scholarly journals Reduction of the incidence of polyspermic penetration into porcine oocytes by pretreatment of fresh spermatozoa with adenosine and a transient co-incubation of the gametes with caffeine

Reproduction ◽  
2004 ◽  
Vol 128 (6) ◽  
pp. 789-800 ◽  
Author(s):  
Hiroaki Funahashi ◽  
Raquel Romar
Keyword(s):  
Incubation Period ◽  
Culture Period ◽  
Sperm Cells ◽  
Free Medium ◽  
Exposure Treatment ◽  
Pretreatment Effect ◽  
The Mean ◽  
Sperm Nuclei

To reduce the incidence of polyspermic penetration, the effects of transient exposure of washed fresh spermatozoa to caffeine in a brief co-culture in vitro fertilization (IVF) system were examined. A pretreatment effect of spermatozoa with adenosine was also examined. When 5 mmol caffeine/l was supplemented during periods of co-culture and additional culture periods until 8 h after insemination, a shortened co-incubation period of gametes (30 denuded oocytes in 100 μl modified Medium 199-suspended spermatozoa at 2.5 ×105 sperm/ml) from 30 to 5 min increased the monospermy rate in total mature oocytes examined. The number of spermatozoa binding to the zona surface was significantly lower in oocytes co-cultured for 5 min (33.1 ± 2.2) than 8 h (207.6 ± 13.7). A limited exposure of gametes to 5 mmol caffeine/l only during a transient co-culture period for 5 or 30 min significantly reduced the mean number of sperm cells that penetrated into the oocyte. Transient exposure of spermatozoa to caffeine for only 5 min increased the percentage of capacitated cells but not acrosome-reacted cells, as compared with a whole exposure treatment. Furthermore, preincubation of spermatozoa with 10 μmol adenosine/l for 90 min increased both the incidence of capacitated cells and the monospermy rate and consequently decreased the number of sperm cells that penetrated into the oocyte. In conclusion, these results have demonstrated that a new transient co-incubation IVF system, in which denuded oocytes are co-cultured with spermatozoa in medium containing caffeine for 5 to 30 min and then continuing the culture in caffeine-free medium, will reduce the incidence of polyspermic penetration. Preincubation of fresh spermatozoa with adenosine before the transient co-incubation IVF can also improve the monospermy rate. Furthermore, asynchrony in the morphology of sperm nuclei in polyspermic oocytes was reduced by the pretreatment with adenosine and a brief exposure to caffeine.

scholarly journals Perioperative Prophylactic Chemotherapy of Echinococcus granulosus: Determination of Minimum Effective Length of Albendazole Therapy in In Vitro Protoscolex Culture

HPB Surgery ◽  
1990 ◽  
Vol 2 (3) ◽  
pp. 159-164 ◽  
Author(s):  
D. H. Taylor ◽  
D. L. Morris ◽  
K. S. Richards
Keyword(s):  
Continuous Culture ◽  
Echinococcus Granulosus ◽  
Effective Length ◽  
Culture Period ◽  
Free Medium ◽  
Continuous Therapy ◽  
Prophylactic Chemotherapy ◽  
Drug Free

Protoscoleces of Echinococcus granulosus were cultured in vitro in 500, 250 or 100 μg/1 albendazole sulphoxide for 1, 3, 7, 10, 14d and then ‘recued’ (R) into drug-free medium for the remainder ofthe culture period. Successful minimum lengths of therapy were much longer than for praziquantel, and only at 500μg/1 was the 10dR treatment as effective as continuous therapy for 28d. Treatment with 100 μg/1 both in continuous culture and in the ‘R’ experiments was ineffective over a 35d period. The results are compared with those from similar experiments using praziquantel.

scholarly journals Morphologic changes in boar sperm nuclei with reduced disulfide bonds in electrostimulated porcine oocytes

Reproduction ◽  
2006 ◽  
Vol 131 (3) ◽  
pp. 603-611 ◽  
Author(s):  
Michiko Nakai ◽  
Naomi Kashiwazaki ◽  
Akiko Takizawa ◽  
Naoki Maedomari ◽  
Manabu Ozawa ◽  
...  
Keyword(s):  
Electrical Stimulation ◽  
Blastocyst Stage ◽  
Blastocyst Formation ◽  
The Mean ◽  
Sperm Nuclei ◽  
Morphologic Changes ◽  
Number Of Cells ◽  
Stimulation Of ◽  
Nuclear Decondensation

