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PubMed1995

Developmental potential of microbiopsied murine blastocysts.

OBJECTIVE: To evaluate the frequency of mouse pups born following blastocyst biopsy and embryo transfer compared to nonbiopsied controls. METHODS: ICR Swiss albino and C57B1/6 mice served as embryo donors. Pregnant mare serum gonadotropin treatment was followed 46-52 hours later by hCG, when donors were paired with fertile males. Mating was confirmed the following day and embryos were collected on the afternoon of day 4. After overnight culture, hatching trophoblast was excised by micromanipulation with a fine-pulled glass pipette. Embryos (206 controls, 206 biopsied) were transferred to 26 pseudopregnant recipients. Alternate mouse strains were used to identify pups born from control or biopsied embryos. RESULTS: The end point was percentage of pups born ((number born/number transferred) x 100), using angular transformation before analysis. There was no significant difference (P > .1) between percent live-born in control (27.7%) or biopsied (34.5%) embryos, nor were there any strain differences. CONCLUSION: These data support the hypothesis that the developmental potential of murine blastocysts is not affected adversely by the biopsy procedure.

Animals↗
PubMed1996

Electroejaculation and assisted reproductive techniques for anejaculatory infertility.

OBJECTIVE: To report our experience with electroejaculation combined with intrauterine inseminations (IUI) and other assisted reproductive technologies. METHODS: Anejaculatory men desirous of fertility were evaluated in our Assisted Reproductive Program. Between March 1991 and December 1994, 26 men (age 24-48) underwent a total of 84 electroejaculations. Causes of anejaculation included spinal cord injury (n = 23) and retroperitoneal lymph node dissection (n = 3). Female partners were super-ovulated to improve the pregnancy rate. The success in sperm retrieval and pregnancy rates were determined, and the quality of electroejaculates was evaluated. RESULTS: Seventy-seven of 84 (92%) electroejaculations were successful, defined by retrieval of more than 10 x 10(6) total and more than 10(6) motile spermatozoa. Mean sperm count was 65 million/mL (range 0-569), but mean motility was only 16% (range 0-66). Mean normal morphology was 27% (range 0-71). Ten couples attempted conception. Fifty cycles of IUIs were performed, resulting in four normal term infants and one spontaneous abortion (pregnancy rate 10% per IUI). One patient failed to conceive with eight cycles of IUIs but became pregnant with in vitro fertilization-embryo transfer with micromanipulation using electroejaculates; she delivered a set of healthy twins. Two couples elected donor sperm insemination after failing to conceive by IUI with electroejaculates; both became pregnant. CONCLUSION: Electroejaculation offers an encouraging pregnancy opportunity for anejaculatory men who otherwise are considered infertile. Marked asthenospermia is observed in electroejaculates, the etiology of which remains obscure. Further studies to elucidate the cause may improve pregnancy rates.

Adult↗
PubMed1996

Prenatal diagnosis of the fetal RhD blood type using a single fetal nucleated erythrocyte from maternal blood.

OBJECTIVE: To develop a method that allows prenatal diagnosis of the fetal RhD blood type from maternal blood. METHODS: Maternal blood was obtained at 8-31 weeks' gestation, and nucleated erythrocytes were separated with Percoll using a discontinuous density gradient method, then collected individually by micromanipulation under microscopic observation. After whole genome amplification with primer extension pre-amplification, exon 7 of the RhD and RhCE as well as the ZFX/ZFY loci were further amplified by a nested polymerase chain reaction (PCR). RESULTS: Nucleated erythrocytes were detected in nine of ten maternal blood samples, and sex was determined in 13 of 21 nucleated erythrocytes. RhD genotype could be diagnosed in 12 of the 13 nucleated erythrocytes in which sex could be determined. The results of RhD blood type and sex in nucleated erythrocytes obtained from maternal blood were identical with those of newborns. Fetal RhD blood type could be determined in six of ten maternal blood samples. CONCLUSION: A new method for noninvasive prenatal diagnosis of the fetal RhD blood type using a single nucleated erythrocyte isolated from maternal blood was demonstrated. This diagnostic method offers extremely useful information for the management of Rh-negative pregnant women. Furthermore, this method of prenatal diagnosis can be applied to other genetic disorders and is expected to become the preferred method of noninvasive prenatal diagnosis of DNA.

Base Sequence↗
PubMed1991

Validity of digital subtraction of transcranial plain films in quantification of positional changes of the mandibular condyle.

