proportions of HBV cccDNA positivity in PBMC in three groups were 76.1％ (51/67),5/18 and 13.0％ (3/23),respectively.Comparing with patients with lower HBV DNA (group B and C),the proportion of HBV cccDNA positivity was higher in patients with higher HBV DNA (group A; x2=14.751,P＜0.05 and x2 =28.384,P＜0.05,resepectively).The HBV cccDNA quantitation in PBMC was positively correlated with the serum HBV DNA level and HBsAg quantification (r=0.554,P＜0.05 and r=0.497,P＜0.05,respectively).The proportion of HBV cccDNA positivity in PBMC of patients with liver histology ≥G2 and/or ≥S2 was significantly higher than that in patients with liver histology ＜ G2/S2 (x2 =9.159,P＜0.05).Conclusions HBV cccDNA exists in PBMC of hepatitis B patients.The HBV cccDNA quantitation in PBMC is positively correlated with the serum level of HBV DNA and HBsAg quantification,and is also associated with liver histology injury.

Detection and clinical significance of circulating tumor cells in pa-tients with gastric cancer / 中国肿瘤临床

Objective: To detect circulating tumor cells (CTCs) in patients with gastric cancer and evaluate the relationship among CTCs, clinico-pathological characteristics, and prognosis of gastric cancer. Methods: Peripheral blood samples (10 mL in EDTA) were obtained from 45 patients with gastric cancer. CTCs were detected using density-gradient centrifugation and immunofluo-rescence staining. The clinical significance of the two methods were also compared and investigated. Results:CTC-positive case was defined by the presence of at least one CK19 (+)-CTC per 10 mL of the sample. CTCs were found in 27 of the 45 patients with gastric cancer. The presence of CTCs was significantly correlated with lymph node metastasis, distant metastasis, and recurrence (P=0.007, 0.035, 0.035, respectively). However, CTCs were not significantly correlated with sex, age, tumor location, TNM staging, and tumor differentiation (P>0.05). Conclusion:CTCs were associated with poor prognosis of gastric cancer.

Density gradient centrifugation for isolation of umbilical cord blood stem cells：Screening of separation medium / 中国组织工程研究

10.3969/j.issn.2095-4344.2013.23.002

Evaluación de las técnicas de capacitación espermática y su efecto en la fragmentación del ADN / Assesment of sperm capacitation techniques and their effect on DNA fragmentation

Objetivo: evaluar el efecto de las técnicas de capacitación espermática en la fragmentación del ADN. Materiales y métodos: estudio experimental en el cual se describió el efecto de las técnicas de capacitación espermática swim up y gradientes de densidad de Isolate en la fragmentación del ADN. Se utilizaron las muestras de 41 pacientes remitidos a espermograma a la Unidad de Fertilidad del Tolima (Unifertil). Requisitos: 1) abstinencia sexual de 3 a 5 días, 2) no haber consumido medicamentos como antibióticos o antiparasitarios, 3) obtención de la muestra por masturbación, y 4) no presentar procesos virales o febriles. Después de realizar la primera evaluación de las muestras dentro de una hora posterior a su obtención se incluyeron aquellas que habían sido recolectadas completamente, que tenían una concentración mínima de 2 millones de espermatozoides/ml, y que no tenían contaminación con sangre. Se evaluaron la concentración, la movilidad, la morfología y la fragmentación del ADN de los espermatozoides, midiendo la longitud de los cometas resultantes del ensayo. Para el análisis estadístico se utilizaron análisis de varianza (ANOVA) y el coeficiente de correlación de Pearson, teniendo un valor de p < 0,05 como significativo estadísticamente. Resultados: la media de edad de los pacientes fue 35,1 ± 7,9 años. Se encontró que las técnicas de capacitación espermática, tanto swim up como gradientes de densidad de Isolate, disminuyen la fragmentación del ADN de los espermatozoides (p < 0,0001). No se encontraron diferencias significativas en cuanto a la fragmentación del ADN entre las dos técnicas de capacitación estudiadas. No se observaron correlaciones significativas entre los niveles de fragmentación del ADN espermático y los diferentes parámetros del espermograma (concentración, movilidad y morfología) (p = 0,9061). El ensayo cometa detectó diferencias en el grado de la fragmentación del ADN de los espermatozoides entre las muestras frescas y capacitadas. Conclusión: la capacitación espermática tanto por swim up como por gradientes de densidad de isolate tiene un efecto positivo al disminuir la fragmentación del ADN de los espermatozoides.