In pigs, failure of sperm nuclear decondensation has been reported after injection into oocytes. We examined the effects of pretreating sperm heads with Triton X-100 (TX-100) and dithiothreitol (DTT) and of electrical stimulation of oocytes after sperm head injection on time-dependent morphologic changes in sperm nuclei andin vitrodevelopment to the blastocyst stage. In experiment 1, spermatozoa were pretreated with 1% TX-100 and 5 mM DTT (T + D) or not treated, and then injected intoin vitromatured oocytes. Electrical stimulation (1.5 kV/cm, 20 μs DC pulse) was applied to the oocytes 1 h after injection (stimulated group) or was not applied (unstimulated group). Some of the oocytes in each group were evaluated at hourly intervals until 10 h after injection for morphologic changes in the sperm nuclei. Unstimulated oocytes injected with untreated spermatozoa showed a delayed peak in the rate of nuclear decondensation (39.4–44.1%, 3–6 h after injection) compared with oocytes injected with T + D-treated spermatozoa (57.0% and 52.6%, 1 and 2 h, respectively). The rate of male pronucleus formation peaked 6 h after stimulation (by 40–60%) after injected oocytes had been stimulated with an electrical pulse, irrespective of whether or not the spermatozoa had been pretreated. In unstimulated oocytes, the rate of male pronucleus formation did not increase and stayed at the basal level (less than 20%) throughout the culture period, regardless of the sperm treatment. Thus, T + D treatment of spermatozoa did not affect completion of fertilization. In experiment 2, we evaluated the effects of electrical stimulation and sperm treatment with T + D on the rate of blastocyst formation and the mean number of cells per blastocyst. Oocytes stimulated after injection with either T + D-treated or untreated spermatozoa showed significantly higher percentages of blastocyst formation (24.8% and 27.1% respectively) than did unstimulated oocytes (1.1% and 4.1% for T + D-treated and untreated respectively;P< 0.01 by Duncan’s multiple-range test). The rate of blastocyst formation did not differ between the T + D-treated and untreated groups. The mean number of cells per blastocyst did not differ among any of the groups (14.0–29.4 cells). These results suggest that pretreatment of sperm with TX-100 and DTT shifted the timing of sperm nuclear decondensation forward. However, pronucleus formation and development to the blastocyst stagein vitrowere not improved by sperm treatment. Thus, electrical stimulation of injected oocytes enhancesin vitrodevelopment to the blastocyst stage in pigs.

EFFECT OF GONADOTROPHINS ON THE PRODUCTION OF STEROIDS BY SHEEP OVARIAN FOLLICLES CULTURED IN VITRO

1973 ◽  
Vol 58 (3) ◽  
pp. 599-611 ◽  
Author(s):  
R. M. MOOR ◽  
MARY F. HAY ◽  
J. E. A. McINTOSH ◽  
B. V. CALDWELL
Keyword(s):  
Ovarian Follicles ◽  
Oestrous Cycle ◽  
Culture Period ◽  
Daily Output ◽  
Progesterone Production ◽  
Progesterone Secretion ◽  
The Mean ◽  
Pregnant Mare Serum Gonadotrophin

SUMMARY The main objective of the study was to determine the rate at which Graafian follicles of sheep that had been treated with exogenous gonadotrophin acquire the ability to secrete oestrogen in vitro. Follicles were explanted from sheep 5 min to 24 h after injection of pregnant mare serum gonadotrophin (PMSG) and kept individually in culture for 7 days. The mean daily output of oestrogen by follicles from PMSG-treated sheep was higher than that secreted by follicles from untreated sheep. However, only a certain proportion of the follicles from each sheep secreted significant amounts of oestrogen in vitro; these follicles were called 'stimulated'. The proportion of stimulated follicles was 5% for control sheep, 20–30% for follicles explanted from sheep 5 min to 12 h after injection with PMSG, and 80% for follicles explanted from sheep that had been injected with PMSG 24 h previously. In the second part of the study, the pattern of oestrogen and progesterone secretion by stimulated follicles of different sizes explanted from PMSG-treated sheep at various stages of the oestrous cycle was determined. Up to the 14th day, oestrogen production in vitro by follicles over 4·5 mm in diameter reached a maximum 2 days after PMSG injection and decreased thereafter; progesterone production rose steadily as the oestrogen levels declined. In contrast, follicles of less than 4·5 mm diameter secreted considerable amounts of oestrogen for the first 5 days in culture, but produced only small quantities of progesterone. In follicles explanted on day 15, oestrogen secretion decreased steadily from the beginning of the culture period and was very low by the 4th day. Most follicles explanted at oestrus secreted only small amounts of oestrogen in vitro but secreted large amounts of progesterone.

