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PubMed2002

In vitro fertilization of in vitro-matured equine oocytes: effect of maturation medium, duration of maturation, and sperm calcium ionophore treatment, and comparison with rates of fertilization in vivo after oviductal transfer.

Three experiments were conducted to evaluate the effect of oocyte and sperm treatments on rates of in vitro fertilization (IVF) in the horse and to determine the capacity of in vitro-matured horse oocytes to be fertilized in vivo. There was no effect of duration of oocyte maturation (24 vs. 42 h) or calcium ionophore concentration during sperm capacitation (3 microM vs. 7.14 microM) on in vitro fertilization rates. Oocytes matured in 100% follicular fluid had significantly higher fertilization (13% to 24%) than did oocytes matured in maturation medium or in 20% follicular fluid (0% to 12%; P < 0.05). There was no significant difference in fertilization rate among 3 sperm treatments utilizing 7.14 microM calcium ionophore (12% to 21%). Of in vitro-matured oocytes recovered 40-44 h after transfer to the oviducts of inseminated mares, 77% showed normal fertilization (2 pronuclei to normal cleavage). Cleavage to 2 or more cells was seen in 22% of oocytes matured in follicular fluid and 63% of oocytes matured in maturation medium; this difference was significant (P < 0.05). We conclude that in vitro-matured horse oocytes are capable of being fertilized at high rates in the appropriate environment and that in vitro maturation of oocytes in follicular fluid increases fertilization rate in vitro but reduces embryo development after fertilization in vivo. Further work is needed to determine the optimum environment for sperm capacitation and IVF in the horse.

Animals↗
PubMed2006

Responses of canine oocytes to in vitro maturation and in vitro fertilization outcome.

The potential benefits of assisted reproduction techniques, such as in vitro maturation (IVM) and in vitro fertilization (IVF) in canids, are linked to the protection and saving of species threatened by extinction due to worldwide habitat destruction and pollution. In both domestic and wild species, these technologies will form the basis for the next leap in reproductive performance by improving fertility rates in valuable middle-aged females, by improving pregnancy rate in infertile or sub-fertile populations and by rescuing biological material to replenish populations of endangered species. In vitro techniques are supposed to answer the reproductive questions of canids, to introduce new methods for contraception and to compete with artificial insemination (AI) as the major or predominant method of embryo production, oocyte- and embryo cryopreservation and cloning. The causes affecting in vitro meiosis of dog oocytes are likely to be diverse. Incomplete understanding of the events associated with oocyte developmental competence are imputed to species reproductive physiology, medium composition and source of ovarian oocyte population used for in vitro maturation. This review addresses some issues on the current state of in vitro maturation and in vitro fertilization of canine oocytes.

Animals↗
PubMed2002

In vitro maturation and fertilization of bovine oocytes and in vitro culture of presumptive zygotes in the presence of bovine pestivirus.

A pathogen which has been shown to commonly contaminate in vitro bovine embryo production system is bovine pestivirus (bovine viral diarrhea virus). Three experiments were designed to evaluate the in vitro maturation (experiment I), fertilization (experiment II) and embryo development (experiment III) of immature oocytes, inseminated oocytes and presumptive zygotes in the presence of a bovine pestivirus (non-cytopathic, nCP type 1). The virus inoculum used was derived from a persistently infected cow. In experiment I, follicular oocytes (n=1257) recovered from slaughterhouse derived ovaries were randomly assigned to either a control group (n=578) which did not become exposed to bovine pestivirus and a treatment group (n=679) which was inoculated with bovine pestivirus (2.20-3.69 log(10) TCID(50)/50 microl) at the time of commencement of in vitro maturation. Overall, there was no significant difference between the control and pestivirus inoculated oocytes in either the cumulus cell expansion rate (79+/-7.5% versus 74+/-10.7%) or the nuclear maturation rate (89+/-4.8% versus 85+/-7.4%), respectively. In experiment II, in vitro matured oocytes (n=607) were inseminated either in the absence (control; n=301) or the presence of bovine pestivirus (4-4.6 log(10) TCID(50)/50 microl; n=306). A significant (P<0.01) reduction in the overall number of fertilized oocytes with two well formed male and female pronuclei was observed in the treatment group compared to the control group (58.5+/-5.8% versus 73.3+/-3.6%, respectively). In experiment III, after in vitro maturation and fertilization, presumptive zygotes were randomly assigned to either a control group (n=139) which was not exposed to bovine pestivirus or a treatment group which was inoculated with bovine pestivirus (2.97-4.47 log(10) TCID(50)/30 microl; n=139). The zygotes were then cultured under mineral oil in an atmosphere of 88% N(2), 7% O(2) and 5% CO(2) at 39 degrees C. The morphologic appearance of the embryos was assessed 48 h after the commencement of culture, and then every 48 h up to days 7-8 after insemination. The 22% (31/139) and 3.6% (5/139) of the presumptive zygotes developed to the morula or blastocyst stage in the control and the bovine pestivirus inoculated groups, respectively (P<0.001). This study demonstrates that bovine pestivirus has a significant detrimental effect on in vitro fertilization and early in vitro embryo development.

