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Biomedical subjects

N L First

Publications and source records attributed to N L First.

At least 109 records · Page 6Linked to original sources
PubMed1986

Reprograming of murine blastocoele formation.

The present study shows that there is communication between reaggregated asynchronous cleavage stage blastomeres that regulates blastocoele formation. Individual blastomeres from eight-cell murine embryos were transferred to empty zonae pellucidae, intact two-cell embryos, or enucleated two-cell embryos, and were examined over a period of 75 hours for development of cavitation. It was found that the isolated blastomeres cavitated concurrently with intact control eight-cell embryos, while intact control two-cell embryos cavitated 24 hours later. However, the embryos resulting from combining a two-cell embryo and a blastomere from an eight-cell embryo cavitated at a time in between the eight- and two-cell controls.

Animals↗
PubMed1986

Effect of cytochalasin B and demecolcine on freeze-thaw survival of murine embryos in vitro.

The effects of cytochalasin B (CB), a microfilament inhibitor, and demecolcine (DC), a microtubule inhibitor, on freeze-thaw survival and culture survival of early cleavage stage mouse embryos, was evaluated. In the first experiment, eight-cell mouse embryos were frozen in dimethylsulfoxide (DMSO) or DMSO + 0.1 microgram/ml DC + 7.5 micrograms/ml CB. In the second experiment, eight-cell embryos were dehydrated and cultured in the presence of either DMSO, DMSO + DC, DMSO + CB, or DMSO + DC + CB for 45 min prior to rehydration and culture to stimulate the osmotic and chemical changes encountered during the dehydration and rehydration procedures, but without the consequences of freezing and thawing. In the third experiment, additional eight-cell embryos were frozen in either DMSO, DMSO + DC, DMSO + CB, or DMSO + DC + CB. The survival of embryos frozen in DMSO (75%) was significantly higher (P less than 0.01) than that of embryos frozen in DMSO + DC + CB (55%). No differences (P = 0.55) were observed after a 48-hr culture period in the development of embryos dehydrated, cultured, and rehydrated but not frozen. Embryos frozen in the presence of both DC and CB had a lower (P = 0.06) survival rate (55%) than that of embryos frozen in the presence of DMSO, DC, or CB (approximately 70%). These results suggest that both microfilaments and microtubules have a role in maintaining the structural integrity of the plasma membrane during the freeze-thaw process and that the loss of either loss does not seem to be detrimental to survival, but the loss of both results in lower survival.

Animals↗
PubMed1986

Bovine in vitro fertilization with frozen-thawed semen.

A procedure to obtain high and repeatable fertilization frequencies for bovine in vitro fertilization (IVF) with frozen-thawed sperm was developed. IVF frequency of in vitro matured oocytes was increased by a swimup sperm separation procedure (P=0.01) or treatment of sperm with the glycosaminoglycan heparin (P=0.0001), but the two factors did not interact (P=0.23). Heparin was the most important factor in increasing IVF frequencies. The fertilization frequency was not affected by the batch of oocytes used (P=0.38), but bull effects were present (P<0.05). Within a bull, the IVF system was highly repeatable and varied between trials no more than +/- 12% in fertilization frequency with an overall fertilization frequency of 299 379 (79%) on four trials over four bulls. In vivo matured oocytes fertilized in vitro were transferred to ewe or heifer oviducts. Morulae or blastocysts were recovered from ewes after four to five days, while conceptuses were present in the bovine after 25 days (diagnosed by ultrasound). Embryonic development from the IVF system either pre- or postimplantation was normal.

Journal Article↗
PubMed1986

Nuclear transplantation in mouse embryos: assessment of recipient cell stage.

Enucleated zygotes were compared with enucleated two-cell embryos as recipients for donor nuclei from eight-cell embryos. Only one or two cleavage divisions were observed when eight-cell nuclei were transplanted to enucleated zygotes. Development of enucleated two-cell embryos containing a transplanted eight-cell nucleus was appreciably better with 51% (45/89) of the embryos forming blastocysts in vitro and 42% (25/60) initiating implantation. Of these, eleven implantation sites on Day 10 of gestation were examined histologically and two contained normally developing embryos. No development was observed beyond Day 12 of gestation. These observations indicate that a major transition occurs between the zygote and two-cell stage that results in the two-cell recipient being more compatible with the eight-cell nucleus than with the zygote.

