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PubMed1987

A comparison of the electrophysiological and pharmacological properties of serotonin-containing neurons in the nucleus raphe dorsalis, raphe medianus and raphe pallidus recorded from mouse brain slices in vitro: role of autoreceptors.

The potential role of autoreceptors in regulating the activity of serotonin-containing nucleus raphe dorsalis (RD), raphe medianus (RM) and raphe pallidus (RPA) neurons was examined by recording the activity of these neurons under a variety of conditions both in vivo and in vitro. Raphe neurons recorded in vivo displayed the characteristic slow, rhythmic discharge pattern previously described for rat and cat raphe cells. The activity of these neurons was suppressed in a dose-dependent manner by tryptophan, LSD and chlorimipramine administered intravenously. There were no significant changes in the spontaneous discharge rate of raphe neurons over time when recorded in vitro, even though tissue serotonin and its metabolite, 5-hydroxyindoleacetic acid, decreased dramatically. RPA neurons fired significantly faster than either RD or RM neurons both in vivo and in vitro. Prior depletion of brain serotonin by p-chlorophenylalanine administration resulted in no significant change in raphe unit activity recorded in vitro. Elevation of brain serotonin by monoamine oxidase inhibition produced a total inhibition of raphe unit activity in vitro. Similarly, increasing the concentration of serotonin in the tissue slice by adding serotonin directly to the incubation medium resulted in a profound, though transitory, depression of unit activity. This depressant effect of serotonin was rapidly reversible upon drug wash-out. Serotonin receptor blockers, methiothepin, cypoheptadine, and methysergide, produced no significant change in unit activity. The serotonin reuptake blocker, fluoxetine, produced a total inhibition of raphe unit activity in all three nuclei in vitro. These data suggest that excess serotonin suppresses the activity of raphe neurons, apparently by an action on autoreceptors, but that a deficiency, or normal concentration, of serotonin does not influence the spontaneous activity of these cells. The data also show that RD and RM are much more sensitive to the depressant effects of serotonin than the caudal RPA neurons. More generally, these studies provide a data base for examining the electrophysiological and pharmacological characteristics of serotonergic neurons in the three major serotonin-containing nuclei in mouse brain. The mouse has proven to be a much easier species than the rat to use in these types of studies, based on the finding that mouse brain slices are more viable in vitro than are rat brain slices.

Action Potentials↗
PubMed1994

Magnetic resonance imaging of the uterus in vivo and in vitro at an ultra low magnetic field (0.02 T): assessment of its normal structure and of leiomyomas.

The purpose of this investigation was to analyze the normal anatomy and leiomyomas of the uterus with an ultra low field (0.02 T) magnetic resonance imaging (MRI) device. MR imaging was performed on 18 uteri, 11 of which were imaged both preoperatively (in vivo) and as an operative specimen (in vitro), 6 only as an operative specimen, and 1 only preoperatively. All uteri were examined histologically after imaging. The junctional zone was much better delineated in vivo than in vitro, indicating that its appearance on MR is partly due to blood flow. No structures contributing to its visibility in vitro could be demonstrated histologically. Twenty leiomyomas (size range 7-79 mm) in 12 uteri were found with MRI. They were slightly better discerned in vivo than in vitro. The leiomyomas, having no degenerative changes, had a signal intensity which was the same or lower than that of the myometrium. On images obtained in vitro the signal intensity of these leiomyomas relative to that of myometrium correlated directly with their muscular content (R = 0.74, p = .002). The authors conclude that the junctional zone is a sum of physiological and structural factors, the latter being responsible for its in vitro delineation. MR imaging of the uterus in vitro did not give more information than MR imaging in vivo. All leiomyomas larger than 10 mm could be detected, indicating that MR imaging at 0.02 T is an accurate method for the imaging of the uterine leiomyomas.

Female↗
PubMed2004

Results of in vitro fertilization in patients with endometriomas: is surgical removal beneficial?