Animals↗
PubMed2002

Vitrification of in vitro produced bovine embryos: in vitro and in vivo evaluations.

The efficacy of different vitrification solutions to cryopreserve in vitro produced bovine blastocysts was evaluated based upon in vitro development of embryos in culture and on in vivo development of embryos transferred into recipients. In the first experiment, ethylene glycol + glycerol (Eg + Gly) + different sucrose concentrations were evaluated. There were no significant differences in development rates among solutions. As for hatching, the Eg + Gly + 0.1 M sucrose group had a greater rate as compared with Eg + Gly + 0 M sucrose and Eg + Gly + 0.5 M sucrose groups in the evaluations of Day 6, Day 7 and Day 6 + Day 7 embryos; and, Eg + Gly + 0.3 M sucrose group had a greater rate as compared with the Eg + Gly + 0 M sucrose and Eg + Gly + 0.5 M sucrose groups in evaluations of Day 6 and Day 6 + Day 7 embryos. There were no significant differences in development and hatching rates between Day 6 and 7 in in vitro produced bovine embryos within each treatment group. There were significant differences in nuclei number after vitrification between Eg + Gly + 0.1 M and Eg + Gly + 0 M sucrose groups and the Eg + Gly + 0.5 M sucrose group. Pregnancy after 60 days of transfer and calving rates showed a difference between in vivo produced embryos freshly transferred and in vitro produced embryos vitrified with Eg + Gly + 0.3 M. There were no significant differences in gestation length and sex ratio between treatments. As for birth weight, there were significant differences between fresh in vivo produced embryos and all treatments of in vitro produced embryos. There were significant differences in dystocial parturition between in vivo produced embryos and all treatments with in vitro produced embryos. These results demonstrate that vitrification can be used successfully in the cryopreservation of in vitro produced bovine embryos, and that it might be considered for use in commercial programs.

Animals↗
PubMed2002

Atrazine effects on in vitro maturation and in vitro fertilization in the bovine oocyte.

The effect of low levels of atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) on in vitro oocyte maturation, in vitro capacitation of sperm, or in vitro fertilization of bovine oocytes and on the quality of blastocyst formation was studied. Bovine oocytes collected from abattoir ovaries were matured, fertilized, and developed to the blastocyst stage in vitro. Embryos that reached a morula or blastocyst stage were stained with Hoechst 33258 stain to determine the number of blastomeres per embryo. Three bulls whose fertilization rates were proven consistent among straws were used for this study. Atrazine was tested at concentrations of 0.01, 0.1, 1, and 10 microM in either the maturation medium, sperm capacitation medium, or the fertilization medium. Because atrazine was dissolved in ethanol, an ethanol control was used to determine any possible effects of ethanol on the in vitro process. The addition of atrazine to both the maturation and fertilization media did not result in any significant difference in fertilization rates between the controls and the treatments. In the capacitation medium, a significant difference between the controls and the atrazine levels of 0.1, 1, and 10 microM was noted for one bull. Atrazine did not affect the number of blastomeres per embryo. There was not a significant difference (p>0.05) in the number of blastomeres per embryo between the controls and the different levels of atrazine in each medium. This study indicates that low levels of atrazine do not have an effect on in vitro fertilization rates or the number of blastomeres per embryo produced in vitro.