scholarly journals Comparative Studies on Cellular Behaviour of Carnation (Dianthus caryophyllusLinn. cv. Grenadin) GrownIn VivoandIn Vitrofor Early Detection of Somaclonal Variation

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Jamilah Syafawati Yaacob ◽  
Rosna Mat Taha ◽  
Arash Khorasani Esmaeili
Keyword(s):  
Somaclonal Variation ◽  
Ploidy Level ◽  
Dianthus Caryophyllus ◽  
Nuclear Dna Content ◽  
Culture Period ◽  
Root Tip ◽  
Root Cells ◽  
The Mean

The present study deals with the cytological investigations on the meristematic root cells of carnation (Dianthus caryophyllusLinn.) grownin vivoandin vitro. Cellular parameters including the mitotic index (MI), chromosome count, ploidy level (nuclear DNA content), mean cell and nuclear areas, and cell doubling time (Cdt) were determined from the 2 mm root tip segments of this species. The MI value decreased when cells were transferred fromin vivotoin vitroconditions, perhaps due to early adaptations of the cells to thein vitroenvironment. The mean chromosome number was generally stable (2n=2x=30) throughout the 6-month culture period, indicating no occurrence of early somaclonal variation. Following the transfer to thein vitroenvironment, a significant increase was recorded for mean cell and nuclear areas, from 26.59 ± 0.09 μm2to 35.66 ± 0.10 μm2and 142.90 ± 0.59 μm2to 165.05 ± 0.58 μm2, respectively. However, the mean cell and nuclear areas ofin vitrogrownD. caryophylluswere unstable and fluctuated throughout the tissue culture period, possibly due to organogenesis or rhizogenesis. Ploidy level analysis revealed thatD. caryophyllusroot cells contained high percentage of polyploid cells when grownin vivoand maintained high throughout the 6-month culture period.

In vitro ovicidal activity of poly lactic acid curcumin-nisin co-entrapped nanoparticle against Fasciola spp. eggs and its reproductive toxicity

2018 ◽  
Vol 29 (1) ◽  
pp. 73-79 ◽  
Author(s):  
Oyetunde Oyeyemi ◽  
Odunayo Adegbeyeni ◽  
Ifeoluwa Oyeyemi ◽  
Jairam Meena ◽  
Amulya Panda
Keyword(s):  
Reproductive Toxicity ◽  
Entrapment Efficiency ◽  
Negative Control ◽  
Poly Lactic Acid ◽  
Sperm Cells ◽  
Ovicidal Activity ◽  
Egg Hatching ◽  
The Mean

AbstractBackground:Curcumin and nisin have been widely reported for their antibacterial and anticancer potency. However, their therapeutic applications are hampered by several factors, which necessitate their development into nanosize ranges for improved delivery and activities. Their incorporation into a single nanosynthesized form may suggest desirable efficacy on parasites. The aim of the study was to assess the ovicidal activity of the curcumin-nisin polylactic acid (PLA) entrapped nanoparticle on theFasciolaeggs and its reproductive toxicity.Methods:The nanoparticle was formulated by double emulsion method. The eggs of the adultFasciolaspp. were exposed to different concentrations (0.3125–5 mg/mL) of the nanoparticle to monitor hatchability. Mice were exposed to 0.5 mL of the formulated drug at varying concentrations (10–20 mg/kg) and then sacrificed for sperm morphology assay.Results:The mean particle size, polydispersity index, and drug entrapment efficiency of the formulated drug were 288.4±24.3 nm, 0.232, and 51.7%, respectively. The highest nanoparticulate concentration (5 mg/mL) showed the least percentage egg hatching (41.7%) compared with the other treatment groups and positive control (albendazole) (45.1%). The aberrations observed in sperm cells were not concentration-dependent and no significant differences were observed in the mean aberrations between the nanoparticulate drug-exposed groups and the negative control (p>0.05).Conclusions:The results confirmed the ovicidal activity of the curcumin-nisin nanoparticulate drug against theFasciolaspecies. The formulation also showed no toxicity to sperm cells. More robust studies on anti-fascioliasis activity of the drug on adultFasciolaspp. andin vivoandin vitrotoxicity studies are recommended.