Objective: This study was aimed at evaluating the effect of sperm capacitation techniques on DNA fragmentation. Materials and methods: This was an experimental study which observed and described the effect of two sperm capacitation techniques on DNA fragmentation (i.e. swim up and isolate density gradients). Samples from 41 patients were used; these were sent for spermogram analysis at the Tolima Fertility Unit (Unifertil), taking the following requirements into account: sexual abstinence for 3 to 5 days, not having taken drugs such as antibiotics or antiparasitics, samples obtained by masturbation and not presenting viral or febrile processes. The first evaluation of the samples was made one hour after they had been obtained; those which had been collected completely, which had a minimum 2 million spermatozoid/ml concentration and which were not contaminated by blood were included. Concentration, mobility, morphology and DNA spermatic fragmentation were evaluated by measuring comet length in the comet assay in fresh samples. Analysis of variance (ANOVA) and Pearson’s correlation coefficient were used for statistical analysis (p < 0.05 being statistically significant). Results: It was found that sperm capacitation techniques reduced spermatozoid DNA fragmentation (p < 0.0001). No significant differences were found between the two different capacitation techniques studied regarding DNA fragmentation. No significant correlation was observed between sperm DNA fragmentation levels and spermogram parameters (concentrations, mobility and morphology) (p = 0.9061). The comet assay detected differences regarding the degree of spermatozoid DNA fragmentation between fresh and capacitated samples. Conclusion: Both swim up and isolate density gradient sperm capacitation had a positive effect by reducing spermatozoid DNA fragmentation.

Assessment of swim-up and discontinuous density gradient in sperm sex preselection for bovine embryo production / Avaliação do swim-up e do gradiente descontinuo de densidade na pré-seleção do sexo de espermatozoides na produção de embriões bovinos

The purpose of this work was to associate the modified swim-up method with centrifugation in density gradient for the separation of X-bearing spermatozoa. Sperm viability and integrity were evaluated through the Trypan Blue/Giemsa staining method. Quality control of centrifuged spermatozoa was performed in in vitro produced embryos. The results were validated by the sex ratio of in vitro produced embryos using PCR by Y- specific sequences present in bovine male genomic DNA. After determining genetic sex of in vitro produced embryos, the results showed difference (P<0.05) in deviation of sex ratio when comparing the control group (45.2% females) with the other spermatozoa selection procedures (60.6% females) (P<0.05). The sperm selection methods are capable of selecting X-bearing spermatozoa without compromising the spermatozoa fertility (cleavage and blastocyst rates, 70% and 26%, respectively) and were considered relevant methods to be introduced in bovine in vitro produced embryo programs.

O objetivo do presente trabalho foi associar o método de swim-up modificado à centrifugação em gradiente de densidade para a separação de espermatozoides portadores do cromossomo X. A viabilidade e a integridade espermática foram avaliadas pelo método de coloração Azul de Tripan e Giemsa. O controle de qualidade dos espermatozoides centrifugados foi realizado por meio da produção in vitro de embriões bovinos. Os resultados foram validados pela técnica de PCR para verificar a proporção sexual dos embriões produzidos in vitro, com o uso de sequências Y especificas presente no DNA genômico de machos bovinos. Após determinar o sexo genético dos embriões produzidos in vitro, os resultados não mostraram diferença (P<0,05) no desvio da proporção do sexo quando comparou o grupo controle (45,2% de fêmeas) com os outros processos de seleção de espermatozoides (60,6% de fêmeas) (P<0,05). Os métodos de seleção de espermatozoides são capazes de selecionar espermatozoides portadores do cromossomo X sem comprometer a fertilidade, medida pelas taxas de clivagem e blastocisto de 70% e 26%, respectivamente, e foram considerados métodos de relevância para serem introduzidos nos programas de produção in vitro de embriões bovinos.