OBJECTIVE: The purpose of this study was to investigate the effect of endometriomas on clinical pregnancy rates of in vitro fertilization-embryo transfer. STUDY DESIGN: Infertile patients with endometriosis who underwent in vitro fertilization-embryo transfer were divided into group 1 patients who had a history of ovarian endometriomas and group 2 patients who did not. The patients in group 1 were further divided into group 1A who had their endometriomas removed by laparoscopic ovarian cystectomy before the start of in vitro fertilization-embryo transfer and group 1B who underwent in vitro fertilization-embryo transfer with the presence of endometriomas. The clinical pregnancy rates of these groups were compared. RESULTS: Clinical pregnancy rates per transfer were similar in group 1 and group 2 (40% vs 47%; P =.38) and similar in patients in group 1A and group 1B (47% vs 34%; P =.28), although the direction was toward improved pregnancy rates in the groups of patients without endometriomas present at the time of in vitro fertilization stimulation. When patients >39 years of age were excluded, the overall pregnancy rate was higher in those patients who had no current endometriomas than in those patients who had endometriomas present at the time of stimulation (65% vs 39%; P =.05). For patients with unilateral endometriomas, similar numbers of mature oocytes were aspirated between the affected and unaffected ovaries. CONCLUSION: Although the history of endometriomas does not appear to affect in vitro fertilization-embryo transfer outcomes adversely, there may be a benefit in the removal of endometriomas before in vitro fertilization.

Adult↗
PubMed2005

Perinatal outcomes of in vitro fertilization twins: a systematic review and meta-analyses.

OBJECTIVE: Uncontrolled studies suggest that in vitro fertilization twins have increased rates of preterm birth and low birth weight and would warrant increased antenatal monitoring. The objective of this meta-analysis was to determine whether the incidence of poor obstetric outcomes is higher for in vitro fertilization twins than for spontaneously conceived twins who were matched for maternal age. STUDY DESIGN: Medline and EMBASE were searched with comprehensive search strategies. Case-control and cohort studies of twins who were conceived by in vitro fertilization or in vitro fertilization/intracytoplasmic sperm injection, with the transfer of fresh embryos or cryopreserved (frozen) in women with infertility, and/or whose partners were subfertile or infertile, compared with naturally (spontaneously) conceived twins who were matched for maternal age (case-control studies) or which were controlled for it (cohort studies). Two reviewers independently assessed titles, abstracts, and study quality and extracted the data. Statistical analysis was performed with commercial statistical software. Dichotomous data were meta-analyzed with odds ratios as measures of effect size, and continuous data was meta-analyzed with mean differences. Interstudy variation was incorporated with the assumption of a random effects model for the treatment effect. RESULTS: Compared with spontaneously conceived twins who were matched for maternal age, in vitro fertilization twins have an increased risk of preterm birth between 32 and 36 weeks of gestation (odds ratio, 1.48; 95% CI, 1.05-2.10), and an elevated risk of preterm birth at <37 weeks of gestation when parity is also matched for an odds ratio of 1.57 (95% CI, 1.01-2.44). There was an increased rate of cesarean delivery among in vitro fertilization twins (odds ratio, 1.33; 95% CI, 1.06-1.67). There were no significant differences in incidences of perinatal death, low birth weight infants, or congenital malformations. CONCLUSION: In vitro fertilization twins have increased rates of preterm birth compared with spontaneously conceived twins who were matched for maternal age, despite the fact that their outcomes would be expected to be better because of the decreased proportion of monochorionic twins.

Case-Control Studies↗
PubMed2006

Thorough sonographic oocyte retrieval during in vitro fertilization produces results similar to ovarian wedge resection in patients with clomiphene citrate-resistant polycystic ovarian syndrome.