Animals↗
PubMed1986

Effect of asynchronous superinduction on embryo survival and range of blastocyst development in swine.

The importance of uniform development of blastocysts was examined by comparing the effects of asynchronous superinduction (Day 6 embryos into Day 7 pregnant recipients and Day 7 embryos into Day 6 pregnant recipients) on the range of embryo development at Days 12 and 13 to subsequent survival to Day 30. Twenty gilts were used to produce five Day 7 recipients that received Day 6 embryos and five Day 6 recipients that received Day 7 embryos. Embryos from the Day 7 and Day 6 recipients were examined 6 days later. Recovered embryos ranged morphologically from spherical to filamentous blastocysts. This range of embryos was within the limits of that previously observed for naturally mated sows. However, recovered blastocysts from the Day 6 embryos transferred into Day 7 recipients were morphologically more variable and proportionately less developed than the blastocysts from the Day 7 embryos transferred into Day 6 recipients. Forty additional gilts were subsequently utilized to generate 20 recipients (10 recipients per transfer group) that were examined on Day 30. More Day 7 embryos transferred into Day 6 recipients survived (p less than 0.05) than Day 6 embryos transferred into Day 7 recipients. These experiments suggested that greater variation in early development of embryos, within litters, subsequently resulted in greater mortality of embryos.

Animals↗
PubMed1986

Effects of fetal calf serum and bovine serum albumin on in vitro maturation and fertilization of bovine and hamster cumulus-oocyte complexes.

Bovine serum albumin (BSA) and fetal calf serum (FCS) were evaluated as protein supplements for in vitro maturation and fertilization of oocytes from cows and hamsters. BSA and low doses of FCS (0.1 or 1.0%) did not support viability or maturation of the cumulus-oocyte complex as well as higher doses of FCS (5, 10, or 20%) for either species. BSA failed to support cumulus expansion for bovine or hamster cumulus-oocyte complexes. All doses of FCS examined supported cumulus expansion in bovine cumulus-oocyte complexes, whereas the hamster complexes required at least 1.0% FCS to induce cumulus expansion. The addition of a serum filtrate, Solcoseryl, with BSA improved viability of the cumulus in the bovine but did not support cumulus expansion or completion of Meiosis I in bovine complexes. In vitro fertilization could be accomplished in media containing FCS by increasing the heparin concentration in the bovine system or reducing FCS for the hamster system. Polyspermy was increased when FCS was the protein supplement. It is not known whether this is an interaction of FCS with the sperm or oocyte. In conclusion, FCS was found necessary for follicle-stimulating-hormone (FSH)-induced cumulus expansion. It also improved cumulus cell viability and completion of the first meiotic division in complexes of both species compared with BSA.

Animals↗
PubMed1986

Intrauterine migration of the porcine embryo: coordination of bead migration with estradiol.

Forty crossbred gilts were used in three experiments to examine the effects of estradiol on embryo migration. Small, spherical beads of Silastic glue containing either cholesterol or estradiol-17 beta were used to mimic embryo migration. In the first experiment, 10 cholesterol- and 10 estradiol-impregnated beads were injected into the tip of the uterine horns, either on the same side (n = 5) or opposite from each other (n = 5). The second experiment consisted of a localized release of cholesterol or estradiol and observing migration of cholesterol-containing beads inserted 10 cm anterior and posterior to this site (n = 5). In the third experiment, 10 cholesterol-impregnated beads were injected into either the tip or base of one uterine horn. Additionally, these gilts were exposed to vehicle or exogenous estradiol in a 2 X 2 factorial arrangement of treatments (n = 5). Results of these experiments indicated that cholesterol-impregnated beads migrated further (P less than .05) when adjacent to estradiol-containing beads than when in an opposite uterine horn. Localized release of estradiol failed to induce movement of beads away from the site of steroid release. Finally, beads inserted at the base of the uterus moved anteriorly following treatment of gilts with estradiol. We suggest from these experiments that the porcine uterine horn cannot discriminate between estradiol- and cholesterol-releasing beads and, further, lacks a coordinated ability to displace adjacent beads. A site-dose dependent mechanism(s) of estrogenic induction of migration may exist such that porcine embryos become bilaterally intermixed following posterior, then anterior, waves of uterine contractions.