Condylar positioning problems have been associated with temporomandibular joint signs and symptoms. The position of the mandibular condyle is difficult to predict and quantify with existing routinely used radiographic techniques. An investigation was implemented to test the validity and reliability of digital subtraction to enhance transcranial plain films and to quantify positional changes of the condyle in a laboratory model. The model consisted of a mounted dried human skull and a mandibular condyle attached to a micromanipulator. Controlled changes in condylar position were made, and the condyle and fossa were imaged. The data were analyzed with descriptive statistics, analyses of variance to evaluate the sources of variability, and linear contrasts to evaluate the differences between observed and expected values. The results indicated that digital subtraction detected posterior positional change within 10% of the expected value. However, inferior positional changes were poorly detected and lateral positional changes could not be evaluated by this technique. We conclude that digitally subtracted transcranial radiographs may be useful for detection of posterior condylar movements, assuming that appropriate head-holding devices are used to fix x-ray source and head geometry.

Analysis of Variance↗
PubMed1981

Sex determination and sex differentiation in Isospora (Toxoplasma) gondii.

Although sexual differentiation in the life-cycle of Eimeriina (Coccidia) becomes morphologically evident during development of the gamonts, there are indications in the literature that pregametogonic schizonts might already be sexually differentiated. On the other hand development of both sexes from cloned postzygotic, presumably haploid stages has been reported. In the present experiments we cloned successive stages of the coccidian representative Isopora (Toxoplasma) gondii to determine the time and stage of onset of sexual differentiation in the life-cycle. Clones of a single proliferative form, of single cysts and of a single cystozoite were made under visual control using a de Fonbrune micromanipulator. Seronegative conventional (CV) and specific pathogen-free (SPF) cats were orally infected, each with brain tissue cysts from a single clone. Periods of oocyst excretion by CV cats were recorded; SPF cats were autopsied at day 5 or 6 after infection (p.i.) and their small intestines were examined histologically. Infectivity of shed oocysts, after sporulation, was determined by mouse inoculation. All clones produced infective oocysts with oocyst shedding for two to nine days between day 4 and 14 p.i., and clones of all three stages produced both types of gamonts and gametes. The present results demonstrate: (i) that all stages of I. (T.) gondii in the intermediate host (as well as the free-living stages) are bisexual, although presumably haploid; (ii) that sexual differentiation is not genetically determined, if the first sporulation fission is indeed meiotic, but is a phenotypic change induced by some final host factor(s). This is the first report of the development of both sexes from a single cyst and cystozoite of I. (T.) gondii.

Animals↗
PubMed1988

Topography of the rat blood-testis barrier after intratubular administration of intercellular tracers.

Intratubular injection of electron-opaque tracers (lanthanum hydroxide, peroxidase) by micromanipulation showed that the intercellular spaces of the adluminal compartment are in continuity with the lumen of the rat seminiferous tubule at all stages of the spermatogenic cycle. This continuity involves the intercellular spaces which surround zygotene spermatocytes and late leptotene spermatocytes in stages X-XI. The present observations would seem to cast doubt on the real existence of a third compartment, or intermediate compartment, in the seminiferous epithelium. In the material which, in addition to intratubular administration, had been treated with intravascular tracer perfusion, we found the presence of a parabasal region of interSertoli junctions where the tracer stopped whatever its direction. Freeze-fracture replicas exhibit interSertoli tight junctions arranged, from basal lamina to lumen, in three well-defined patterns: in the most basal area, which is the zone of free penetration of tracers from the peritubular environment, junctions are very sparse or absent; a belt of closely apposed continuous and parallel junctions, also parallel to the basal lamina, is the exclusion zone which prevents tracer penetration from either direction; the most apical membrane areas display irregularly arranged, loosely spaced and frequently interrupted rows of particles, most tending to be perpendicular to the basal lamina. This area corresponds to the territory where the intraluminally injected tracer permeates the interSertoli space.

Animals↗
PubMed1985

High susceptibility to ADP-induced thrombus formation in mast cell-deficient W/Wv mice.