OBJECTIVE: The purpose of this study was to compare the effectiveness of a thorough sonographic oocyte retrieval to a routine in vitro fertilization retrieval to induce ovulation, pregnancy, and endocrine changes in patients with polycystic ovarian syndrome in their post-in vitro fertilization cycles. STUDY DESIGN: Sixty-four patients from a tertiary infertility clinic, with clomiphene citrate-resistant polycystic ovarian syndrome and who were undergoing in vitro fertilization, were assigned randomly into 2 groups. Group 1 (n = 34) had a thorough (every possible follicle punctured) sonographic oocyte retrieval, and group 2 (n = 30) had a routine (only follicles that were likely to contain oocytes) in vitro fertilization retrieval. These patients who did not conceive in their in vitro fertilization cycle were monitored with ultrasound scanning for evidence of ovulation, pregnancy, before and after day 3 gonadotropin, and steroid hormone levels. Analysis of variance and the Student t test were used for statistical significance. RESULTS: Ovulation rates of 53% (18/34) and cumulative pregnancy rate of 44% (8/18) were observed in group 1, with no ovulations in group 2. Significant decreases in luteinizing hormone/FSH ratio (4.1 to 1.7) and testosterone (1.2 to 0.7 ng/mL) occurred after treatment in the thorough sonographic oocyte retrieval group, with no change after routine in vitro fertilization. Operating time was increased significantly in the thorough sonographic oocyte retrieval group (45 vs 24 minutes) in group 2. CONCLUSION: Thorough sonographic oocyte retrieval during in vitro fertilization cycle can produce significant improvements in the endocrinologic abnormalities, ovulation, and pregnancies that are comparable with ovarian wedge resection in patients with clomiphene citrate-resistant polycystic ovarian syndrome.

Clomiphene↗
PubMed2006

Effect of follicular size on in vitro developmental competence of oocytes and viability of embryos after transfer in the dromedary (Camelus dromedarius).

The aim of this work was to determine the effect of follicle size on camel oocyte quality as measured by developmental competence in vitro and in vivo. Ovaries from a local slaughterhouse were dissected to obtain two classes of follicle size: small (3-6 mm) and large (>6 mm) follicles. Quality of the oocytes was assessed after in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) of cumulus oocyte complexes (COCs). All cultures were done in four replicates at 38.5 degrees C, under 5% CO(2) and high humidity (>95%). Only COCs with cumulus and homogenous (dark) cytoplasm were used. The COCs were matured for 28 h in TCM-199 medium supplemented with 10% heat-treated fetal calf serum (FCS), 10 ng/mL EGF, and 250 microM cysteamine. Nuclear maturation rate for each class of follicle size was determined by contrast phase microscopy in a sample of COCs (n=30) denuded, fixed and stained with aceto-orcein. In vitro fertilization was performed using fresh semen (0.5 x 10(6)spermatozoa/mL in modified TALP-solution). Fertilized oocytes were cultured in mKSOMaa, under 5% O(2) and 90% N(2). The percentage of COCs reaching metaphase II (MII) after 28 h of maturation was 87% (26/30) and 73% (22/30) for oocytes originating from large and small follicles, respectively (P>0.1). The rate of total cleavage (two cells to blastocyst stage) was greater (P<0.05) for oocytes originating from large follicles (72%; 116/162) than for those derived from small follicles (59%; 140/237). The percentage of fertilized oocytes reaching the blastocyst stage was 35% (57/162) and 20% (48/237) for oocytes collected from large and small follicles, respectively (P<0.05). The viability of in vitro-produced hatched blastocyst from the two groups (15 from 3 to 6mm follicle size and 22 from follicles >6 mm) was assessed by transfer to synchronized recipients. None of the hatched blastocysts from small follicles resulted in a pregnancy whereas 68% (15/22) of the transferred hatched embryos from large follicles developed into a 25-day pregnancy. Of the resulting 15 pregnancies, 53% (n=8) aborted (five between 2 and 4 months and three between 5 and 7 months of pregnancy). The remaining seven pregnant females gave birth to normal healthy offsprings (four females and three males). The present study shows that dromedary oocytes developmental competence is acquired late during the final phase of follicular development and this developmental ability translates into greater pregnancy rates after transfer of in vitro produced hatched blastocysts.

Animals↗
PubMed2004

Integrated oral bioavailability projection using in vitro screening data as a selection tool in drug discovery.

