Development of in vitro tests to predict fertility of bulls

2005 ◽  
Vol 85 (1) ◽  
pp. 47-52 ◽  
Author(s):  
G. Giritharan ◽  
N. Ramakrishnappa ◽  
A. Balendran ◽  
K. M. Cheng ◽  
R. Rajamahendran
Keyword(s):  
Acrosome Reaction ◽  
Return Rate ◽  
In Vitro Tests ◽  
In Vitro Test ◽  
Motile Sperm ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Frozen Semen ◽  
Vitro Test

The overall objective was to develop an in vitro test to predict fertility of bulls in the field. We investigated the bull effect on in vitro embryo production, zona binding and acrosome reaction, and the correlation of this effect to field fertility meas ured by 60–90 d non-return rate. Frozen semen from three separate ejaculates of eight unrelated young bulls, obtained from an artificial insemination (AI) center, was used. On thawing, ejaculates from each bull were pooled, motile sperm were selected and (a) subjected to immunofluorescent assay at 0 and 4 h of incubation in capacitation medium to assess acrosome status, (b) used in an in vitro fertilization assay system to assess cleavage and blastocyst production rates, and (c) sperm-zona binding assay was carried out to determine the number of sperm bound to the zona pellucida of mature oocytes. Percentage of pre-freeze motile sperm (PrFM) and non-return rate data were obtained from the AI center. PrFM, percentage of acrosome reacted sperm at 0 h (AR1), increase in percentage of acrosome reacted sperm after 4 h (InAR) and sperm-zona binding rates (ZB) differed (P < 0.05) among sperm samples obtained from different young bulls. Significant correlations (P < 0.05) were observed between PrFM and AR1 (r = -0.31), InAR (r = 0.36), and ZB (r = 0.32). AR1 was negatively correlated to ZB (r = -0.27) and cleavage rate (r = -0.20), InAR was positively correlated with ZB (r = 0.31) and cleavage rate (r = 0.26). None of the in vitro tests was correlated with non-return rate. These findings indicate that along with pre-freeze motility, a combination of in vitro tests including the percentage of spontaneously acrosome reacted sperm at thawing, might be useful in predicting bull field fertility. Such a combination of assays, however, has yet to be determined. Key words: Field fertility, acrosome reaction, zona binding, IVF, fertility assay

scholarly journals Recombinant in vitro test T-SPOT.TB as a screening method for early diagnosis of tuberculosis infection

2020 ◽  
Vol 98 (4) ◽  
pp. 48-52
Author(s):  
E. P. Eremenko ◽  
E. A. Borodulina ◽  
I. A. Sergeeva ◽  
D. A. Kudlay ◽  
B. E. Borodulin
Keyword(s):  
Screening Method ◽  
Latent Tuberculosis ◽  
Tuberculosis Infection ◽  
Mantoux Test ◽  
Skin Tests ◽  
In Vitro Tests ◽  
In Vitro Test ◽  
Healthy Children ◽  
Vitro Test

In addition to standard skin tests (Mantoux test with 2 TU PPD-L and diaskintest) for the diagnosis of tuberculosis infection, in vitro tests are used. One of these tests is T-SPOT.TB being more widely used in recent years.The objective: to evaluate the effectiveness of T-SPOT.TB test for early detection of tuberculosis infection in children and adolescents in Samara Region.Subjects and methods. From 2016 to 2019, results of T-SPOT.TB tests performed in 596 children aged 2 to 17 years inclusive were analyzed; those children had no immunodiagnosis of tuberculosis infection using skin tests since their parents refused to have it.Results. It was found out that the major reason for refusing skin tests was the “fear” of visiting a TB dispensary if the result had been positive — 38.43% (n = 229). The latent tuberculosis infection according to the results of T-SPOT.TB among children with concomitant pathology made 2.6%, among healthy children – 0.7%.Conclusion. T-SPOT.TB test may be used as an alternative method for diagnosis of tuberculosis infection, should the parent refuse to have skin tests. In children with concomitant pathology, T-SPOT.TB test can serve as a leading method for immunodiagnosis of tuberculosis.The authors state that they have no conflict of interests.

