31 research outputs found

    In Vitro Fertility of Post-thawed Epididymal Ram Spermatozoa After Storage at 5 °C Before Cryopreservation

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    This study addressed the effects of storage duration of epididymides at 5 °C before sperm collection and their fertility after cryopreservation in vitro. Spermatozoa from one of the testes pairs were immediately collected, evaluated and frozen (control group). The remaining epididymides were cooled to 5 °C and stored for 24, 48, 72, and 96 h (experimental groups), after which spermatozoa were collected and frozen as in the control group. Before and after thawing, sperm motility, sperm viability and plasma membrane integrity were assessed. The fertilizing ability of frozen-thawed spermatozoa of each group was evaluated by in vitro fertilization of matured sheep oocytes. Sperm quality (sperm motility, viability, and plasma membrane integrity) at collection and after cryopreservation decreased as the duration of the epididymal storage interval increase (P < 0.05). The motility decreased steadily along the studied time periods. Although, the fertilizing ability of post-thawed epididymal spermatozoa gradually decreased as the storage period was prolonged, the spermatozoa collected from the cauda epididymides stored at 5 °C for up to 96 h were able to fertilize 16%-65% of oocytes in vitro. Results of the present study showed that ram epididymal spermatozoa survive in storage at 5 °C for up to 96 h. These spermatozoa maintain their fertilizing ability and may be suitable for use in IVF and other assisted reproductive procedures

    Kemampuan Fertilisasi Spermatozoa Sexing dan Perkembangan Awal Embrio secara In Vitro pada Sapi

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    The aim of the present study was to investigate the fertilization ability of bovine oocytes and early bovine embryonic development in vitro, fertilized by frozen X and Y sperm separated by bovine serum albumin (BSA) gradient column. Oocytes were collected from slaughter house ovarian by flushing and slicing technique. Oocytes were than maturated in tissue culture medium (TCM) 199 supplemented with 10 IU/ml pregnant mare's serum gonadotropin (PMSG), 10 IU/ml human chorionic gonadotropin (hCG) and 10% fetal bovine serum (FBS) for 24 h in 5% CO2 incubator 39oC. Oocytes then fertilized with three kind of different frozen spermatozoa (X,Y and unsexing spermatozoa as control) for 14 h with final concentration 2x106 spermatozoa/mL. Embryos were cultured insynthetic oviductal fluid (SOF) supplemented with essential and non essential amino acid and 0.3% bovine serum albumin (BSA) for 96 h. Results of the experiments revealed that there was no significant difference (P>0.05) in thefertilization ability (49.17%; 51.40%; 53.42%) for X, Y and control group, respectively. No significant difference (P>0.05) in the number of embryos development (47.77%; 48.25%; 54.43%) for X, Y and control group, respectively. Furthermore, only small number of embryos could pass development blockade (23.80%; 26.08%; 23.61%) for X, Y and control spermatozoa with statistically no significant difference (P>0.05). It is concluded that sexed spermatozoa separated by BSA gradient column had comparable fertilization ability with unsexing spermatozoa and had ability to supported early embryonic development

    Efficacy of Leptin Supplementation on Nuclear Maturation and Fertilization Rate of Sheep Oocytes In Vitro

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    The objective of this study was to determine the efficacy of leptin supplementation into maturation medium on nuclear maturation and fertilization rate of sheep oocytes. The maturation process was conducted using a tissue culture medium (TCM) 199 with four supplementation treatments of leptin namely 0 (control), 10, 50, and 100 ng/mL. Fertilization was conducted in the oocytes supplemented with 10 ng/mL and control using 5×106 mL-1 spermatozoa. At the end of maturation and fertilization processes, the oocytes were stained with 2% aceto orcein to determine nuclear maturation rate and pronuclear development. The results showed that the percentage of oocytes reaching metaphase II (MII) stage significantly increased in the oocytes supplemented with leptin at a dose of 10 ng/mL (P<0.05) compared to those supplemented at doses of 50, 100, and 0 ng/mL (93.7±5.9% vs 78.8±4.4%; 72.0±2.6%; 82.1±9.9%). However, fertilization rate of the oocytes supplemented with leptin at a dose of 10 ng/mL and control were similar (72.1±5.5% vs 79.2±7.0%). The data indicated that leptin could improve maturation rate in lower concentration. However, the improved maturation rate of oocytes with leptin supplementation at a dose of 10 ng/mL could not improve the fertilization rate of the oocytes. In conclusion, the supplementation of leptin at a dose of 10 ng/mL could increase the number of oocytes that reached MII stage, but could not increase the fertilization rate