We used mast cell-deficient W/Wv mice to clarify whether mast cell-derived heparin may play a role in inhibiting thrombus formation in the living organism. Small veins in the mesentery of W/Wv or congenic +/+ mice were stretched over an inverted microscope; a micropipette filled with varying concentrations of ADP was set close to the outside of a vein by using a micromanipulator. Thrombus formation was directly examined under the microscope. The concentration of ADP necessary for thrombus formation was significantly lower in the W/Wv mice than in the congenic +/+ mice. Furthermore, the concentration of ADP necessary for aggregation of platelets in platelet-rich plasma (PRP) was significantly lower in W/Wv mice than in +/+ mice. The higher sensitivity of PRP of W/Wv mice is not attributed to the platelets, but to the plasma, since platelets of +/+ mice suspended in platelet-poor plasma (PPP) of W/Wv mice were more sensitive to ADP than platelets of W/Wv mice suspended in PPP of +/+ mice. The present results suggest that plasma of W/Wv mice may lack any inhibitory factor(s) or contain promoting factor(s) for platelet aggregation.

Adenosine Diphosphate↗
PubMed1985

Thrombus formation by the application of thrombin to the outer surface of mouse mesenteric vein: comparison with the application of ADP.

Mesenteries of mice under anesthesia were stretched over an inverted microscope. A micropipette filled with solution containing various concentrations of ADP or thrombin was brought into contact with the outside of a mesenteric vein by micromanipulation, and then poured over the outer surface of the vein. Morphological characteristics of the thrombi and the time needed for thrombus formation were examined. Application of either thrombin or ADP to the adventitia of mesenteric veins caused thrombus formation. Although thrombi by application of ADP seemed to be anchored by direct adhesion of platelets to the exposed subendothelium, thrombi by application of thrombin seemed to be anchored by deposited fibrin.

Adenosine Diphosphate↗
PubMed1987

Beneficial effect of agar for the frozen storage of bisected embryos.

The effect of agar embedding on the viability of intact and bisected goat embryos during freezing and thawing was examined. Blastocysts or hatched blatocysts were bisected into haves by micromanipulation. After embedding with or without agar, they were stored by deep freezing. After thawing, undamaged and some partially damaged embryos were transferred into uteri of recipients. Of 22 demi-embryos embedded with agar, only one was undamaged, but of 58 demi-embryos embedded with agar, 29 were undamaged. Although one set of monozygotic twins was obtained after the transfer of 15 sets of frozen-thawed bisected embryos, the pregnancy rate (47%) and the proportion of young (27%) were lower than those obtained by transfer of frozen-thawed, intact embryos (67 to 80% for pregnancy rate, 45 to 53% for the young).

Journal Article↗
PubMed1988

Quick-splitting of bovine embryos.

Described is a simplified method of bovine embryo bisection amenable to on-farm embryo transfer. Using a microblade operated by a hand-held micromanipulator, Day 7 bovine embryos were bisected while in the zona pellucida. With a vertical motion, the embryo was pinned between the blade and the bottom of a plastic petri dish and bisected. Demi-embryos were transferred nonsurgically (without zonae pellucidae) into synchronized recipients. Pregnancy rates were normal with 5 13 (38%) and 9 20 (45%) of recipients confirmed pregnant 70 to 80 d after receiving either twin or single half embryos, respectively. This compared to 12 28 (43%) of recipients becoming pregnant from transfer of whole embryos. These data confirm that bovine demi-embryos do not need zonae pellucidae on Day 7 and that simplified field methods of bisection give normal pregnancy results.

Journal Article↗
PubMed1988

Production of normal piglets from microsurgically split morulae and blastocysts.

The embryo splitting technique was applied to pig embryos, and the developmental ability of the split embryos was examined by means of in vitro culture and transfer. Morulae, early blastocysts and blastocysts were collected from Landrace x Large White F(1) gilts which had been mated to Duroc boars. The embryos were bisected with a fine glass or alloy (PtIr) needle after the softening of zonae pellucidae. The halved-embryos, which had either been placed in zonae pellucidae or not, were transferred to recipient gilts immediately after the micromanipulation (Experiment 1) or after cultivation for 15 to 20 h (Experiment 2). In Experiment 1, two fetuses were obtained from one of three recipients which had received 12 half-embryos. In Experiment 2, three of five recipients became pregnant, and in one recipient, seven piglets of a litter were obtained from 12 zona-free half-embryos produced from the original seven blastocysts. The results obtained indicate that a simple method not requiring the encasing of split embryos into zonae pellucidae is satisfactory to produce viable half-embryos.

Journal Article↗
PubMed1989

Competition between split and nonmanipulated embryos in the production of identical piglets.