scholarly journals UJI DAYAHAMBAT EUGENOL DARI DAUN CENGKEH HASIL FRAKSINASI TERHADAP PERTUMBUHAN JAMUR PATOGEN Fusarium oxysporum f.sp. Cubense Eugenol Inhibitory Test From Clove Leaf Fractionation Of Fungal Patogen Growth Fusarium oxysporum f.sp. cubense

AgriPeat ◽  
2019 ◽  
Vol 20 (02) ◽  
pp. 107-113
Author(s):  
Admin Journal
Keyword(s):  
Fusarium Oxysporum ◽  
Vacuum Distillation ◽  
Pathogenic Fungus ◽  
Lethal Concentration ◽  
In Vitro Tests ◽  
In Vitro Test ◽  
Microsoft Excel ◽  
Vitro Test

ABSTRACTThis study aims to determine the inhibition of eugenol derived from fractionation clove leaf essentialoils (CLEO) on the growth of pathogenic fungus Fusarium oxysporum f. sp. cubense (Foc) and LC50(Lethal Concentration 50). This research was in vitro, started with purification of clove leaf essentialoil, fractionation by vacuum distillation and bioassay. In vitro tests include exploration of minimuminhibition and preventability tests. Data were analyzed with Microsoft Excel 2010 program. Theresults of minimum inhibition showed at 218,75 ppm concentration of each level was able to inhibitthe growth of Foc fungi. The minimum inhibition exploration was carried out at 218,75 ppm, 109,38ppm, 54,69 ppm and 27,34 ppm. Exploration results showed that fractionated CLEO has been able toinhibit the growth of Foc fungi at 27,34 ppm in the amount of 15,60%. This concentration is used asthe lowest concentration in the inhibitory test. Furthermore, the inhibitory test was carried out startingat the highest concentration of 218,75 ppm, 109,38 ppm, 54,69 ppm and 27,34 ppm. Observationswere made for 7 days after inoculation (DAI). The results showed the best inhibition was at aconcentration of 218,75 ppm at 90,70% and LC50 at 11.17 µL.Keywords: CLEO, fractionation, Foc, in vitro test and LC50

256 EFFECT OF SPERM SELECTION ON THE RATE OF IN VITRO FERTILIZATION IN ALPACA (VICUGNA PACOS)

2015 ◽  
Vol 27 (1) ◽  
pp. 217
Author(s):  
E. Mellisho ◽  
V. Rivas ◽  
J. Ruiz ◽  
G. Mamani
Keyword(s):  
Extended Period ◽  
Cumulus Cells ◽  
Sperm Selection ◽  
Cleavage Rate ◽  
Slow Cooling ◽  
Vitro Fertilization ◽  
Progressive Motility ◽  
Frozen Semen ◽  
Washing Method

