• Journal of Embryo Transfer
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• v.15 no.3
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• pp.237-245
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• 2000

This study was carried out to elucidate whether sperm contain a factor inducing second polar body extrusion and to search for an effective collection method of the sperm factor Thus, sperm extract, dialyzed sperm-extract or liquid chromatographic fractions of sperm extract was microinjected into ovulated oocytes. And the microinjected oocytes were incubated for 24 hours to investigate about the extrusion of second polar body. The results obtained were as follows; 1. Sperm extract significantly increased the second polar body extrusion. 2. Sperm extract showed five major fractions at retention volumes (RVs) 1.25, 1.37, 1.84, 2.10 and 2.67ml after separation with Superose 12 column. These sperm extract fractions did not significantly increase the second polar body extrusion. 3. Dialyzed sperm-extract significantly increased the second polar body extrusion 4. Dialyzed sperm-extract showed three maior fractions at RVs 1.88, 2.14 and 2.77ml after separation with Superose 12 column. Of these fractions, the fraction RV2.14 significantly increased the second polar body extrusion. In conclusion, sperm extract contained a factor inducing the second polar body extrusion and the factor was contained largely in fraction RV2.14 after dialysis and liquid chromatographic fractionation of sperm extract.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.2
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• pp.142-148
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• 2020

The research work was undertaken to determine an effective fertilization medium, sperm separation method and sperm capacitating agent for optimum in vitro fertilization (IVF) rates of indigenous zebu cow oocytes. In experiment 1, tissue culture medium (TCM 199), Tyrode's albumin lactate pyruvate (TALP) and Brackett and Oliphant (BO) medium were used as basic medium for IVF of oocytes of indigenous zebu cows. In experiment 2, three sperm separation methods namely centrifugation, swim up and percoll gradient methods were used for separation of motile and viable spermatozoa for IVF. In experiment 3, for capacitation of spermatozoa, IVF medium supplemented with the heparin, mixture of penicillamine, hypotaurine and epinephrine (PHE) or the combination of heparin with PHE were used for fertilization. In vitro culture (IVC) of presumptive zygotes was done in modified synthetic oviduct fluid (mSOF) medium using standard procedure 24 h after sperm-oocytes co-culture. The cleavage rate was determined to evaluate the efficacy of fertilization medium, sperm separation method and sperm capacitating agent 24 h after IVC. The cleavage rate was higher in oocytes fertilized in TALP (63.3%) than in TCM 199 (47.5%) (p < 0.05). The cleavage rate was higher in oocytes fertilized by spermatozoa separated by percoll gradient method (62.3%) than by centrifugation (51.6%) (p < 0.05). The cleavage rate of oocytes was higher when insemination was done with spermatozoa capacitated in TALP supplemented with heparin and PHE (61.3%) compared to control (40.9%) (p < 0.05). In conclusions, TALP based medium and percoll gradient sperm separation followed by capacitation with combination of heparin and PHE are suitable for IVF of indigenous zebu cow oocytes in Bangladesh.

• Journal of Animal Reproduction and Biotechnology
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• v.36 no.2
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• pp.91-98
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• 2021

The objective of this study was to establish a selection process for high quality sperm in bovine semen using sperm separation by magnetic activation (MACS). For this, semen from 21 Nellore bulls was collected using an artificial vagina. To guarantee the presence of pathologies in the ejaculate, animals previously declassified in four consecutive spermiogram were used. Semen was analyzed in five statuses: (1) fresh semen (fresh); (2) density gradient centrifugation (DGC), percoll column; (3) non-apoptotic fraction after separation by MACS (MAC); (4) apoptotic fraction from the separation (MACPOOR); and (5) MAC followed by DGC (MACDGC). Using a computerized analysis system (CASA), motility was measured. The sperm morphology was evaluated by phase contrast, and the supravital test was completed with eosin/nigrosin staining. For DGC, 20 × 10⁶ cells were used in a gradient of 90% and 45% percoll. MACS used 10 × 10⁶ cells with 20 μL of nanoparticles attached to annexin V, and filtered through the MiniMACS magnetic separation column. Membrane integrity was assessed with SYBR-14/IP and mitochondrial potential with JC-1 by flow cytometry. Processing sperm by MACDGC, was more effective in obtaining samples with high quality sperm, verified by the total of abnormalities in the samples: 35.04 ± 2.29%, 21.50 ± 1.47%, 17.30 ± 1.10%, 30.68 ± 1.94% and 10.50 ± 1.46%, respectively for fresh, DGC, MAC, MACPOOR, and MACDGC. The subpopulation of non-apoptotic sperm had a high number of live cells (82.65%), membrane integrity (56.60%) and mitochondrial potential (83.98%) (p < 0.05). These findings suggest that this nanotechnological method, that uses nanoparticles, is efficient in the production of high-quality semen samples for assisted reproduction procedures in cattle.