The objective of the analysis described herein is to examine the in vitro/in vivo relationship of estimated bioavailability values and also the applicability of the estimated in vitro bioavailability to lead candidate selection in drug discovery. To this end, in vitro ADME data from screening assays as well as in vivo rat pharmacokinetic (PK) data were compiled for 140 compounds across therapeutic areas from the Pfizer library in Ann Arbor. The compounds span a broad range of structural types, including neutral, basic, and acidic compounds. Solubility and Caco-2 permeability data from in vitro ADME screening were used to calculate the fraction dose absorbed (FDp) using the physiologically based IDEA model. In vitro metabolic stability (t(1/2)) from human and rat liver microsomal incubations was converted to an in vitro intrinsic clearance value (CL(int)'), which was then scaled up to reflect in vivo clearance (CL) and hepatic extraction as described by Obach et al. [J. Pharmcol. Exp. Ther. 283 (1997) 46]. Subsequently, the in vitro/in vivo relationship between the measured bioavailability (F(obs)) in rats and the estimated bioavailability (F(est)) from FDp and predicted CL values was examined. The observed data suggest that compounds with low estimated in vitro bioavailability (F(est)<15%) are more likely to have low in vivo bioavailability (F(obs)<30%). Therefore, the present study indicates that in vitro estimation of bioavailability is an efficient tool to eliminate compounds having low bioavailability prior to in vivo characterization and therefore can be used to reduce attrition due to poor ADME properties in drug development.

Administration, Oral↗
PubMed2004

Differences in the incidence of apoptosis between in vivo and in vitro produced blastocysts of farm animal species: a comparative study.

The occurrence of pregnancies and births after embryo transfer (ET) of in vivo produced embryos is generally more successful compared to that of embryos produced in vitro. This difference in ET success has been observed when embryos of morphological equal (high) quality were used. The incidence of apoptosis has been suggested as an additional criterion to morphological embryo evaluation in order to assess embryo quality and effectively predict embryo viability. In this study, equine, porcine, ovine, caprine and bovine in vivo and in vitro produced morphologically selected high quality (grade-I) blastocysts were compared for the occurrence of apoptosis in blastomeres. The total number of cells per embryo and the number of cells with damaged plasma membranes, fragmented DNA and fragmented nuclei per embryo were assessed in selected blastocysts by combining Ethidium homodimer (EthD-1), terminal dUTP nick end labeling (TUNEL) and Hoechst 33342 staining. In general, the level of blastomere apoptosis was low. A higher level of apoptosis was observed in in vitro produced equine, porcine and bovine blastocysts compared to their in vivo counterparts. Interestingly, 4 of the initially selected 29 bovine in vitro produced blastocysts exhibited extensive signs of apoptosis affecting the inner cell mass (ICM), which is not compatible with a viable conceptus. Repeated occurrence of this observation may explain the lower ET outcome of in vitro produced bovine embryos compared to in vivo produced embryos. It is concluded that, although in morphologically high quality blastocysts of several farm animal species a significant difference exists in the percentages of apoptotic cells between in vivo and in vitro produced embryos, the incidence of apoptosis at the blastocyst stage is at such a low level that it cannot reflect the substantial differences in embryo viability that have been described between in vivo and in vitro produced blastocysts following ET.

Animals↗
PubMed2005

Successful piglet production by IVF of oocytes matured in vitro using NCSU-37 supplemented with fetal bovine serum.