Animals↗
PubMed2001

Energy status of nonmatured and in vitro-matured domestic cat oocytes and of different stages of in vitro-produced embryos: enzymatic removal of the zona pellucida increases adenosine triphosphate content and total cell number of blastocysts.

In this study, we evaluated the adenosine triphosphate (ATP) content of individual domestic cat oocytes before and after in vitro maturation and of different stages of in vitro-produced embryos. To investigate the effects of assisted-hatching technique on the ATP content and total cell number, the zona pellucida of in vitro-produced blastocysts and expanded blastocysts (recovered 144 h postinsemination [hpi]) was completely removed by pronase treatment. The average (mean +/- SEM) ATP content of nonmatured oocytes (3.47 +/- 0.18 pmol) was significantly (P < 0.01) higher than that of in vitro-matured oocytes (2.17 +/- 0.10 pmol). After in vitro fertilization and culture, the ATP content of two-cell stages (24 hpi) was 1.17 +/- 0.08 pmol, which increased to 1.47 +/- 0.19 and 1.88 +/- 0.32 pmol at the four- (40 hpi) and eight-cell (48 hpi) stages, respectively. The ATP content then decreased to 1.48 +/- 0.10 pmol in 16-cell embryos (64 hpi), reaching a minimum of 0.49 +/- 0.04 pmol at the morula stage (120 hpi). Blastocysts, expanded blastocysts (both 144 hpi), and hatching blastocysts (192 hpi) revealed ATP levels of 1.05 +/- 0.09, 1.79 +/- 0.01, and 4.17 +/- 0.21 pmol, respectively. After enzymatic removal of the zona pellucida (ERZP) at 144 hpi, ATP content and total cell numbers of blastocysts (4.15 +/- 0.37 pmol of ATP, 328.3 +/- 48.5 cells) and expanded blastocysts (5.81 +/- 0.54 pmol of ATP, 430.1 +/- 29.7 cells) analyzed at 192 hpi were significantly (P < 0.001) higher than in their nontreated counterparts (blastocysts: 1.00 +/- 0.09 pmol of ATP, 65.3 +/- 4.6 cells; expanded blastocysts: 1.79 +/- 0.11 pmol of ATP, 121.4 +/- 6.5 cells). Our study describes, to our knowledge for the first time, changes in the energy status of domestic cat oocytes before and after maturation and during in vitro development after fertilization. The ERZP markedly increased the ATP content and total cell number of blastocyst stages, suggesting that this technique may improve the quality and viability of in vitro-produced domestic cat embryos.

Adenosine Triphosphate↗
PubMed2002

Fertilization of mouse oocytes from in vitro-matured preantral follicles using classical in vitro fertilization or intracytoplasmic sperm injection.

Early preantral mouse follicles with a diameter of 110-160 microm were cultured in vitro for 10 or 12 days. Mature oocytes were retrieved following hCG, and fertilization was attempted either by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Two-cell and blastocyst formation rates and blastocyst cell numbers were compared between 10-day and 12-day in vitro-matured oocytes versus in vivo-matured oocytes. Uncleaved IVF oocytes were subjected to chromosome analysis. The 2-cell formation rate was significantly improved by ICSI compared with IVF both in 10-day (72.1% versus 56.1%; P = 0.03) and 12-day cultures (74.1% versus 54.5%; P = 0.028). Cytogenetic analysis of uncleaved MII oocytes following IVF showed that about 30% of MII oocytes showed no sign of sperm penetration. The blastocyst formation rate was significantly lower in 12-day versus 10-day cultures, whether fertilization was by IVF (40.7% versus 62.4%, P = 0.016) or by ICSI (32.5% versus 57.1%, P = 0.035). Blastocyst cell numbers from IVF and ICSI 10-day groups were similar and both significantly higher (P < 0.001) than from IVF 12-day cultures. All above expressed values were significantly higher for in vivo-matured oocytes. In conclusion, fertilization of oocytes from in vitro-matured mouse preantral follicles can be optimized with ICSI, giving significantly higher 2-cell formation rates than IVF. Blastocyst formation rate was not influenced by the technique of fertilization but rather by the extent of the in vitro culture period. Best results on preimplantation development of oocytes for in vitro-matured preantral follicles were obtained with ICSI on oocytes from 10-day in vitro cultures.