In alpacas, improvement of reproductive efficiency of male camelids is limited by the small size of the testes, extended period of ejaculation, and low quality of semen. This study was designed to determine the effect of 2 sperm preparation treatments before IVF on the cleavage rate. The sperm was obtained by slicing the head of the epididymis of slaughtered male alpacas (n = 8), diluting in Tris-yolk-glycerol, and freezing with the slow-cooling method. Frozen semen straws per each male were thawed in a water bath at 37°C for 15 s and evaluated for percentage of progressive motility (32 ± 8.6%) and concentration (66.5 ± 24 × 106 sperm mL–1) post-thawing. Sperm selection by the swim-up method was performed by centrifugation at 1077 × g for 5 min with washing sperm medium eliminating the supernatant; sperm were settled in inclined tube with fertilization medium (without capacitating agent) for 60 min, after which 100 μL from the surface was recovered for use in IVF. The washing method consisted in repeated washing (twice) of sperm in washing sperm medium and fertilization medium by centrifugation at 1077 × g for 5 and 3 min, respectively, and recovery of 50 μL from the bottom of the tube for use in IVF. Sperm selected by swim-up or washing methods had similar characteristics of progressive motility (18 and 23%); however, the concentration was higher for the washing v. swim-up method (52 v. 14 × 106 sperm mL–1, respectively). Cumulus-oocyte complexes (COC) were recovered from 278 ovaries of alpacas killed at abattoirs and classified (Grade 1 and 2) for in vitro maturation (38.5°C at 5% CO2 in air for 27 h in 50 μL of 10 COC per drop). A total of 839 oocytes cultured for 27 h in maturation medium were partially stripped out of cumulus cells by gentle aspiration with a pipette. Sperm suspensions in Fert TALP medium (5 μL) from each treatment group were added to each fertilization drop with 10 oocytes per drop of 45 μL obtaining a final concentration of 10 × 106 sperm mL–1 and cultivated for 72 h until their evaluation. The data for the 13 repetitions of the rate of cleavage (2 to 8 cells) were converted to angular values (angle = arcsin √%) with the object of normalizing the distribution of the data; the analysis of variance was performed (complete randomised design with sub-sampling, P < 0.05) using SAS® version 8.0 for Windows. The rate of cleavage (cell division) did not show statistical differences (P = 0.67) for the swim-up method (37%; 155/421) v. washing method (35%; 147/418). The methods of sperm selection (swim-up and washing) did not affect the rate of IVF.

322 EFFECT OF SODIUM NITROPRUSSIDE ON BUFFALO SPERM CAPACITATION IN VITRO

2007 ◽  
Vol 19 (1) ◽  
pp. 276 ◽  
Author(s):  
L. Boccia ◽  
L. Attanasio ◽  
A. De Rosa ◽  
G. Pellerano ◽  
R. Di Palo ◽  
...  
Keyword(s):  
Sodium Nitroprusside ◽  
Acrosome Reaction ◽  
Production Efficiency ◽  
Control Group ◽  
Sperm Capacitation ◽  
Promoting Effect ◽  
Vitro Fertilization ◽  
Domestic Species ◽  
Frozen Semen

The overall in vitro embryo production efficiency in buffalo is hampered by the poor fertilization rate. It is known that the quality of the frozen semen may affect fertilization efficiency. However, it is not possible to rule out that the process of capacitation, required by spermatozoa to acquire the fertilizing ability, is impaired in the in vitro fertilization (IVF) system. Although several agents have been proven to induce sperm capacitation in vitro, heparin treatment is still the most efficient method in most of the domestic species. There is evidence that capacitation is part of an oxidative process and that nitric oxide (NO) acts as a capacitation inducer in human (Herrero et al. 1999 Biol. Reprod. 61, 575–581) and bovine (Rodriguez et al. 2005 Anim. Reprod. Sci. 85, 231–242) spermatozoa. The aim of the present study was to evaluate whether sodium nitroprusside (SNP), a well-known generator of NO in vitro, improves buffalo sperm capacitation in vitro. Frozen–thawed sperm from a bull previously tested for IVF were treated by swim-up in order to select only the motile population. Spermatozoa were incubated in the presence of 0.01 mM heparin (control group) for 1 h (n = 266), 2 h (n = 270), and 3 h (n = 306), and in the presence of 10 �M SNP for 1 h (n = 302), 2 h (n = 286), and 3 h (n = 260). The concentration of SNP was chosen on the basis of a preliminary dose-response trial (0.1 �M, 1 �M, and 10 �M). Following incubation with these agents, sperm were exposed for 15 min to 60 �g mL-1 of lysophosphatidylcholine, an agent known to induce acrosome reaction only on capacitated spermatozoa. Trypan blue was used first to differentiate live from dead spermatozoa and the dried smears were then fixed in 37% formaldehyde and stained with Giemsa for acrosome evaluation by microscopic examination. The proportion of acrosome-reacted spermatozoa in each group was used to assess the efficiency of capacitation under different incubation conditions. Differences between groups were analyzed by chi-squared test. No dead spermatozoa were found in all groups. Following 1-h sperm treatment with either heparin or SNP, the proportion of acrosome-reacted spermatozoa was similar (35.3% vs. 28.5%, respectively). However, extending the incubation time to 2 h, SNP significantly (P &lt; 0.01) increased the incidence of acrosome reaction compared to heparin (60.1% vs. 44.1%, respectively). Analogously, when the sperm treatment was prolonged to 3 h, SNP gave a significantly (P &lt; 0.01) higher percentage of acrosome reaction compared to the control (68.8% vs. 36.6%, respectively). In conclusion, sperm treatment with SNP for either 2 or 3 h significant improved the efficiency of buffalo sperm capacitation in vitro compared with heparin, that is, the capacitating agent currently used in the IVF system. The promoting effect of SNP indirectly indicates that NO acts as a capacitation inducer in buffalo spermatozoa. Finally, these results suggest the need to evaluate the effect of SNP on the fertilizing capability of buffalo spermatozoa in vitro.