Meiotic behaviour of the pollen mother cells in cultured anthers of rye excised at early meiotic prophase

Genome ◽  
1988 ◽  
Vol 30 (2) ◽  
pp. 161-165
Author(s):  
J. Rueda ◽  
A. M. Vázquez
Keyword(s):  
Acetic Acid ◽  
Chiasma Formation ◽  
Free Medium ◽  
Pollen Mother Cells ◽  
The Mean ◽  
Mother Cells ◽  
Pollen Sacs ◽  
Indole 3 Acetic Acid

Anthers of rye were excised during leptotene and zygotene and cultured in vitro on media with and without indole-3-acetic acid to study the behaviour of pollen mother cells during meiosis. Percentages of pollen sacs in which pollen mother cells continued meiosis increased from approximately 20% to 60–70% when anthers were excised at early and late leptotene. The highest values were about 90% when the excision was during zygotene. The results were similar on both media. The timing of pollen mother cells through meiosis on the hormone-free medium was similar to that described in vivo. The mean numbers of chiasmata per cell at metaphase I from the cultured pollen sacs were compared with those from a random population of pollen sacs grown in the field. A significant decrease was observed when anthers were excised during leptotene and cultured on both media, as well as when anthers were excised at early zygotene and cultured on the hormone-supplemented medium. We concluded that at early zygotene there are processes related to chiasma formation that seem to be influenced by hormone balance.Key words: Secale cereale, anther culture, meiosis development, chiasma formation, indole-3-acetic acid.

Effect of Temperature on ADP-Induced Platelet Aggregation

1973 ◽  
Vol 29 (01) ◽  
pp. 183-189
Author(s):  
C. A Praga ◽  
E. M Pogliani
Keyword(s):  
Platelet Aggregation ◽  
Incubation Period ◽  
Room Temperature ◽  
Important Variable ◽  
Practical Significance ◽  
Effect Of Temperature ◽  
Induce Platelet Aggregation ◽  
Cuvette Holder ◽  
Exact Temperature

SummaryTemperature represents a very important variable in ADP-induced platelet aggregation.When low doses of ADP ( < 1 (μM) are used to induce platelet aggregation, the length of the incubation period of PRP in the cuvette holder of the aggregometer, thermostatted at 37° C, is very critical. Samples of the same PRP previously kept at room temperature, were incubated for increasing periods of time in the cuvette of the aggregometer before adding ADP, and a significant decrease of aggregation, proportional to the length of incubation, was observed. Stirring of the PRP during the incubation period made these changes more evident.To measure the exact temperature of the PRP during incubation in the aggre- gometer, a thermocouple device was used. While the temperature of the cuvette holder was stable at 37° C, the PRP temperature itself increased exponentially, taking about ten minutes from the beginning of the incubation to reach the value of 37° C. The above results have a practical significance in the reproducibility of the platelet aggregation test in vitro and acquire particular value when the effect of inhibitors of ADP induced platelet aggregation is studied.Experiments carried out with three anti-aggregating agents (acetyl salicyclic acid, dipyridamole and metergoline) have shown that the incubation conditions which influence both the effect of the drugs on platelets and the ADP breakdown in plasma must be strictly controlled.

Influence of Platelet Membrane Sialic Acid and Platelet-Associated IgG on Ageing and Sequestration of Blood Platelets in Baboons

1993 ◽  
Vol 70 (04) ◽  
pp. 676-680 ◽  
Author(s):  
H F Kotzé ◽  
V van Wyk ◽  
P N Badenhorst ◽  
A du P Heyns ◽  
J P Roodt ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Life Span ◽  
Blood Platelets ◽  
Senescent Cell ◽  
Platelet Membrane ◽  
Antigen Complex ◽  
The Mean ◽  
Aggregation Of Platelets

SummaryPlatelets were isolated from blood of baboons and treated with neuraminidase to remove platelet membrane sialic acid, a process which artificially ages the platelets. The platelets were then labelled with 111In and their mean life span, in vivo distribution and sites of Sequestration were measured. The effect of removal of sialic acid on the attachment of immunoglobulin to platelets were investigated and related to the Sequestration of the platelets by the spleen, liver, and bone marrow. Removal of sialic acid by neuraminidase did not affect the aggregation of platelets by agonists in vitro, nor their sites of Sequestration. The removal of 0.51 (median, range 0.01 to 2.10) nmol sialic acid/108 platelets shortened their life span by 75 h (median, range 0 to 132) h (n = 19, p <0.001), and there was an exponential correlation between the shortening of the mean platelet life span and the amount of sialic acid removed. The increase in platelet-associated IgG was 0.112 (median, range 0.007 to 0.309) fg/platelet (n = 25, p <0.001) after 0.79 (median, range 0.00 to 6.70) nmol sialic acid/108 platelets was removed (p <0.001). There was an exponential correlation between the shortening of mean platelet life span after the removal of sialic acid and the increase in platelet-associated IgG. The results suggest that platelet membrane sialic acid influences ageing of circulating platelets, and that the loss of sialic acid may have exposed a senescent cell antigen that binds IgG on the platelet membrane. The antibody-antigen complex may then provide a signal to the macrophages that the platelet is old, and can be phagocytosed and destroyed.