Recently, piglets have been obtained from in vitro-produced blastocysts by using in vitro maturation systems in which oocytes have been matured in North Carolina State University (NCSU) solution supplemented with porcine follicular fluid (PFF). However, PFF is not available commercially. To prepare PFF from the ovaries required time and effort and there is substantial variation in quality among batches. Furthermore, PFF is considered a potential source of infectious agents. We evaluated another commercially available potential protein source, fetal bovine serum (FBS), for in vitro maturation, to produce embryos and piglets. Cumulus-oocyte complexes were matured in NCSU-37 with PFF or with one of four batches of FBS. The proportions of oocytes with expanded cumulus cells were lower in all FBS batch groups (P < 0.05, 15-41%) than that in the PFF group (74%). The proportions of oocytes that matured were also lower in all FBS batch groups (P < 0.05, 26-41%) than in the PFF group (73%), irrespective of cumulus expansion. However, the proportions of oocytes that underwent germinal vesicle breakdown were almost the same in all groups (76-96%). After in vitro fertilization, the rate of sperm penetration into matured oocytes was higher in the PFF group (P < 0.05, 63%) than in one batch of FBS (22%) and removal of the compacted cumulus cells after maturation did not affect fertilization status (21%). Subsequent in vitro embryo culture of the PFF and FBS groups for 6 day resulted in similar rates of blastocyst formation (17 and 19%, respectively) and similar numbers of cells per blastocyst (43 and 46 cells, respectively). When blastocysts obtained from oocytes matured with FBS were transferred into two recipients, one became pregnant and farrowed seven piglets. Transfer of blastocysts obtained from oocytes matured with PFF into two other recipients resulted in one pregnancy and production of four piglets. These data suggested that porcine in vitro maturation in NCSU-37 supplemented with FBS reduced the maturational ability of oocytes, but once oocytes have matured, they have the same ability to develop to term after in vitro fertilization and embryo transfer as those matured with PFF.

Animal Husbandry↗
PubMed1997

Coculture of human embryos with buffalo rat liver cells for women with decreased prognosis in in vitro fertilization.

OBJECTIVE: The coculture of human embryos with epithelial cells may improve both embryo quality and pregnancy rates. In this current study we tested the efficacy of coculture with the buffalo rat liver cell line on pregnancy rates in women with a potentially poor prognosis for success with in vitro fertilization (previous in vitro fertilization failure, advanced maternal age, increased early follicular follicle-stimulating hormone levels, and anovulation). STUDY DESIGN: This prospective controlled study evaluated a total of 203 women (135 coculture, 68 controls) undergoing in vitro fertilization. Implantation rates per embryo, clinical pregnancy rates, and continuing/delivered pregnancy rates were analyzed. RESULTS: Buffalo rat liver cells, which are commercially available, are stable in coculture. Implantation rates (number of sacs with fetal heart motion per embryos transferred) were similar for coculture (19%) and control (18%) embryos. No difference in the rate of continuing/delivered pregnancies per retrieval was noted (17% coculture vs 14% control) in the group with advanced maternal age, but coculture caused a trend toward improved pregnancy rates in the group with ovulatory dysfunction (43% coculture vs 14% control) and the group with previous in vitro fertilization failure (34% coculture vs 28% control). CONCLUSION: This is the first published controlled study to our knowledge that reports the use of the buffalo rat liver cell coculture for human in vitro fertilization in a large number of patients. Our data support consideration of buffalo rat liver coculture for in vitro fertilization for women with previous in vitro fertilization failure and possibly for patients with oocyte or ovulatory dysfunction.

Adult↗
PubMed1999

In vitro models to predict the in vivo mechanism, rate, and extent of placental transfer of dideoxynucleoside drugs against human immunodeficiency virus.