• Clinical and Experimental Reproductive Medicine
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• v.20 no.1
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• pp.57-63
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• 1993

From September 1988 to August 1992, two different methods of preparing human sperm before intrauterine insemination(IUI) were compared using the semen samples of seventy-three infertile couples. The sperms were prepared by a swim-up after sperm washing or by a continuous percoll gradient technique. Fourteen of 35 women conceived during IUI cycles using a sperm washing and swim-up method (40%), and 12 of 38 women conceived during IUI cycles using a percoll gradient technique(31.6%). Among the group with male infertile etiologic factor only, one of 5 women conceived during sperm washing and swim-up cycles(20%); one of 4 women conceived during percoll gradient cycles(25%). On the contrary, among the group with cervical factor only, six of 10 women conceived during sperm washing and swim-up cycles (60%) ; Five of 17 women conceived during percoll gradient cycles(29.4%). It is suggested that sperm separation by sperm washing ar -up is a useful technique for intrauterine insemination in cervical infertility, and sperm separation in percoll gradient appears to be more valuable for intrauterine insemination of male subfertility.

• Transactions of the Korean Society of Mechanical Engineers A
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• v.32 no.12
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• pp.1115-1122
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• 2008

This paper presents a new microchip which can separate motile sperm by chemotaxis. The microchip was developed to create longitudinal concentration gradient in the microchannel due to diffusion. Linearly good concentration gradient of chemoattractant was generated without any fluid control devices. In sperm separation experiment with the developed microchip, mouse sperm was used as sample and acetylcholine was selected as chemoattractant. Human tubal fluid (HTF), buffer solution, was introduced into the microchannel of the microchip and attractants diluted in ratio of 1, 1/2, 1/4, 1/8, 1/16, 1/32 and 1/64 including control (DI water) were dropped in each outlet by $2\;{\mu}l$ volume with micropippet. After 5min, $1\;{\mu}l$ sperm solution was dropped into inlet of the chip. After 10 min, when sperms reached to the outlet by chemotaxis, we counted sperms in each outlet by using microscopy. Consequently, we could separate progressive motile sperm with the new microchip. In the experiment, the most sperms were isolated at the outlet dropped with 1/16 diluted solution. The optimal concentration gradient to induce chemotaxis was about 0.625 mg/ml/mm.

• Journal of Embryo Transfer
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• v.14 no.1
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• pp.23-29
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• 1999

The objectives of the study were to establish sperm separation method and duration of insemination for bovine IVF. Oocytes from slaughterhouse ovaries were matured and fertilized using general protocol. After 18 or 42 h of insemination, six to ten embryos were placed into a 30${mu}ell$ drop of each medium, and the embryos were examined 7~10d post in semination without medium renewal. First, we compared Percoll gradient will swim-up technique for sperm separation. There was no difference in cleavage rates between them, but the development rates over morula stage of oocytes fertilized with sperm separated by Percoll gradient was significantly higher than that sperm selected by swim-up technique (p<0.05). Second, we evaluated development of bovine embryos derived from the IVF procedure with different durations(18 vs 42 h) of fertilization. There was also no difference in cleavage rates, but the development to blastocyst stage of oocytes exposed in cleavage rates, but the development to blastocyst stage of oocytes exposed to sperm for 42 h was significantly higher than that exposed for 18 h (p<0.05). In conclusion, Percoll gradient can be used for sperm selecton, improving of embryonic development. Also, 42h of IVF may improve the development of bovine embryos.

