ABSTRACT
SUMMARY: Freeze/thawing process reduces sperm survival and fertilizing ability of cat spermatozoa, with sperm motility being the most sensitive sperm parameter altered, due to cryo-damage. In this context, swim-up and density gradient processing methods can help to recover high motile and normal spermatozoa. Maximizing the use of frozen semen sample is essential, especially in endangered felids or high value cats in which sample size, number of samples or access to semen collection is reduced. To our knowledge, there is no previous report describing an in depth analysis of sperm motility improvement, after sperm selection techniques in frozen cat semen. Accordingly, we evaluated the effect of percoll gradient (PG) and swim up (SU) sperm selection techniques on sperm motility parameters and sperm recovery rate in frozen/thawed spermatozoa of domestic cat. Next, we evaluated the individual effect of the cat over sperm motility after PG sperm selection of frozen/thawed spermatozoa. SU and PG improved significantly all sperm motility parameters of frozen/thawed cat spermatozoa compared to simple washing. However, PG allows better sperm recovery from the original frozen sample and works mostly homogeneously among individual cats. This new information could help to maximize the use of frozen semen in endangered felids or high value domestic cats for its subsequent application on in vitro fertilization and artificial insemination.
RESUMEN: El proceso de congelación/descongelación reduce la sobrevivencia espermática y la habilidad para fertilizar en los espermatozoides de gato, siendo la motilidad espermática el parámetro más sensiblemente alterado debido al daño por frío. En este contexto, los métodos de procesamiento de swim-up y gradiente de densidad pueden ayudar a recuperar los espermatozoides normales y de alta motilidad. Maximizar el uso de una muestra de semen congelado es esencial, especialmente en felinos amenazados o en gatos de alto valor en los cuales el tamaño de muestra, número de muestras o el acceso a la colecta de semen son reducidos. Para nuestro conocimiento, no hay reportes previos que describan un análisis profundo del mejoramiento de la motilidad luego de técnicas de selección espermática en semen congelado de gato. De acuerdo a esto, evaluamos el efecto de las técnicas de selección espermática gradiente de percoll (PG) y swim up (SU) sobre los parámetros de motilidad y porcentaje de recuperación de espermatozoides congelados/descongelados de gato doméstico. Luego, evaluamos el efecto individual del gato sobre la motilidad espermática luego de la selección espermática con PG en espermatozoides congelados/descongelados. SU y PG mejoraron significativamente todos los parámetros de motilidad espermática de los espermatozoides congelados/descongelados comparado con el lavado simple. Sin embargo, PG permitió una mejor recuperación de espermatozoides desde la muestra congelada original y funcionó en su mayoría de manera homogénea entre los gatos individualmente. Esta nueva información puede ayudar a maximizar el uso del semen congelado en felinos amenazados o en gatos de alto valor para su posterior aplicación en fecundación in vitro e inseminación artificial.
Subject(s)
Animals , Male , Cats , Sperm Motility , Cryopreservation , Sperm Retrieval/veterinary , Semen Analysis/veterinary , Semen Preservation , Image Processing, Computer-Assisted , Centrifugation, Density Gradient , Semen Analysis/methodsABSTRACT
The aim of this study was to compare the percentage of sperm tail membrane swelling under hypoosmotic conditions with those with and without pentoxifylline supplements in sperm prepared by the two-layer Percoll gradient method. Twenty five normal semen samples were collected from male partners of infertile couples attending the Infretility Clinic at Siriraj Hospital. After the process of sperm preparation by the two-layer Percoll gradient method, the final samples were divided into 2 tubes, 0.5 ml was added into one tube and another tube was kept as control. The hypoosmotic swelling test was performed on both specimens. The percentage of swollen spermatozoa in the pentoxifylline supplement group was significantly higher than the control group (82.8 + 7.7 vs 70.8 + 12.7; p < 0.00). It was concluded that the addition of pentoxifylline to the sperm prepared by the two- layer Percoll gradient method can enhance the sperm membrane integrity, and it may be beneficial to add pentoxifylline to sperm preparation for use in IUI or IVF.