Animals↗
PubMed1986

Glycosaminoglycans in ewe reproductive tracts and their influence on acrosome reactions in bovine spermatozoa in vitro.

Glycosaminoglycans (GAG) promote acrosome reactions (AR) in bovine and rabbit spermatozoa in vitro. Female bovine reproductive tract secretions contained GAG and the concentrations and composition of those GAG varied with different regions and stages of the estrous cycle. This study was designed to evaluate the types of GAG found in reproductive tract secretions of ewes at different stages of the estrous cycle and the ability of the secretions to promote AR. Ewes (n = 48) were slaughtered at 0, 12, 24, 36, 72 and 144 h following observations of standing estrus. Reproductive tracts were flushed with a modified Tyrode's medium (TALP: 7 ml) not containing calcium. Concentrations and composition of GAG in the tract were determined by high performance liquid chromatography. Concentrations of GAG decreased anterior to the cervix: 2.28 to .74 mg/100 mg protein (P less than .05). Increases in heparin-like GAG during the estrual phase and chondroitin sulfate GAG during the luteal phase were noted. Bovine sperm were incubated in tract flushings standardized to contain 4 micrograms/ml of GAG and supplemented with calcium. Sperm incubated in estrual flushings for 9 h exhibited a higher incidence of AR than those incubated in luteal flushings or control, 50% vs 36% and 29%, respectively (P less than .0001). It is concluded that during the estrous cycle there were changes in concentrations and composition of GAG in ewe reproductive tracts and the potencies of those female secretions causing AR varied.

Acrosome↗
PubMed1986

Dose-response shift in the ability of gilts to remain pregnant following exogenous estradiol-17 beta exposure.

Sixty mated gilts were assigned to a 2 X 6 factorial arrangement (n = 5) of day of injection (d 9 and 10 vs 12 and 13; d 0 = first day of estrus) and dose of estradiol-17 beta (0, .125, .5, 2, 8 and 32 mg X gilt-1 X d-1). Gilts were subsequently slaughtered on d 30; pregnancy was verified and percent embryonic survival calculated. A 64-fold shift in the dose-response curve for percent embryonic survival illustrated that the adverse effects of exogenous estradiol-17 beta were less when administered on d 12 and 13 as compared with d 9 and 10 (day X dose, P less than .01). This experiment demonstrated that the uterine-embryonic environment of d 12 and 13 pregnant gilts was more tolerant of exogenous estrogen alterations than that of d 9 and 10 pregnant gilts.

Animals↗
PubMed1986

Use of a fluorescent stain for visualization of nuclear material in living oocytes and early embryos.

Hoechst dyes 33342 and 33258 were used to visualize pronuclei and nuclei of early preimplantation embryos. Murine one-cell zygotes exposed to dye stained rapidly over a range of concentrations (0, 0.02, 0.04, 0.1 or 0.2 micrograms/50 microliter of media). Development to morula and blastocyst in vitro was reduced (39/70, 56%; p less than 0.05) compared to controls (44/57, 77%) but not completely blocked. Porcine and bovine zygotes and embryos could also be stained but required incubation times up to 4 hr. Porcine embryos exposed to Hoechst 33342 had limited (p less than 0.01) in vitro development (29/74, 39%) compared to unstained controls (49/64, 76%). Hoechst dyes stain embryos from different species but suitably adjusted incubation times are required. Limited preimplantation development in vitro may be expected following staining and exposure to ultraviolet light.

Animals↗
PubMed1985

Effect of heparin and chondroitin sulfate on the acrosome reaction and fertility of bovine sperm in vitro.