    Pengendalian Folikulogenesis Ovarium dengan Pemberian Ekstrak Biji Kapas

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    Gosipol is a substances contained in extracted cotton seed which is thought to have the antifertility ability therefore it is often used as a herbal contraceptive. The aim of this study were to assess the folliculogenesis in mice after administrated with cottonseed extract. 60 female mice strain DDY which was 14-15 weeks old and 30-35 g body weight were divided into five groups and given cottonseed extract each 0; 1,5; 2,1 and 2,7 g/kg BW for 5, 10, 15, 24, and 24 + 10 days (without cottonseed treatment). At the end of the treatment period, mice was euthanasia to observe follicular development histomorphology (each three mice of each treatment). Mice estrous status were evaluated based on the description of the vaginal smear cells with Giemsa staining. The results showedthat the number of developing follicles was low (P < 0.05) compared with control after 5 days cottonseed extract administration at dose 2,7 g/kg BW that were 23 ± 3,6. At dose 1,5 and 2,1 g/kg BW the number of follicles was low after 24 days that were 25 ± 10,4 and 27 ± 3,5. Recovery effects of follicle number after cottonseed extract administration for 24 days was the best at a dose of 1,5 g/kg BW. Prolonge of estrous cycle occured in mice which were administrated the cottonseed extract of at all dose treatment. In conclusion, although the decrease in the number of developing follicles and prolonge of estrous cycles occurred after cottonseed extract administration, but these effects are reversible after the administration ended

    Cryopreservation of Swamp Buffalo Semen in Skim Milk Yolk-based Diluent with Two Different Cryoprotectants

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    The successful of artificial insemination (AI) in buffalo is still low. The quality of frozen semen determines the successful of AI program. Glycerol is widely used for cryopreservation of buffalo semen with unsatisfactory results due to its toxicity and antifertility properties. This study aimed to investigate the effect of glycerol and dimethylformamide (DMF) concentrations in skim milk egg yolk (SMEY)–based diluent on the quality of frozen-thawed swamp buffalo bull semen. Fresh semen was divided into eight aliquots. Each aliquot was diluted in SMEY containing 4%, 5%, 6%, and 7% glycerol and 4%, 5%, 6%, and 7% DMF. Diluted semen was packed into mini straw (0.25 mL), equilibrated at 4 °C for 4 hours, frozen in liquid nitrogen vapor for 10 minutes, and stored at liquid nitrogen container. The straws were thawed after 24 hours at 37 °C for 30 seconds for evaluation. The results showed that the post thawed sperm motility and recovery rate in SMEY extender containing 7% glycerol and 5% DMF were higher than the other glycerol or DMF concentrations. Post thawed sperm viability and plasma membrane integrity in SMEY containing 4%-6% DMF did not differ significantly, however both values were higher than those in 7% DMF. Post thawed sperm viability and plasma membrane integrity did not differ between SMEY containing 6% and 7% glycerol. However, both values were higher than those in 4% and 5% glycerol. It is concluded that 7% glycerol or 5% DMF is considered to be the most suitable cryoprotective agent for swamp buffalo semen cryopreservation.The successful of artificial insemination (AI) in buffalo is still low. The quality of frozen semen determines the successful of AI program. Glycerol is widely used for cryopreservation of buffalo semen with unsatisfactory results due to its toxicity and antifertility properties. This study aimed to investigate the effect of glycerol and dimethylformamide (DMF) concentrations in skim milk egg yolk (SMEY)–based diluent on the quality of frozen-thawed swamp buffalo bull semen. Fresh semen was divided into eight aliquots. Each aliquot was diluted in SMEY containing 4%, 5%, 6%, and 7% glycerol and 4%, 5%, 6%, and 7% DMF. Diluted semen was packed into mini straw (0.25 mL), equilibrated at 4 °C for 4 hours, frozen in liquid nitrogen vapor for 10 minutes, and stored at liquid nitrogen container. The straws were thawed after 24 hours at 37 °C for 30 seconds for evaluation. The results showed that the post thawed sperm motility and recovery rate in SMEY extender containing 7% glycerol and 5% DMF were higher than the other glycerol or DMF concentrations. Post thawed sperm viability and plasma membrane integrity in SMEY containing 4%-6% DMF did not differ significantly, however both values were higher than those in 7% DMF. Post thawed sperm viability and plasma membrane integrity did not differ between SMEY containing 6% and 7% glycerol. However, both values were higher than those in 4% and 5% glycerol. It is concluded that 7% glycerol or 5% DMF is considered to be the most suitable cryoprotective agent for swamp buffalo semen cryopreservation