IN VITRO UPTAKE OF TRITIATED OESTRADIOL BY THE ANTERIOR HYPOTHALAMUS AND HYPOPHYSIS OF THE RAT

1970 ◽  
Vol 64 (4) ◽  
pp. 687-695 ◽  
Author(s):  
Junzo Kato
Keyword(s):  
Incubation Medium ◽  
Posterior Hypothalamus ◽  
Anterior Hypothalamus ◽  
Ovariectomized Rats ◽  
Free Medium ◽  
Cortex Cerebri ◽  
Anterior Hypophysis ◽  
In Vitro Uptake

ABSTRACT The anterior, middle, and posterior hypothalamus, the cortex cerebri, the anterior hypophysis as well as the diaphragm of adult ovariectomized rats were incubated in vitro with tritiated 17β-oestradiol. The uptake of tritiated oestradiol was differentially distributed intracerebrally with higher accumulation in the anterior hypothalamus and the hypophysis. Lowering the temperature of the incubation medium caused a reduction in the uptake of radioactivity by the anterior hypothalamus as compared to that found in other brain tissues. Tritiated oestradiol taken up in vitro by the anterior hypothalamus and the hypophysis tended to be retained after further incubation in a steroid-free medium. The addition of non-radioactive 17β-oestradiol to the medium inhibited the uptake of tritiated oestradiol by these tissues. Moreover, pretreatment with non-radioactive 17β-oestradiol in vivo prevented the preferential accumulation of tritiated oestradiol in vitro in the anterior hypothalamus and the hypophysis. These results indicate that oestradiol is preferentially taken up in vitro by the anterior hypothalamus and the hypophysis of the rat.

The Safety and Antimicrobial Properties of Stabilized Hypochlorous Acid in Acetic Acid Buffer for the Treatment of Acute Wounds—a Human Pilot Study and In Vitro Data

2021 ◽  
pp. 153473462110156
Author(s):  
Ewa A. Burian ◽  
Lubna Sabah ◽  
Klaus Kirketerp-Møller ◽  
Elin Ibstedt ◽  
Magnus M. Fazli ◽  
...  
Keyword(s):  
Pilot Study ◽  
Hypochlorous Acid ◽  
Treatment Time ◽  
Donor Site ◽  
Antimicrobial Properties ◽  
Prolonged Treatment ◽  
Wound Irrigation ◽  
The Mean ◽  
And Performance

Acute wounds may require cleansing to reduce the risk of infection. Stabilized hypochlorous acid in acetic buffer (HOCl + buffer) is a novel wound irrigation solution with antimicrobial properties. We performed a first-in-man, prospective, open-label pilot study to document preliminary safety and performance in the treatment of acute wounds. The study enrolled 12 subjects scheduled for a split-skin graft transplantation, where the donor site was used as a model of an acute wound. The treatment time was 75 s, given on 6 occasions. A total of 7 adverse events were regarded as related to the treatment; all registered as pain during the procedure for 2 subjects. One subject had a wound infection at the donor site. The mean colony-forming unit (CFU) decreased by 41% after the treatment, and the mean epithelialization was 96% on both days 14 (standard deviation [SD] 8%) and 21 (SD 10%). The study provides preliminary support for the safety, well-tolerance, and efficacy of HOCl + buffer for acute wounds. The pain was frequent although resolved quickly. Excellent wound healing and satisfying antimicrobial properties were observed. A subsequent in vitro biofilm study also indicated good antimicrobial activity against Pseudomonas aeruginosa with a 96% mean reduction of CFU, when used for a treatment duration of 15 min ( P < .0001), and a 50% decrease for Staphylococcus aureus ( P = .1010). Future larger studies are needed to evaluate the safety and performance of HOCl + buffer in acute wounds, including the promising antimicrobial effect by prolonged treatment on bacterial biofilms.

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