ABSTRACT
The aim of this study was to compare thepercentages of sperm with an acrosome reaction between those with and without calcium ionophore A23187 induction after two-layer Percoll gradient separation. Thirty normal semen samples were obtained from the male partners of infertile couples attending the Infertility Clinic at Siriraj Hospital. After the process of sperm separation by two-layer Percoll gradient technique, the final samples samples were divided into 2 portions. An aliquot of 10 ตM of calcium ionophore A23187 was added to one portion to induce an acrosome reaction, while the other portion was used as a control. Fluorescein isothiocyanate-conjugated Pisum sativam agglutinin (FITC-PSA) staining was performed on both specimens and the acrosome reated-sperm were evaluated. The percentage of acrosome-reated sperm in the calcium ionophore A23187 induced group was significantly higher than those of the control group (24.8+6.6vs 15.4+6.0;p < 0.001). It is concluded that calcium ionophore can significantly induce an acrosome reaction on sperm separated by two-layer Percoll gradient technique, and it may be beneficial to add calcium ionophore A23187 to sperm preparation for use in IUI or IVF.
ABSTRACT
In order to compare the Percell gradient and IxaPrep gradient methods, the percentage of progressively motile sperm and the percentage of sperm with normal morphology were examined. Thirty five normal semen sample were collected from the male partners of infertile couples attending the Infertility Clinic. The samples were divided into two fractions of 1 ml each. Both fractions were processed using the Percoll gradient and IxaPrep gradient methods, and sperm motility and normal morphology were evaluated. The percentages of spermatozoa that showed progressive motility and normal morphology from the sperm preparation using the IxaPrep gradient method were significantly higher than those prepared by the Percoll gradient method (77.7 + 12.0 vs 73.7 + 12.3 and 64.4 + 15.1 vs 61.1 + 13.3 respectively ; p<0.05). It was concluded that sperm preparation using IxaPrep gradient method showed better sperm quality with respect to progressive motility and normal morphology than those using the Percoll gradient method, The IxaPrep gradient method can be considered as an alternative to the Percoll gradient for sperm preparation.
ABSTRACT
In order to assess the efficiency of Percoll gradient centrifugation(PGC) as a method of sperm selection, we have examined morphological characteristics of spermatozoa from 40 teratozoospermic patients attending the Infertility Clinic of Inchon Gil Gerneral Hospital. Patients were divided into three groups according to percentage normal morphology in the fresh sample : group A(n=5), 14% normal morphology. Morphology slides were perpared using Diff-Quik staining techniques and evaulated by Kruger strict criteria, under oil immersion at a magnification of X 1000, specific defects, head, neck and tail were assesed individualy. The results were as follows. 1. Following PGC, sperm samples with enhanced morphology were recovered for all groups. 2. For group A, PGC did not select a sample with significantly improved morphological characteristics. 3. Usually, sperm defects affected by PGC was head and neck. No significant difference was found for tail abnormality. In conclusion, Percoll gradient centrifugation is an efficient sperm preparation technique when the semen sample exhibits teratozoospermia, especially head or neck abnormality. However, in sample with < 5% normal form or tail abnormality, There is not significant improvement following PGC.
Subject(s)
Humans , Centrifugation , Equidae , Head , Immersion , Infertility , Neck , Semen , SpermatozoaABSTRACT
Highly pure lysosomes were isolated from buffalo (Bubalus bubalis) kidney cortex by a procedure involving differential and isopycnic Percoll density gradient centrifugations. Arylsulphatase, N-acetyl-ß-glucosamindase and cathepsin D in the lysosomal preparation were 26-45-fold enriched over the homogenate. The purified lysosomes contained less than 0·06% of mitochondrial, microsomal and peroxisomal marker enzymes. In the electron micrographs the particles appeared as large dense granules of size 0·3–1·9 μm with no apparent structural features belonging to mitochondria or microsomes. The isolation procedure was also found to be suitable to obtain highly pure lysosome particles from renal cortex of other sources such as rat, lamb and beef. No ultracentrifugation steps were involved in the procedure.
ABSTRACT
Objective To explore an applicable method for isolation and purification of Cryptosporidium parvum oocysts with high purity, recovery and vigor from mouse feces. Methods Four techniques were used for isolating and purifying C.parvum oocysts from mouse feces: modified saturated saline flotation, percoll gradient centrifugation, CsCl gradient centrifugation and the classical discontinuous sucrose gradient centrifugation. Oocysts received from the methods were used respectively to infect in vitro bovine fallopian tube epithelial cells (BFTE) and the development of the oocysts was examined under microscope after 48 h and 72 h cultivation. Results The number of oocysts received by the classical discontinuous sucrose gradient centrifugation [(2.86?0.08)?107] was significantly higher than that of percoll gradient centrifugation [(1.52?0.08)?107] (P0.05). Oocysts received from CsCl gradient centrifugation showed higher purity than those by discontinuous sucrose gradient centrifugation. Conclusion In comparison to the classical discontinuous sucrose gradient centrifugation, operation of the modified saturated saline flotation is easier and faster, and the purity of oocysts isolated by CsCl gradient centrifugation is higher.