Animals↗
PubMed2004

In vitro testing of drug absorption for drug 'developability' assessment: forming an interface between in vitro preclinical data and clinical outcome.

Drug 'developability' assessment has become an increasingly important addition to traditional drug efficacy and toxicity evaluations, as pharmaceutical scientists strive to accelerate drug discovery and development processes in a time- and cost-effective manner. The fraction of drug absorbed and the maximum absorbable dose (MAD) can be estimated from in vivo clinical pharmacokinetics, mass balance studies or in vivo drug permeability in humans by different calculation methods. Unfortunately, in vivo data are usually unavailable at the early stages of drug discovery and development, and in vitro screening for the permeability, solubility, activity and toxicity of a drug has become a routine measurement in drug discovery and development. These in vitro data could be used to predict drug 'developability' with different calculation methods before selecting candidates for clinical evaluation. The fraction of drug absorbed in human could be predicted by in vivo human permeability or in vitro Caco2 permeability. For example, if drug permeability in Caco2 cells reaches 13.3 to 18.1 x 10(-6) cm/s, its predicted in vivo permeability in humans would reach 2 x 10(-4) cm/s, and its predicted fraction of drug absorbed would be > 90%, which is defined as highly permeable. The MAD could also be predicted with in vitro permeability, or calculated absorption rate constant. In addition, in vitro solubility and permeability data can also be used for the biopharmaceutics classification system (BCS) and, subsequently, to direct formulation optimization strategies. If drug 'developability' becomes an obstacle for drug delivery based on these in vitro data and predictions at the early stages of drug discovery and development, options such as prodrug approaches could be explored to enhance drug 'developability', in addition to different formulation methods. Therefore, in vitro absorption testing is a highly valuable tool in the decision-making process to select candidates for in vivo clinical studies at early-stage drug discovery and development.

Biological Transport↗
PubMed1975

The failure to show a necessary role for C3 in the in vitro antibody response.

The in vitro antibody response of mouse spleen cells to TNP coupled to both T-dependent and T-independent carriers as well as to sheep erythrocytes has been studied to investigate the possible role of complement activation in the induction of antibody formation. The following has been found. (1) In vitro responses of both IgM and IgG can be obtained to both T-dependent and T-independent antigens in serum-free media, although they are smaller than those found in serum-containing media. This shows that no exogenous source of complement is necessary for in vitro antibody formation by spleen cells. (2) Similarly, normal antibody responses are obtained if the cultures are grown in human serum depleted of C3b-inactivator, which contains high concentrations of C3b. (3) In the presence of antibody to mouse C3 the response to the T-independent antigen is reduced, the IgM responses being more affected than the IgG. However, purified F(ab')2 anti-C3 has no inhibitory effect and it therefore seems likely that it is the formation of intact immune complexes containing Fc rather than the interference with C3 function that is responsible for the inhibition seen. (4) The conventionally purified anti-complementary factor from cobra venom has no effect on the antibody response in serum-free culture or when human or fetal calf sera are used. In no experiment was any potentiation of T-dependent responses observed. However, the presence of quite small concentrations (2%) of cobra veno

Animals↗
PubMed1984

Induction of unscheduled DNA synthesis in primary rat hepatocytes by benzidine-congener-derived azo dyes in the in vitro and in vivo/in vitro assays.