Glycosaminoglycans (GAGs) were reported to induce acrosome reactions (AR) in epididymal and ejaculated bovine sperm (4,5). The GAGs chondroitin sulfate A (CS-A) and heparin were tested on ejaculated bovine sperm for their ability to increase in vitro fertilization (IVF) frequencies. Regardless of treatment, a sperm-egg incubation time of 18 hr was sufficient to achieve maximal rates of fertilization. The IVF frequency of sperm incubated 6 hr with 10 mug/ml heparin (116 173 , 67%) was increased (P<0.05) above control levels (56 181 , 31%); however, 10 mug/ml CS-A (56 164 , 34%) was without effect (P>0.05). In contrast to previous reports, CS-A did not (P>0.05) induce AR in ejaculated (9.5-hr incubation) or epididymal sperm (22.5-hr incubation). Linear increases in fertilization frequency (40% to 81%; P=0.001) and AR (9% to 32%; P</=0.05) occurred with time of sperm exposure to heparin (15 sec to 6 hr) suggesting a direct effect of heparin on sperm. Glucose interfered with the effect of heparin on sperm. These data show heparin can prepare sperm for AR and fertilization in vitro and suggest that heparin-like material present in the female bovine reproductive tract may play a role in vivo in sperm capacitation and fertilization.

Journal Article↗
PubMed1985

Acrosome reaction of bovine spermatozoa in vivo: sites and effects of stages of the estrous cycle.

Thirty-two cows were inseminated near the uterotubal junction at various stages of the estrous cycle and slaughtered 16 h later to determine the effects of stage of the estrous cycle and tubal site of sperm recovery on the frequency of acrosome-reacted bull spermatozoa. Slaughter times were 46, 70, 144, or 168 h after each cow was injected with prostaglandin (PG) F 2 alpha or during the luteal phase of the estrous cycle. Sperm were recovered from the upper uterus and the isthmus and ampulla of the oviducts and stained for both viability and acrosome reaction. The highest frequency of acrosome-reacted sperm was found in the ampulla ipsilateral to a dominant follicle (largest follicle present) or recent ovulation and primarily at 70 h after PGF2 alpha (P less than 0.05). Also, fewer sperm were acrosome reacted prior to (46 h post-PGF2 alpha) and well after (168 h post-PGF2 alpha) estrus than during or immediately postestrus (70, 90, and 144 h post-PGF2 alpha; P less than 0.05). Except for two cows, one at 46 h and one at 70 h, all cows with more than 50% acrosome-reacted sperm in the ampulla had ovulated before slaughter. These data suggest that capacitated sperm become localized in the ampulla of the oviduct of the ovulatory side around the time of ovulation.

Acrosome↗
PubMed1985

Manipulation of gametes and embryos in the pig.

Several manipulation techniques including nuclear injections, nuclear transplantation, embryo splitting, chimaera production and sperm injection are discussed with special reference to their application in the pig. The nuclear injection technique is likely to be of greatest use for gene transfer. Gene transfer is feasible for pig embryos, but it is very inefficient. Efficiencies of various steps in the successful production of transgenic offspring as summarized from several published references are as follows: immediate survival of embryos following injection (60%), offspring produced from injected embryos (15%), offspring with an integrated foreign gene (24%) and offspring expressing the integrated foreign gene (60%). The overall efficiency is about 1%. Potential uses for nuclear transplantation are for the production of gynogenetic and androgenetic embryos and for cloning embryos. The feasibility of producing androgenetic and gynogenetic embryos will depend on whether in the pig, as in the mouse, the paternal and maternal genomes function differently. The feasibility of cloning by nuclear transplantation will depend upon the timing of developmental events in pig embryos and the development of methods for reprogramming nuclei. Pig embryos have been cloned by embryo splitting. Chimaera production and fertilization by sperm injection are likely to be feasible for pig embryos but have not yet found application.

Animals↗
PubMed1984

Developmental potential of isolated blastomeres from early murine embryos.