    Prognostic impact of peritumoral lymphocyte infiltration in soft tissue sarcomas

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    <p>Abstract</p> <p>Background</p> <p>The purpose of this study was to clarify the prognostic significance of peritumoral lymphocyte infiltration in the capsule of soft tissue sarcomas (STS). Multiple observations in preclinical and clinical studies have shown that the immune system has a role in controlling tumor growth and progression. Prognostic markers in potentially curable STS should guide therapy after surgical resection. The immune status at the time of resection may be important, but the prognostic significance of peritumoral lymphocytes is unknown.</p> <p>Methods</p> <p>Tissue microarrays from 80 patients with STS were constructed from duplicate cores of tissue from the tumor and the peritumoral capsule. Immunohistochemistry was used to evaluate the CD3+, CD4+, CD8+ and CD20+ lymphocytes in the tumor and the peritumoral capsule.</p> <p>Results</p> <p>In univariate analyses, increasing numbers of CD20+ (<it>P </it>= 0.032) peritumoral lymphocytes were associated with a reduced disease free survival (DSS). In multivariate analyses, a high number of CD20+ peritumoral lymphocytes (<it>P </it>= 0.030) in the capsule was an independent negative prognostic factor for DSS. There were no such associations of lymphocyte infiltration in the tumor.</p> <p>Conclusions</p> <p>A high density of CD20+ peritumoral lymphocytes is an independent negative prognostic indicator for patients with STS. Further research is needed to determine whether CD20 cells in the peritumoral capsule of STS may promote tumor invasion in the surrounding tissue and increase the metastatic potential.</p

    The Quality of Frozen Friesian Holstein Semen after Long-Term Storage

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    Semen cryopreservation is the long-term storage at very low temperatures in liquid nitrogen for future use. This study investigates the quality of frozen Friesian Holstein (FH) semen after long-term storage. Samples of FH semen stored for 25, 20, 15, 10, 5, and 1 years were collected from one of the national centers for artificial inseminations. Frozen semen was stored in containers with liquid nitrogen at -196 ᵒC in a room with a temperature of 20 ᵒC The variables used after thawing were sperm motility, viability, and abnormalities, as well as plasma membrane integrity (IPM) using computer-assisted sperm analysis (CASA), eosin-nigrosine staining, and hypoosmotic swelling (HOS) test, respectively. Data were analyzed by one-way ANOVA and expressed as mean ± SEM. The result showed no significant differences in sperm viability, abnormalities, and IPM. Furthermore, sperm motility was &gt;40%, consistent with the Indonesian standard for frozen bovine semen. CASA analysis showed that all variables of the motility pattern have no significant difference, except linearity (LIN). The Lin of sperm was lower in frozen semen after one and five years than after 20 and 25 years of storage. The overall quality of semen after 25, 20, 15, 10, 5, and 1 years of storage met the standard and was suitable for artificial insemination