OBJECTIVE: We tested the hypothesis that the mechanism, rate, and extent of in vivo placental transfer of dideoxynucleoside drugs against human immunodeficiency virus can be predicted by the in vitro perfused human placenta and the drug octanol-water partition coefficient. STUDY DESIGN: Near-term pregnant macaques (Macaca nemestrina ) underwent long-term catheterization for the administration of 4 dideoxynucleosides against human immunodeficiency virus: zidovudine, didanosine, zalcitabine, and stavudine. Maternal plasma, fetal plasma, and amniotic fluid concentrations were determined frequently after intravenous bolus and/or infusion of the drugs administered into the maternal or fetal circulation on separate occasions. Antipyrine was included in all experiments as a marker of placental blood flow. The mechanism, rate, and extent of placental transfer of the 4 dideoxynucleosides in perfused human placenta were determined and compared with the findings obtained by others. RESULTS: The mechanism and rate of the antipyrine-normalized placental transfer of the 4 dideoxynucleosides in perfused human placenta were highly correlated with those observed in vivo. The extent of placental transfer (fetal/maternal steady-state plasma concentration ratio) was also highly correlated with both the antipyrine-normalized placental transfer clearance (clearance index) determined in the in vitro perfused human placenta model (r 2 = 0.95, in vitro clearance-index model) and the drug octanol-water partition coefficient (r 2 = 0.99, in vitro partition-coefficient model). To determine the predictive capacity of these correlative models, we predicted the fetal/maternal steady-state plasma concentration ratio of each drug after excluding the data on that drug from the model fit. Both in vitro models to predict in vivo placental transfer of drug models resulted in good predictions of the observed fetal/maternal steady-state plasma concentration ratio (mean error: in vitro clearance-index model = -1. 2%; in vitro partition-coefficient model = 3.9%). CONCLUSIONS: We propose that our models will accurately predict the extent of placental transfer of dideoxynucleoside drugs against human immunodeficiency virus. The models may also be applicable to other classes of drugs, regardless of therapeutic category, provided that these drugs passively diffuse across the placenta. Such a result will expedite phase 1 clinical trials of drugs in pregnant women.

Animals↗
PubMed1988

Criteria for in vitro susceptibility testing of cefotetan. Correlations with clinical and bacteriologic responses.

Standardized in vitro susceptibility testing criteria and quality-control guidelines for cefotetan have been well documented in the scientific literature. Cefotetan and cefoxitin differ in their in vitro spectrums of activity and cannot be used interchangeably for in vitro tests. In vitro susceptibility information failed to correlate with clinical or bacteriologic responses of treated patients when 314 anaerobic bacteria from 145 patients were evaluated. All cefotetan-resistant anaerobic bacteria were eradicated by cefotetan therapy, and 93 percent of patients with resistant strains were clinically curved. Most members of the Bacteroides distasonis-ovatus-thetaiotaomicron group of pathogens were resistant in vitro, but only one strain persisted after cefotetan therapy for a 96.6 percent cure rate. There was better correlation when 2,336 aerobic bacteria from 1,630 infected body sites were considered. Microorganisms susceptible or moderately susceptible by in vitro tests were equally responsive to therapy (95 percent of patients clinically cured and 94 percent of isolates eradicated). However, strains that were resistant by in vitro test criteria were less likely to respond clinically. Development of resistance during therapy or superinfections (1.7 percent) were not found to be clinically important problems with cefotetan chemotherapy.

Bacteria, Aerobic↗
PubMed2003

Differential in vitro response of the human radial artery versus left internal thoracic artery to cerivastatin: implications to bypass grafting.

BACKGROUND: This study investigated acute (in vitro) and long-term (in vivo) effects of statins on the vascular function of human radial artery (RA) and left internal thoracic artery (LITA). METHODS: RA and LITA specimens were divided into vascular rings, which were incubated in the absence or presence of 10(-6) mol/L Cerivastatin for 2 or 24 hours. In terms of preoperative statin treatment, four groups included: group 1 [preop statin(-)/in vitro cerivastatin(-)]; group 2 [preop(-)/in vitro(+)]; group 3 [preop(+)/in vitro(-)]; and group 4 [preop(+)/in vitro(+)]. Endothelial function was assessed with acetylcholine (10(-9) to 10(-5) mol/L) following contraction by 3 x 10(-8) mol/L endothelin-1. RESULTS: Although endothelium-dependent vasodilatation was higher in RA (57.7% +/- 3.5%) than in LITA (46.5% +/- 3.8%, p = 0.046), there was no significant evidence that it depended on the preoperative use of statins or incubation period. In vitro incubation with cerivastatin significantly increased endothelium-dependent vasodilatation by 14.2% +/- 2.4% (p < 0.0001) independent of artery types (RA/LITA). There was no significant evidence that endothelium-dependent vasodilatation depended on the preoperative use of statins or incubation period. CONCLUSIONS: In vitro incubation with cerivastatin preserved endothelial function more effectively than preoperative use of statins. This could have implications to perioperative use of statins for patients undergoing coronary surgery.