Experiments were designed to evaluate the effect of blastomere separation on blastocoele formation and development of viable fetuses. Two-cell and four-cell murine embryos were dissociated into individual blastomeres and cultured to the blastocyst stage. For embryos of both stages, zona removal and blastomere separation reduced (P<0.05) the number of viable embryos at the onset of culture and reduced (P<0.01) the frequency of continuation of development of blastomeres to the blastocyst stage. Attempts to repeatedly split two-cell stage embryos decreased in vitro development to blastocysts. The number of cells in two-cell embryos that were cultured to blastocyst was not different for control (64.8 +/- 11.5) or for two-cell embryos cultured without the zona pellucida (60.9 +/- 10.1) but was reduced (P<0.01) for one-half embryos that were cultured to blastocysts (35.6 +/- 10.6). The cell number of blastocysts obtained from dissociated four-cell (1/4) embryos (17.4 +/- 1.4) was similarly reduced (P<0.01). In vivo development was assessed after cultured embryos were transferred to the uteri of day 3 pseudopregnant females. Zona free intact embryos (2/36, 6%) and zona free half embryos (7/36; 19%) developed less frequently (P<0.05) than intact controls (45/100). Noncultured morula briefly exposed to pronase to thin the zona had similar impaired development. Embryos with thinned zona or no zona developed less frequently (21/82, 2/72 respectively, P<0.05) than nonpronase-treated controls (50/83).

Journal Article↗
PubMed1984

The effect of semen extension, cAMP and caffeine on in vitro fertilization of bovine oocytes.

A previously reported in vitro system that used epididymal spermatozoa for fertilizing bovine follicular oocytes (1) has been expanded to include ejaculated semen as the sperm source. Frequency of fertilization was higher when semen was extended 1:1 prior to transport to the laboratory rather than transport as neat semen. Pretreatment of spermatozoa with cAMP, caffeine or both prior to insemination of oocytes did not increase frequency of either acrosome reactions or fertilization after sperm/oocyte incubation.

Journal Article↗
PubMed1984

Role of calcium and the calcium-calmodulin complex in resumption of meiosis, cumulus expansion, viability and hyaluronidase sensitivity of bovine cumulus-oocyte complexes.

The necessity of calcium (Ca2+) and the Ca2+-calmodulin complex for resumption and completion of meiosis, expansion of cumulus cells, viability and hyaluronidase sensitivity of in vitro cultured bovine cumulus-oocyte complexes was examined by inhibition of the Ca2+-calmodulin complex with eight graduated doses of trifluoperazine (TFP) and by Ca2+ deficiency or depletion. Doses of TFP greater than 2.5 microM decreased the percent of cumulus complexes surviving culture and oocytes completing meiosis, whereas cumulus expansion was unaffected until the cultures contained a near lethal dose (greater than 10 microM). Hyaluronidase caused dispersion of cumulus cells whenever they were expanded regardless of TFP dose. In TC-199 media the completion of meiosis I was suppressed by 0.1 to 1 mM ethylenediaminotetraacetic acid (EDTA) (P less than 0.05) and drastically reduced by 1.0 mM (P less than 0.05). Viability of the cumulus-oocyte complex was not reduced until the dose of EDTA was increased to 1.0 mM (P less than 0.0001). Cumulus expansion was also not suppressed until the dose of EDTA reached 1.0 mM (P less than 0.05). In Ca2+-free (CF) basal media Eagles, completion of meiosis I was reduced by all doses of EDTA (P less than 0.05), whereas viability of the cumulus-oocyte complex was decreased by Ca2+ deficiency or by EDTA addition to basal media Eagles (P less than 0.01). Cumulus expansion was unaffected by Ca2+ removal or chelation. In all experiments, oocytes which were not degenerate underwent germinal vesicle breakdown regardless of treatment.

Animals↗
PubMed1984

Maturation and fertilization of bovine oocytes in vitro.

This report is to review and summarize various aspects of maturation and fertilization of bovine oocytes. Reference also is made to other species where pertinent data for the bovine are unavailable. Finally, factors important to success of in vitro maturation and fertilization as well as applications of these procedures to the animal industry are addressed.

Animals↗