Comparison of methods for measurement of the pressure exerted by knitted fabrics

2016 ◽  
Vol 87 (17) ◽  
pp. 2117-2126 ◽  
Author(s):  
Małgorzata Cieślak ◽  
Agnieszka Karaszewska ◽  
Ewa Gromadzińska ◽  
Izabela Jasińska ◽  
Irena Kamińska
Keyword(s):  
In Vitro Tests ◽  
Knitted Fabric ◽  
In Vitro Test ◽  
Knitted Fabrics ◽  
Weft Knitted Fabric ◽  
In Vivo Tests ◽  
Vitro Test ◽  
Warp Knitted Fabric

The article presents the results of measurements of pressure exerted by two model knitted products – bands with different structure (WI jersey weft-knitted fabric and WII openwork warp-knitted fabric). The tests were carried out with using the I-Scan system (in vivo and in vitro tests) and the STM 579 device (in vitro test). A comparative analysis of the in vivo and in vitro results for the I-Scan method and in vitro results for the I-Scan and STM 579 method was performed. It was found that the pressure values are lower for openwork warp-knitted fabric than for jersey weft-knitted fabric both in the case of the in vitro and in vivo tests, and the values of pressure for the same band are higher in the case of the in vitro tests.

353 IN VITRO FERTILIZATION WITH FLOW-SORTED BUFFALO SPERM

2006 ◽  
Vol 18 (2) ◽  
pp. 283 ◽  
Author(s):  
M. Zhang ◽  
X. W. Liang ◽  
Y. Q. Lu ◽  
K. H. Lu
Keyword(s):  
Mineral Oil ◽  
Motile Sperm ◽  
Percoll Gradient ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Blastocyst Rate ◽  
Maximum Humidity ◽  
Cattle And Sheep