Acetylcholine↗
PubMed1984

Ultrastructure of ovarian follicles in in vitro perfused rabbit ovaries: response to human chorionic gonadotropin and comparison with in vivo observations.

Ovulation may be achieved and studied in an isolated perfused rabbit ovary upon inclusion of human chorionic gonadotropin (hCG) in the perfusion fluid. The ultrastructural features of the rabbit ovarian follicle prior to ovulation in vitro were compared with those in vivo. The perifollicular vasculature was also examined in in vitro perfused rabbit ovaries during the preovulatory interval. Granulosa cells of the preovulatory follicle share many ultrastructural features in vivo and in vitro; however, only small amounts of smooth endoplasmic reticulum (sER) were observed in granulosa cells in vitro after hCG. Ovulation after hCG in the in vitro preparation tends to occur earlier (6 hours) than in vivo (12 hours). Thus, there may be insufficient time and/or gonadotropin exposure to permit full functional development of granulosa cells, as reflected by reduced amounts of sER. Degradation of collagen fibrils was less prominent in the theca externa and tunica albuginea in vitro than in in vivo. Perifollicular capillaries became dilated after hCG, but interendothelial gaps were not observed. Disappearance of surface epithelium in the apex of follicles was similar in vitro and in vivo.

Animals↗
PubMed2003

In vitro fertility and motility characteristics of frozen-thawed boar epididymal spermatozoa separated by Percoll.

Frozen-thawed epididymal spermatozoa from four boars were separated through a Percoll gradient, and motility characteristics and in vitro fertility were assessed. Percoll-separated spermatozoa had a significantly higher percentage of motile and progressively motile spermatozoa than those that were not separated (P < 0.0001). However, there were no clear differences in other motility parameters between Percoll-separated and un-separated spermatozoa. Furthermore, sperm agglutination was decreased by Percoll separation (P < 0.05). The effects of Percoll separation on in vitro fertility of spermatozoa differed among boars. In addition, there were large differences in fertility between sperm samples in vitro. Sperm samples, which indicate highly motile and progressively motile, did not always show high in vitro fertility. Furthermore, there was no distinct pattern between fertility in vitro and motility parameters. There was no difference in fertility in vitro between Percoll-separated and un-separated spermatozoa from two of the four boars. However, in vitro fertility of Percoll-separated spermatozoa was higher than that of un-separated spermatozoa from the other two boars.

Animals↗
PubMed1998

Comparison of preimplantation developmental competence after mouse oocyte growth and development in vitro and in vivo.

Culture systems for oocytes are essential for the experimental analysis of the basic mechanisms of oocyte development and, moreover, they will eventually find wide application in agriculture, the clinic, and wildlife preservation. Here, progress in mouse oocyte growth and development in vitro using oocyte-granulosa cell complexes from preantral follicles is reviewed. Oocyte-granulosa cell complexes were isolated from preantral (secondary) follicles of 12 day old mice, grown in vitro for 10 days, then matured and fertilized in vitro. The developmental competence of these oocytes was compared with oocytes grown in vivo and isolated from 22 day old mice, then matured and fertilized in vitro. In vitro-grown oocytes did not achieve the same size as their in vivo-grown counterparts. However, when oocytes were grown in medium containing fetal bovine serum, their preimplantation developmental competence was equivalent to that of in vivo-grown oocytes. Surprisingly, more blastocysts per animal were produced when oocytes were grown in vitro than in vivo. There was no correlation between oocyte size and either preimplantation developmental competence or number of cells per blastocyst. Oocytes grown in serum-free medium did not achieve the same developmental competence as oocytes grown in medium supplemented with serum. Lastly, the health status as an adult of the only animal born after complete oocyte development in vitro is described and discussed.