The genotoxicity of the benzidine-congener-derived azo dyes. Direct Blue 1 ( DB1 ), Direct Blue 14 ( DB14 ), Direct Brown 95 ( DB95 ), and Direct Red 46 ( DR46 ) was studied in the in vitro and in vivo/in vitro unscheduled DNA synthesis (UDS) assays in primary rat hepatocytes to determine if in vivo metabolism of these compounds was required for induction of UDS. Hepatocytes were isolated, cultured, and treated with the azo dyes and [3H]thymidine (in vitro assay); alternatively, in the in vivo/in vitro assay, rats were intubated with the azo dyes, the hepatocytes isolated at 17 h after dosing and incubated in a medium containing [3H]thymidine. UDS was quantified by an autoradiographic method. None of the azo dyes induced UDS in the in vitro assay. However, DR46 did induce marginal, but significant UDS in 1 experiment (1.2 net grains at 500 micrograms/ml media). No significant UDS was observed when DR46 was tested in a subsequent in vitro assay. In the in vivo/in vitro assay, DB95 (100 mg/kg), DB14 (125 mg/kg), and DR46 (100 mg/kg) induced significant UDS (12, 2.1, and 3.5 net grains, respectively). None of the azo dyes tested was mutagenic in the Salmonella/microsome assay in the presence and absence of rat liver enzymes. Therefore, in vivo reduction of azo dyes, presumably by the gut microflora, is a requirement for the genotoxicity of these azo dyes in the primary rat hepatocyte UDS assay.

Animals↗
PubMed2001

An in vitro DNA virus for in vitro protein evolution.

In vitro virus is a molecular construct for in vitro protein evolution, which requires some mechanism to link phenotype to genotype. The first in vitro virus was realized by bonding a nascent protein with its coding mRNA via puromycin in in vitro translation. We report a new construct of in vitro DNA virus. The virion was a covalent cDNA-protein fusion, and virion formation did not require any modification of mRNA. Due to intactness of mRNA, this type of in vitro DNA virus will take the next step toward in vitro autonomous evolution, just like in vivo viral evolution in a cellstat.

DNA Primers↗
PubMed2003

In vitro susceptibility testing of ciclopirox, terbinafine, ketoconazole and itraconazole against dermatophytes and nondermatophytes, and in vitro evaluation of combination antifungal activity.

BACKGROUND: With the development of newer antifungal agents with activity against both yeasts and filamentous fungi, there is an increased need to develop and standardize in vitro assays that will evaluate the activity of antimycotics against filamentous fungi. In vitro analysis of antifungal activity of these agents would also allow for the comparison between different antimycotics, which in turn may clarify the reasons for lack of clinical response or serve as an effective therapy for patients with chronic infection. OBJECTIVES: To determine the in vitro susceptibility of fungal organisms to ciclopirox, terbinafine, ketoconazole and itraconazole and to evaluate the in vitro activity and mode of interaction of ciclopirox in combination with either terbinafine or itraconazole. MATERIALS AND METHODS: In the minimum inhibitory concentration (MIC) study 133 strains were evaluated, including dermatophytes (110 strains; 98 from Trichophyton spp.), Candida spp. (14 strains) and nondermatophyte moulds (nine strains). In vitro susceptibility testing was conducted in microbroth dilutions based on the National Committee for Clinical Laboratory Standards (NCCLS) M27-A proposed standard. The testing MIC ranges were 0.003-2 microg mL-1 for ciclopirox and terbinafine, and 0.06-32 microg mL-1 for itraconazole and ketoconazole. For inoculum preparation, dermatophytes were grown on Heinz oatmeal cereal agar slants. Inoculum suspensions of dermatophytes were diluted in RPMI 1640 (Sigma-Aldrich) with the desired final concentration being 2-5 x 103 c.f.u. mL-1. Once inoculated, the microdilution plates were set up according to the NCCLS M27-A method, incubated at 35 degrees C, and read visually following 7 days of incubation. For azole agents, the MIC was the lowest concentration showing 80% growth inhibition; for terbinafine and ciclopirox, the MIC was the lowest concentration showing 100% growth inhibition. In the synergy studies, 29 strains from nondermatophyte species were evaluated using a checkerboard microdilution method. The concentrations tested were: 0 and 0.06-32 microg mL-1 for itraconazole, and 0 and 0.003-4 microg mL-1 for both terbinafine and ciclopirox. Modes of interaction between drugs were classified as synergism, additivism, antagonism or indifference based on fractional inhibitory concentration index values (FIC index). Synergism was defined as an FIC index of < or = 0.50, additivity as an FIC index of < or = 1.0, and antagonism as an FIC index of > or = 2.0. The drug combination was interpreted as indifferent if neither of the drugs had any visible effect on the presence of the other drug. RESULTS: In the MIC study, the dermatophyte MIC values (microg mL-1) (mean +/- SEM) were: ciclopirox (0.04 +/- 0.02), terbinafine (0.04 +/- 0.23), itraconazole (2.28 +/- 7.42) and ketoconazole (0.83 +/- 1.99). The yeast MIC values (microg mL-1) (mean +/- SEM) were: ciclopirox (0.05 +/- 0.02), terbinafine (1.77 +/- 0.58), itraconazole (0.18 +/- 0.27) and ketoconazole (0.56 +/- 0.60). The non-dermatophyte fungi MIC values (microg mL-1) (mean +/- SEM) were: ciclopirox (1.04 +/- 2.62), terbinafine (1.04 +/- 0.95), itraconazole (17.87 +/- 16.75) and ketoconazole (10.69 +/- 13.09). In the synergy study, with ciclopirox in combination with terbinafine, mainly a synergistic or additive reaction was observed; there were no cases of antagonism. For ciclopirox in combination with itraconazole, there were some instances of additivism or synergism, with indifference in the majority of instances; there were no cases of antagonism. CONCLUSIONS: In vitro susceptibility testing indicates that ciclopirox may have a broad antimicrobial profile including dermatophytes, yeasts and other nondermatophytes. Terbinafine is extremely potent against dermatophytes. In vitro evaluation of activity of ciclopirox and terbinafine suggests many instances of synergy or additivism; for ciclopirox and itraconazole there may be indifference, synergy or additivism.