Flow cytometrically sorted X and Y sperm have been successfully used for IVF and the production of offspring in cattle and sheep (Maxwell et al. 2004 Anim. Reprod. Sci. 82–83, 79–95).The objective of this study was to test the feasibility of flow sorted buffalo sperm used in IVF systems and to establish a suitable IVF system for sorted buffalo sperm. Oocytes were aspirated from 2–6 mm follicles on the buffalo ovaries from a slaughterhouse and matured for 22–24 h in a 1-mL dish containing TCM199 + 10% OCS + 3% BFF (bovine folliciular fluid) + hormones at 38.5°C, 5% CO2 in air with maximum humidity. Semen was sorted by a flow-sorter (Clontech, Mountain View, CA, USA) into X- and Y-chromosome bearing sperm at 90% accuracy and stored at 4°C for later use with IVF. Sorted sperm were prepared for IVF by centrifugation (700g, 20 min) through a Percoll gradient (90%:45%), and washed (centrifugation at 700g, 5 min) in mTALP-BSA. For IVF, groups of 10–15 oocytes were transferred to 40-μL drops of mTALP-BSA and incubated with motile sperm at a concentration of 2 × 106 sperm mL−1 in each fertilization drop for 8–10 h under mineral oil. Presumptive zygotes were cultured until Day 8 in 25-µL drops of TCM–199 supplemented with 0.33 mM pyruvate and 10% NCS with granulosa cells at 38.5°C under 5% CO2 in air. Cleavage and blastocyst rates per oocyte insemination were recorded on Day 2 and Days 6–8 after insemination, respectively. Data were analyzed by ANOVA procedures with replicates and treatments in the model. There were significant differences in cleavage rate (42.23% vs. 52.28%) and blastocyst rate (20.62% vs. 27.67%) between sorted and unsorted sperm, respectively (Table 1). There were no significant differences in the proportions of blastocyst development rates on Days 6, 7, or 8 after insemination with sorted and unsorted sperm. The results indicate that sorted buffalo sperm from two bulls have been successfully used in an IVF system to produce sex-controlled embryos. Table 1. Cleavage and blastocyst rates with different sperm types This research was supported by grants from the National Natural Science Foundation of China (30360073) and the Guangxi Department of Science and Technology (0330004–13). The authors (M. Z. and X.W. L.) contributed equally to this work.

In Vitro Toxicology and the Test Guidelines

1996 ◽  
Vol 24 (3) ◽  
pp. 435-438
Author(s):  
Kimmo Louekari
Keyword(s):  
Cost Effective ◽  
Test Methods ◽  
In Vitro Tests ◽  
In Vitro Test ◽  
In Vitro Methods ◽  
Effective Manner ◽  
Genotoxic Carcinogens ◽  
Vitro Test

Ethical, economical and scientific considerations should encourage the development of alternative and in vitro test methods. Before their adoption, in vitro methods need to be validated and scientifically justified. Demand for rigorous validation schemes for in vitro tests must be emphasised, even more than in the case of in vivo tests. The OECD has adopted in vitro guidelines for testing genotoxicity; several endpoints and mechanisms can be studied in a cost-effective manner in vitro. Similar advantages could be afforded if acute irritation and corrosion, as well as the non-genotoxic carcinogenic effects of chemicals, could be studied in vitro. Evaluation of the validation status of various methods used to study non-genotoxic carcinogens was begun by the Nordic Working Group on In Vitro Methods for Non-genotoxic Mechanisms in 1996. In some established OECD test guidelines (for example, the dermal irritation/corrosion test), there is already room for the application of in vitro methods which have not been formally validated. In January 1996, the OECD Workshop on Harmonisation of Validation and Acceptance Criteria for Alternative Toxicological Test Methods set the basis for internationally acceptable principles to be followed in the validation of in vitro test methods.

In Vitro Toxicity Testing: Purpose, Validation and Strategy

1990 ◽  
Vol 18 (1_part_1) ◽  
pp. 11-18 ◽  
Author(s):  
Oliver P. Flint
Keyword(s):  
Cell Function ◽  
Toxicity Testing ◽  
In Vitro Toxicity ◽  
In Vitro Tests ◽  
In Vitro Test ◽  
Vitro Test ◽  
Basal Cytotoxicity ◽  
Biological Endpoint

The fullest potential for in vitro evaluation of toxicity will be realised in the context of the process of assessing the risk of human toxicity. This article is an attempt to clarify what contributions can be made by in vitro tests and what types of in vitro test can best be used. In vitro tests are clarified according to the type of biological endpoint evaluated, first into tests for general (‘basal’) cytotoxicity and, secondly, into tests for differentiated cell function. The role of each type of test is analysed and it is suggested that tests for general cytotoxicity, as opposed to differentiated function, are difficult to interpret in terms of in vivo toxicity. A general approach to evaluating in vitro tests is described, and a strategy for using these tests is proposed.