The effect of nonmanipulated embryos on in utero survival of split porcine embryos was examined in this study. Previously, only limited success in the production of identical twin piglets has been reported. Embryos were collected from slaughtered donors (4 to 7 d post estrus) and were either split with the aid of a micromanipulator or left as whole embryos. Monozygotic pairs of split embryos were then surgically transferred to recipients with a complement of either split or nonmanipulated embryos. A total of 217 split embryos and 60 nonmanipulated embryos were transferred to 19 recipients. Nine of these recipients farrowed. In the two recipients that received only split embryos and farrowed, 31% of the split embryos survived to term, including two sets of monozygotic twins. In the remaining seven recipients, only 10% of the split embryos that were transferred along with nonmanipulated embryos survived to farrowing. This difference in split embryo survival (31 vs 10%) was significantly different (P<0.005). Sixty-nine percent of the nonmanipulated embryos survived to term in recipients that maintained pregnancy. Data presented in this study suggest that competition occurs between split and nonmanipulated embryos transferred to the same uterine environment.

Journal Article↗
PubMed1989

Survival of DNA-injected cow embryos temporarily cultured in rabbit oviducts.

Holstein or Angus cows were superovulated, inseminated with fresh bull semen, and necropsied about 12 h after estimated time of ovulation. Ova were centrifuged at 15,600 G for 3 to 8 min to reveal pronuclei. In Experiment 1, pronuclear bovine embryos were transferred to ligated or unligated oviducts of 1-d pseudopregnant rabbits for 7 d; 30 of 32 embryos were recovered from ligated oviducts but only 2 of 26 from oviducts and uterine horns of unligated oviducts. In Experiment 2, a Rous sarcoma virus-chloramphenicol acetyl transferase fusion gene was injected into one pronucleus of about half of 404 fertilized bovine ova, using a micromanipulator and interference contrast optics. Injected and noninjected embryos were then transferred to opposite ligated rabbit oviducts. Embryos were recovered after 7, 8 or 9 d. Of 120 centrifuged but uninjected embryos recovered from rabbit oviducts, 66 (55%) were in the morula to hatching blastocyst stage of development. Of 105 embryos centrifuged and injected with foreign DNA, 55 (52%) were in the morula to hatching blastocyst stage. In Experiment 3, centrifuged bovine embryos, noninjected or DNA-injected, were cultured in rabbit oviducts for 7 d then transferred nonsurgically to the uterus of recipient cows. Embryos were also flushed from superovulated cows 8 d after estrus and transferred directly to recipient cows. After 7 d, the uterus of recipient cows was flushed nonsurgically to recover embryos. The proportion of transferred embryos recovered with normally elongated trophoblastic membranes and the proportion of recipient cows with developing embryos were 14 of 25 DNA-injected embryos, 5 of 8 cows; 6 of 15 centrifuged but noninjected embryos, 4 of 6 cows; and 11 of 29 embryos transferred directly, 5 of 8 cows. Results indicate that bovine embryos can be cultured in rabbit oviducts and survive after transfer to cow uteri and that injection of foreign DNA may not increase embryonic loss within the first 2 wk after injection.

Journal Article↗
PubMed1992

The freezability of biopsied bovine embryos.

A biopsy of 1-5 blastomeres was taken from 86 bovine compacted morulae, using a technique, that did minimal damage to the zona pellucida. As soon as possible after the micromanipulation the embryos were frozen, without sealing the penetrated zona pellucida. In order to evaluate the viability of the biopsied frozen-thawed embryos, half of them were transferred to synchronized recipients while the remaining half were co-cultured to evaluate the developmental capacity. A control group of 43 intact embryos was subjected to the same procedures. This study revealed a slight, but not significant, decrease in the pregnancy rate of the biopsied embryos after freezing and thawing, although the embryos by co-culture with bovine oviduct epithelial cells revealed normal morphology and developmental rate. It is concluded that the described biopsy technique did not compromise the freezability of 7-day-old bovine embryos.

Journal Article↗
PubMed1992

Sex determination and milk protein genotyping of preimplantation stage bovine embryos using multiplex PCR.