Animals↗
PubMed1998

Washing and trypsin treatment of in vitro derived bovine embryos exposed to bovine viral diarrhea virus.

Gametes, somatic cells and materials of animal origin in media are potential sources for introducing bovine viral diarrhea virus (BVDV) into systems for production of IVF bovine embryos. Further, the efficacy of washing and trypsin treatment for removal of BVDV from IVF embryos is questionable. Washing and trypsin treatments recommended by the International Embryo Transfer Society for in vivo-derived embryos were applied to in vitro-derived, virus-exposed, bovine embryos in this side-by-side comparison of treatments. Embryos for the study were produced in a virus-free system in which follicular oocytes were matured and fertilized in vitro and presumptive zygotes were co-cultured with bovine uterine tubal cells for 7 d. A total of 18 trials was performed, 9 using a noncytopathic BVDV and 9 using a cytopathic BVDV. In each trial, 4 equal groups of 10 or less, zona pellucida-intact embryos/ova were assembled, including 2 groups of morulae and blastocysts (M/B) and 2 groups of nonfertile or degenerated ova (NFD). Each group was prewashed and exposed to 10(4) to 10(6) TCID50/mL of either noncytopathic (SD-1) or cytopathic (NADL) BVDV for 2 h. Following in vitro viral exposure, one group of M/B and one group of NFD were washed. The other groups of M/B and NFD were trypsin-treated. Both treatments were consistent with IETS guidelines. After in vitro exposure to noncytopathic BVDV and washing, viral assays of 100% (9/9) and 78% (7/9) of the groups of M/B and NFD ova, respectively, were positive. After in vitro exposure to cytopathic BVDV and washing, viral assay of 33% (3/9) of the groups of both M/B and NFD ova were positive. After in vitro exposure to noncytopathic BVDV and trypsin treatment, viral assay of 44% (4/9) of groups of M/B and 67% (6/9) of groups of NFD ova were positive. Finally, after in vitro exposure to cytopathic BVDV and trypsin treatment, viral assay of 22% (2/9) of the groups of M/B and 44% (4/9) of the groups of NFD ova were positive. Contingency table analysis, in which data was stratified by embryo type and virus biotype, was used to compare results. While a difference existed between results of the 2 treatments of groups of M/B within the noncytopathic biotype (P = 0.01, Mantel Haenszel Chi-square), no difference was observed between comparison of treatment between all groups in both biotypes (P > 0.05).

Animals↗
PubMed1998

Noncytopathogenic bovine viral diarrhea virus (BVDV) reduces cleavage but increases blastocyst yield of in vitro produced embryos.

The growing application of in vitro embryo production systems that utilize slaughterhouse tissues of animals of unknown health status conveys the risk of disease transmission. One pathogen of concern in this regard is bovine viral diarrhea virus (BVDV), and the objective of this study was to investigate the effect of BVDV on in vitro embryonic development. A bovine in vitro embryo production system was experimentally infected with BVDV at 2 stages: prior to in vitro maturation by incubating cumulus-oocyte complexes (COC) with virus (strain Pe515; titer 10(6.2) tissue culture infective dose (TCID)50/mL) or vehicle for 2 h, and then during in vitro culture by the use of BVDV infected granulosa cells. Exposure to BVDV throughout in vitro production reduced cleavage rates (P = 0.01) but increased (P = 0.05) the number of embryos that reached the 8-cell stage when expressed as a percentage of cleaved oocytes. Blastocyst yield was increased by the presence of virus when expressed as a proportion of oocytes (P = 0.0034) or of those cleaved (P < 0.0001). The percentage of total blastocyst yield on Days 7, 8 and 9 for the control and virus treatments was 20, 51, 29 and 29, 41, and 29%, respectively, indicating that the rate of blastocyst development was nonsignificantly faster in the virus-treated group (P = 0.06). These results indicate that the presence of non-cytopathogenic BVDV in an in vitro production system may reduce cleavage rates but allow those cleaved to develop to blastocysts at a higher rate.

Animals↗