scholarly journals Effect of sperm preparation method on in vitro fertilization in pigs

Reproduction ◽  
2003 ◽  
pp. 133-141 ◽  
Author(s):  
C Matas ◽  
P Coy ◽  
R Romar ◽  
M Marco ◽  
J Gadea ◽  
...  
Keyword(s):  
Acrosome Reaction ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Sperm Preparation ◽  
Male Pronucleus ◽  
Penetration Time

This study was designed to determine the effect of different sperm preparation treatments before IVF on the acrosome reaction, oocyte penetration time, early embryo development and timing of female and male pronucleus formation. Pooled sperm-rich fractions were (i) washed in PBS, (ii) left unwashed, or (iii) layered in a Percoll gradient. In Expt 1, the proportion of acrosome-reacted spermatozoa, determined by staining with fluorescein isothyocyanate-labelled peanut agglutinin lectin and propidium iodide, was highest after treatment with Percoll (P < 0.001). In Expt 2, oocytes matured in vitro were co-cultured with spermatozoa for 2, 4 or 6 h. Attached spermatozoa were then removed and the oocytes were cultured in fresh IVF medium for 16 h. Both sperm treatment and co-culture time were found to affect penetrability and monospermy rates (P < 0.001); spermatozoa treated with Percoll showed fastest oocyte penetration and highest penetrability. In Expt 3, matured oocytes were co-incubated with spermatozoa pretreated by the three above mentioned procedures (i, ii, iii) for 2, 6 and 2 h respectively. Putative zygotes were then washed and transferred to medium NCSU-23 until the blastocyst stage. In this experiment, sperm treatment had a significant effect on the cleavage rate (P < 0.001) and rate of blastocyst formation (P < 0.05); the group treated with Percoll showed the highest rate of blastocyst formation. Finally, in Expt 4, timing of female and male pronucleus formation for each sperm treatment was determined 4, 6 and 8 h after insemination. The time of female and male pronucleus formation was affected by the sperm treatment and was faster for the Percoll group (P < 0.05). The findings of the present study indicate that treatment with Percoll yields the best results in this in vitro pig embryo production system.

scholarly journals Effect of Powder/Water Ratio Variation on Viscosity, Tear Strength and Detail Reproduction of Dental Alginate Impression Material (In Vitro and Clinical Study)

Polymers ◽  
2021 ◽  
Vol 13 (17) ◽  
pp. 2923
Author(s):  
Rasha M. Abdelraouf ◽  
Rania E. Bayoumi ◽  
Tamer M. Hamdy
Keyword(s):  
Strength Test ◽  
In Vitro Tests ◽  
In Vitro Test ◽  
Tear Strength ◽  
Ratio Variation ◽  
Clinical Difference ◽  
Reproduction Test ◽  
Vitro Test ◽  
Water Ratio

Background: Alginate impression is a common dental polymeric material, presented as powder to be mixed with water. Aim: 1. To analyze the effect of alginate powder/water ratio variation on viscosity, tear strength and detail reproduction by in vitro tests, and 2. To evaluate this variation’s effect on patients’ impressions. Materials and methods: Two commercial alginate products were mixed in different viscosities. Viscosity was measured by a viscometer. For the tear strength test, V-shaped specimens were used. For detail reproduction, a die with three scribed lines was used. Clinical dental impressions were examined by stereomicroscope. Results: The alginate specimens mixed with a higher powder/water ratio showed a higher viscosity and tear strength compared to those with a lower powder/water ratio. Both alginate mixtures reproduced two scribed lines in a detail reproduction test. On the other hand, no clear clinical difference was detected when examining dental impressions mixed with a different powder/water ratio. Conclusion: Although increasing the powder/water ratio of mixed alginate raised the resultant viscosity and tear strength by an in vitro test, clinically, no clear difference in tearing was detected. Detail reproduction was minimally affected by the variation in powder/water ratio.

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