Antifungal Agents↗
PubMed2004

Embryonic development of in vitro matured and in vitro fertilized dog oocytes.

In several studies, early cleavage stage canine embryos have been derived from in vitro fertilized oocytes cultured under various conditions. Despite these results, IVF protocols for canine oocytes have yielded low fertilization rates. In this study, Experiment I compared the effects of tissue culture medium (TCM)-199 supplemented with either (A) 1 microg/ml estradiol or (B) 20 microg/ml estradiol + 1 microg/ml human somatotropin (hST) on the in vitro nuclear maturation rate of canine oocytes. Meiotic progression to the metaphase I and II (MI/MII) stages at 72 hr of in vitro culture (IVC) was 10.2% (11/108) in medium A versus 14.1% (30/142) in medium B (P = 0.802). In Experiment II, cleavage rate was determined among oocytes recovered from ovaries of bitches at different reproductive stages. Oocytes (n = 888) were retrieved from bitches at the follicular, anestrous, and luteal stages and selected for high morphological quality. Oocytes were matured for 48 hr in TCM-199 supplemented with 1 microg/ml hST + 20 microg/ml estradiol. Oocytes were in vitro fertilized with fresh canine spermatozoa that had been isolated on a Percoll gradient, and were cultured in synthetic oviduct fluid (SOF) medium with bovine serum albumin (BSA; 4 mg/ml) up to 5 days in 5% CO(2) in air at 37 degrees C. A proportion of oocytes (30.6%) with identifiable nuclear material had cytoplasm penetrated or fertilized by sperm. The percentage of oocytes developing into early stage embryos was 10.1% (27/267). Although pronuclear development was observed to be higher for oocytes recovered at the follicular phase, the cleavage rate was similar among oocytes recovered from bitches at the follicular, anestrus, and luteal stages. There was no correlation between the proportion of capacitated or acrosome reacted spermatozoa and pronuclei formation and/or percent cleavage. It was concluded that TCM-199 supplemented with 1 microg/ml hST and estradiol (20 microg/ml) supports nuclear and cytoplasmic maturation of canine oocytes. In this study, meiotic competence was verified by the in vitro production (IVP) and development of embryos up to the 8 cell-stage. Furthermore, the results indicate that, under the described conditions and despite the influence of reproductive status of the bitch on the developmental competence of in vitro fertilized oocytes to the pronuclei stage, cleavage was independent of donor's reproductive estrous cycle stage.