    L-carnitine Supplementation Enhances Nuclear and Cytoplasmic Maturation Rates of Sheep Oocytes In Vitro

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    The aim of the present study was to determine the effectiveness of l-carnitine (LC) supplementation on nuclear and cytoplasmic maturation rates of sheep oocytes. In experiment 1, oocytes were maturated for 24 hours in tissue culture medium 199 supplemented with LC at doses of 0.3 mg/mL, 0.6 mg/mL, and 0.9 mg/mL. In experiment 2, oocytes were maturated and fertilized in a media supplemented with LC at a dose of 0.3 mg/mL and incubated with 5x106 sperm/mL for 12 hours. The treatment group consisted of LC supplementation only in maturation medium (P1), only in fertilization medium (P2), and in both maturation and fertilization media (P3). In experiment 3, sperm motility patterns were assessed using CASA after being exposed to fertilization medium supplemented with LC at a dose of 0.3 mg/mL for 0 and 3 hours. Our results showed that supplementation of LC at a dose of 0.3 mg/mL significantly (p&lt;0.05) increased the percentage of oocytes reaching metaphase II (86.7±4.1%) compared to those supplemented with LA at doses of 0, 0.6, and 0.9 mg/mL (73.6±1.2, 81.4±1.3%, and 70.5±1.6%, respectively). The LC treatment in the fertilization medium only did not influence the number of two pronuclear formations (62.1±2.5%) compared to supplementation either in the maturation medium only (72.0±4.7%) or a combination of both in maturation and fertilization media (68.2±2.7%) (p&lt;0.05). Further results after 3 hours of incubation compared to the control group showed the total motility (24.8±2.04% vs. 17.49±2.37%), progressive motility (14.17±2.03% vs. 6.49±1.64%), and curvilinear velocity (VCL) (119.70±3.73% vs. 71.15±10.59%) (p&lt;0.05) were increased in the fertilization medium containing LC but it did not improve the fertilization rate. It is concluded that supplementation of LC at a dose of 0.3 mg/mL in the maturation medium only could better improve the nuclear and cytoplasmic maturation rates of sheep oocytes

    Comparison of Different Lecithin Diluents for Cryopreservation of Toraya Buffalo Semen

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    The objective of this study was to compare the quality of frozen semen from Toraya buffalo with different lecithins from different commercial diluents. Fresh semen from two 6-9 years old Toraya buffaloes were collected once a week in the morning through an artificial vagina. Fresh semen was examined macroscopically and microscopically. Semen with more than 70% sperm motility was divided into three tubes, each diluted with Andromed (soy lecithin), Steridyl (egg yolk lecithin), and Bovifree (synthetic lecithin) diluents at a concentration of 100 x106 mL-1 motile sperm. The diluted sperm were placed in 0.25 mL straws and allowed to equilibrate for 4 hours. The sperm was frozen above liquid nitrogen favour for 15 minutes and stored in liquid nitrogen containers for further evaluation. The quality of frozen semen was assessed 24 hours after freezing. The parameters tested were motility, viability, abnormalities, plasma membrane integrity, and sperm recovery rate (RR). The results showed no significant differences in the values of motility, viability, abnormalities, plasma membrane integrity, and sperm RR in the three diluents used. Sperm motility values ranged from 45.93% to 47.09% after freezing. Sperm viability ranged from 56.21% to 60.27%. The values of membrane integrity of the three diluents used ranged from 57.73% to 61.36%. The values of sperm abnormalities after freezing and thawing ranged from 2.74% to 3.18%. In conclusion, three commercial diluents containing animal, vegetable, and synthetic lecithin bases can be used as diluents for freezing Toraya buffalo semen with similar results
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