• Journal of Embryo Transfer
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• v.13 no.3
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• pp.277-289
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• 1998

To establish a system for sperm swim-up separation through sucrose layer, indiscreet sperm migration should sufficiently to block but movement of sperm shouldn't inhibit. Thus, the effects of sucrose levels in sucrose layer, incubation times and types of sucrose layer on sperm separation were examined. And the results obtained were as follows; 1. Layer of 10mM sucrose inhibited sperm swim-up migration through sucrose layer. 2. Incubation for 25 minutes without sucrose layer significantly increased sperm swim-up migration. However, incubation for 10 minutes to induce swim-up through sucrose layer significantly stimulated sperm migration and maintained sperm movement. 3. There was no significant difference between Type I and Type II in barrier effect of sucrose layer. However, sucrose layer of Type II with shorter distance of barrier was efficient for sampling. To elucidate a function of follicular fluid on sperm chemotaxis using in vitro system of sucrose layer of Type II and incubation for 10 minutes, the effects of dilution, heat treatment, and protein and lipid extracts of follicular fluid on sperm swim-up separation were examined. And the results obtained were as follows; 4. Follicular fluid stimulated sperm migration and movement, and significantly-attracted capacitated-sperm at 10% level. 5. Follicular fluid heated at 55$^{\circ}C$ for 30 minutes maintained the effect of follicular fluid stimulating sperm migration and movement. 6. Follicular protein stimulated sperm movement that was reduced by filtration of the protein. 7. Follicular lipid didn't significantly stimulate sperm migration and movement. 8. Both of follicular protein and lipid reduced the effect of follicular fluid stimulating sperm migration and movement. In conclusion, sucrose layer could be used for a barrier against indiscreet sperm migration by swim-up. And follicular fluid stimulated migration and movement of sperm and attracted capacitated-sperm through sucrose layer. Especially, heat-resistant protein of follicular fluid stimulated sperm migration.

• Korean Journal of Veterinary Research
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• v.29 no.1
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• pp.57-60
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• 1989

This study was carried out to evaluate the possibility of sex preselection by gradients methods using bovine serum albumin in rabbits. Artificial insemination was performed with sperm from the top and bottom layer of rabbit semen separated by bovine serum albumin gradients. Various characteristics of separated sperm, and the conception rate and secondary sex ratio at artificial insemination with separated sperm were compared. The results obtained were as follows. 1. The sperm from the bottom layer showed significanty high value in motility, percent of normal sperm and progressive motility as compared with control sperm and the sperm and the sperm from the top layer. 2. The conception rate of sperm from the bottom layer was higher than that of the top layer. But secondary sex ratio was not altered by this methods.

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.219-226
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• 1997

Follicular fluid influxed into the oviduct during ovulation may affect movement of sperm for fertilization Thus, in this study, the effect of follicular fluid, obtained from follicles of l0mm in diameter, on number and quality of sperm recovered by swim-up separation was investigated and sperm-movement stimulating components extracted from follicular fluid with methanol and isooctane were separated by gel filtration with Sepadex G-1O, G-25 and G-1OO gels, and were isolated by electrophoresis with SDS-PAGE mini gel. The results obtained were as follows; 1. Diluted follicular fluid stimulated sperm movement. 2. Sperm-movement stimulating factors were in methanol extract. 3. Sperm-movement stimulating effect of methanol extract appeared in fraction I among fractions recovered after gel filtration. And the fraction I contained proteins indicating 4 major bands as about 47, 43, 25 and 14 kilodaldons and 5 minor bands as about 67, 58, 23, 22 and 21 kilodaldons. 4. The fraction I recovered from G-100 gel showed significantly low percentage of motile sperm and had no protein indicating the band of 67 kilodaldons among the minor bands.

• Journal of Embryo Transfer
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• v.13 no.3
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• pp.301-312
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• 1998

To efficiently separate a protein stimulating sperm swim-up migration and movement from follicular proteins, the effect of paper chromatography and liquid chromatography with reverse phase column and superose column on protein separation was examined. And the results obtained were as follows; 1. The band component that was separated with paper chromatography stimulated sperm migration and movement depending on its additional levels. Especially, band I component significantly increased sperm migration. But, all components of bands 1, 2 and 3 showed lower sperm migration and movement, compared to follicular fluid at the same additional level. 2. Among the components separated from follicular protein of 2~5mm follicles with reverse phase column ($\mu$RPC), components at retention time (RT) of 3.33, 7.00, 13.87, and 16.6A minutes stimulated sperm migration within a limited range. 3. All components separated from follicular protein of 10mm follicles with $\mu$RPC column didn't stimulate sperm migration and movement. 4. Among the components separated from follicular protein of 2~5m follicles with superose column, components at retention volume (RV) of 1.35 and 0.82 ml significantly stimulated sperm migration and movement. In conclusion, protein components stimulating sperm migration and movement were efficiently separated with superose column in Smart system. Especially, components of RV 1.35 and RV0.82 stimulated sperm swim-up separation.