Animals↗
PubMed1996

In vivo interpretation of in vitro effect studies with a detailed analysis of the method of in vitro transcription in isolated cell nuclei.

In vitro experimental approaches are of central importance to contemporary molecular and cellular biology and toxicology. However, the scientific value or impact of in vitro results depends on their relevance in vivo. In vitro effect studies address inobservable in vivo phenomena through experiments on analogous in vitro phenomena. We present a theoretical basis developed to evaluate the in vivo relevance of in vitro effect studies. As a case study, the procedure for measuring specific gene transcription in isolated cell nuclei (¿nuclear run-off method¿) is analyzed. It is concluded that current evidence fails to justify in vivo interpretations of nuclear run-off experiments within the framework of theoretical models of transcription, implying that quantitative in vivo interpretations are unwarranted. Qualitative interpretations of nuclear run-off experiments may be justified by inferring ¿the best explanation¿, especially when significant in vitro effects follow in vivo perturbations. Elements of a general theory are proposed. It is concluded that quantitative in vivo interpretations are warranted primarily in biochemical quantitation of biomolecules, while studies on biological function should be interpreted qualitatively in terms of causal explanations. Inferences to the best explanations are strengthened through additional evidence and the creation of experimental differences (effects).

Animals↗
PubMed2002

Development of in vitro embryo production systems for red deer (Cervus elaphus). Part 3. In vitro fertilisation using sheep serum as a capacitating agent and the subsequent birth of calves.

The following experiments investigated the use of sheep serum (SS) as a capacitating agent for red deer (Cervus elaphus) sperm during in vitro fertilisation. Red deer oocytes were collected at slaughter and matured in vitro for 24h in TCM-199 supplemented with 10% foetal calf serum, 10 microg ml(-1) FSH and LH, and 1microg ml(-1) of oestradiol. Fertilisation medium was IVF-SOF modified to contain 5mM Ca(2+) and no glucose. Experiment 1 investigated the addition of heparin, BSA (8 mg ml(-1)) or 20% SS. All oocytes were penetrated when IVF-SOF was supplemented with SS compared to 10 and 0% penetration when either heparin or BSA was present (P<0.01). However, 43.8% of these oocytes were polyspermic when the medium contained SS. In Experiment 2, the effect of sperm concentration on penetration rates during in vitro fertilisation was investigated. Total sperm penetration and monospermic penetration rates increased with increased sperm concentrations in a log linear manner (P<0.001) and both approached an asymptote at 0.4 x 10(6) sperm ml(-1) with 93.6 and 77% for total and monospermic penetration, respectively. Polyspermic fertilisation also increased with increasing sperm concentrations (P<0.05) but was variable (range 3.5+/-4.2 to 42.3+/-10.6%), especially at the lower sperm concentrations. Experiment 3 investigated the viability of these oocytes after transfer into red deer recipients. Fifteen 2- and 4-cell embryos were transferred into the oviducts of synchronized recipients 28 h post in vitro insemination. An additional fourteen embryos (8-10 cell) were transferred into synchronised recipients after 48 h of in vitro culture in either SOFaaBSA (n=10) or on red deer epithelial oviduct monolayers (n=4). Five (33% 5/15) of the recipients that received 2- and 4-cell embryos were pregnant at Day 45 (verified by ultrasonography) and four recipients subsequently calved. One recipient receiving an embryo cultured in SOFaaBSA was pregnant at Day 45 and subsequently calved. The birth of five normal calves indicate that full developmental competence of red deer oocytes matured and fertilised in vitro can be achieved by the techniques described.

Animals↗
PubMed2003

In vitro parthenogenetic development of mouse oocytes following reciprocal transfer of the chromosome spindle between in vivo-matured oocytes and in vitro-matured oocytes.