A method for determining the sex and milk protein genotypes (RFLPs) of preimplantation stage bovine embryos using multiplex polymerase chain reaction (PCR) is described. Day 6 to 7 embryos were micromanipulated to isolate 5 to 6 cells. These cells were then dried in reaction tubes for transport to the laboratory. Subsequently, two sets of PCRs were performed using Y chromosome, k-casein and beta-lactoglobulin gene specific primers, followed by electrophoretic analysis of the PCR products. The presence or absence of the Y chromosome was ascertained in 90 of 92 embryos. Moreover, the k-casein specific fragment was amplified and detected in all these embryos. The PCR products were digested in order to genotype the k-casein gene. In 70% of the embryos, the beta-lactoglobulin specific fragment was amplified, although together with some unspecific fragments.

Journal Article↗
PubMed1995

A rapid method for mRNA detection in single-cell biopsies from preimplantation-stage bovine embryos.

Major questions concerning the control of development and gene expression at the cellular level are still unanswered. Nowhere is this more evident than during the earliest stages of development and embryogenesis. This study describes the detection of specific gene transcripts in single cells derived from bovine embryos. Following in vitro fertilization (IVF) and in vitro culture (IVC) of bovine embryos, small groups of cells and even single blastomeres from 32 to 64-cell embryos were micromanipulated into individual tubes for analysis of cytoplasmic RNAs. Reverse transcriptase-PCR was applied to cell lysates for the amplification of beta-actin mRNA transcripts. Primers were designed to flank an intron expected to be present within genomic DNA sequences, thus allowing for simple differentiation between DNA- and RNA-derived amplification products. Using a 50-cycle amplification profile, a 260 bp band could be seen as a PCR product derived from a single blastomere following electrophoresis in an ethidium bromide-stained agarose gel. The identity of the band was verified by DNA sequence determination and diagnostic restriction digestion. Lysates derived from single blastomeres in this way have been used for simultaneously phenotyping multiple RNA products. This capability allows the spatial analysis of gene expression and development within embryos from the earliest stages of cellular differentiation.

Journal Article↗
PubMed1995

PCR-sexing of bovine embryos: a simplified protocol.

To make bovine embryo sexing under farm conditions more feasible we developed a simplified protocol utilizing manual biopsy and detection of the Y chromosome directly from polymerase chain reaction (PCR) reaction tubes. Twenty-four embryos (morulae and blastocysts) were biopsied manually into 2 to 4 samples. One sample of each original embryo was diagnosed for sex, based on restriction fragment length polymorphism of PCR-amplified DNA of the ZFX/ZFY locus. The remaining 44 samples were diagnosed using the tube detection assay. In this assay the biopsies were pipetted into 0.5 -ml reaction tubes containing lysis mixture, incubated 10 to 60 min at 37 degrees C and inactivated 10 min at 98 degrees C. Then the PCR mixture was added containing buffer, DNA polymerase, ethidium bromide and primers designed to amplify the highly repeated btDYZ-1 region of the bovine Y chromosome. After 50 cycles of PCR, the reaction tubes were examined under UV illumination for pink fluorescence indicating the presence of Y-chromosomal DNA. All sexing results from the replicates were in agreement with the ZFX/ZFY assay, with 12 of the original embryos diagnosed as females and 12 as males. We conclude that highly efficient and accurate PCR-sexing of embryos can be accomplished without the use of micromanipulators, control primers and electrophoresis. The 2 reaction mixtures needed for sex diagnosis can be stored at -20 degrees C and -196 degrees C, respectively. The tube detection assay minimizes the risk of carryover contamination by previously amplified products as there is no need to open the tubes following PCR.

Journal Article↗
PubMed1990

Determination of oxygen profiles in biocatalyst particles by means of a combined polarographic oxygen microsensor.

When studying the effect of immobilization of enzymes or whole cells on the conversion of substrate, more information is gained if measurements of substrate inside the biocatalyst particles are possible. With the methods used until now, only measurements outside the particle can be performed. In this article a method for measuring oxygen profiles in a biocatalyst particle under steady state conditions is described. The biocatalyst particle was made of agarose and contained the enzyme L-lactate 2-monooxygenase. This enzyme decarboxylates lactic acid to acetic acid in the presence of oxygen. The experiments were carried out in a flow chamber with the use of a micromanipulator and a stereomicroscope. The data were sampled by means of a computer. Four different profiles were measured using four different enzyme concentrations. The measured oxygen profiles were reproducible and the signal was very stable. It was also possible to measure the boundary layer around the particle. With the use of the oxygen microsensor, measurements in a biocatalyst particle could be performed accurately, giving way for model validation.

Biosensing Techniques↗