Mouse follicles grown in vitro from preantral to mature stages yield oocytes that can be fertilized in vitro, but embryonic development is poor. To investigate whether this poor development is due to a nuclear or a cytoplasmatic factor, we designed an experiment in which the MII chromosome spindle was exchanged between in vitro-matured oocytes and in vivo-matured oocytes by electrofusion. Subsequent embryo development was evaluated by blastocyst formation rate and blastocyst cell number after parthenogenetic activation. Electrofusion was successful in 62-78% of the oocytes. Transfer of the spindle apparatus from in vitro-matured oocytes to the in vivo MII cytoplasmic environment resulted in a high rate of blastocyst development, whereas in the reverse situation (transfer of the nucleus from in vivo-matured oocytes into in vitro-matured MII cytoplasm) poor quality embryos and a low rate of blastocyst formation was observed. These results indicate that the low developmental competence of in vitro-matured oocytes from mouse preantral follicles after activation is caused by the cytoplasmic component rather than the nuclear component.

Animals↗
PubMed2003

Blastocyst formation rates in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection.

This study was conducted to evaluate in vivo and in vitro development of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection. Oocytes were collected from slaughterhouse-derived ovaries, matured in vitro, and injected with frozen-thawed stallion sperm. In vivo development was assessed after transfer of injected oocytes to the oviducts of recipient mares. Mares were killed 7.5-8.5 days after transfer and the uterus and oviducts flushed for embryo recovery. Of 132 injected oocytes transferred, 69 (52%) were recovered; of these, 25 (36%) were blastocysts with a blastocoele and capsule. In vitro development was assessed in three culture systems. Culture of zygotes in modified Chatot, Ziomek, Bavister medium with BSA containing either 5.5 mM glucose for 7.5 days or 0.55 mM glucose for 3 days, followed by 3 mM glucose for 2 days, then 4.3 mM glucose for 2.5 days, did not result in blastocyst formation. Culture of zygotes in Dulbecco modified Eagle medium (DMEM)/F-12 with 10% fetal bovine serum with and without coculture with equine oviductal epithelial explants yielded 16% and 15% blastocyst development, respectively. Development to blastocyst was significantly lower in G1.3/2.3/BSA than in DMEM/F-12/BSA or in either medium with 10% added serum (2% vs. 18%, 18% or 20%; P < 0.05), suggesting that requirements for equine embryo development differ from those for other species. These results indicate that in vitro-matured equine oocytes are sufficiently competent to form 36% blastocysts in an optimal environment (in vivo). While we identified an in vitro culture system that provided repeatable blastocyst development without coculture, this yielded only half the rate of development achieved in vivo.

Animals↗
PubMed1995

Cysteamine enhances in vitro development of porcine oocytes matured and fertilized in vitro.

Most porcine oocytes matured and fertilized in vitro fail to develop normally due to abnormal fertilization. The aim of this study was to determine the effect of cysteamine on pronuclear formation and developmental competence in pig embryos produced in vitro. Follicular oocytes were matured in vitro with or without cysteamine in Medium 199 supplemented with sodium pyruvate, FSH, LH, 17 beta-estradiol, antibiotics, and 25% porcine follicular fluid. Matured oocytes were then inseminated for 10 h. In the first experiment, pronuclear formation was assessed immediately after the insemination period. The addition of 50 microM or 500 microM cysteamine to the maturation medium increased male pronuclear development, resulting in a higher proportion of oocytes with synchronously formed male and female pronuclei. The frequency of synchronous pronuclear formation was increased in monospermic oocytes from 10% in the control group to 43% (p < 0.001) and 45% (p < 0.001), respectively, and in polyspermic oocytes from 43% in the control group to 68% (p < 0.05) and 75% (p < 0.001), respectively. In the second experiment, development of the in-vitro produced embryos was assessed after 7 days of culture in vitro. The addition 500 microM cysteamine to the maturation medium increased the percentage of cleaving embryos developing to the 8-cell (37% vs. 16%; p < 0.001), morula (19% vs. 6%; p < 0.001), and blastocyst (12% vs. 1%; p < 0.001) stages. These results demonstrate that the addition of cysteamine to maturation medium increases synchronous pronuclear formation and normal embryonic development in porcine oocytes matured and fertilized in vitro.

Animals↗