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Biomedical subjects

U Besenfelder

Publications and source records attributed to U Besenfelder.

At least 19 recordsLinked to original sources
PubMed2005

In vivo culture of IVM/IVF embryos in bovine oviducts by transvaginal endoscopy.

This study was conducted to establish a new approach for in vivo culture of in vitro produced embryos in the bovine oviduct by transvaginal endoscopy. Embryos were in vitro matured, fertilized and cultured for 1-4 days and assigned to groups consisting of 10-30 embryos. Embryos were transferred unilaterally into oviducts of 24 heifers by the means of transvaginal endoscopy. After 3-6 days of in vivo incubation embryos were re-collected. Experiment I aimed to evaluate the capability of embryos to migrate to the uterus. The uterine horns of four animals were flushed first, followed by a combined flushing of both oviducts and uterine horns resulting in collection rates of 31 and 34%, respectively. In experiment II, the transfer of embryos into the oviduct close to ovulation (day 1-2--experiment IIA) or at a more advanced cyclic stage (day 3--experiment IIB) succeeded in the collection of 46 and 34% of the transferred complexes, of which 13 and 37% showed the blastocyst stage. This is the first report of successful recovery of transferable blastocysts by transvaginal endoscopy after tubal in vivo culture in the homologous species of originally in vitro produced embryos.

Animals↗
PubMed2004

Effect of morphological properties of transferred embryonic stages on tubal migration Implications for in vivo culture in the bovine oviduct.

In cattle, there is no practical method, which allows tubal transfer of pre-implantation embryos for routine in vivo culture as it has been established in sheep. The aim of our study was to perform tubal transfer by transvaginal endoscopy in synchronized heifers, in order to expose embryos at various embryonic stages to the physiological mechanisms of migration in the non-ligated oviducts. Various embryonic stages were transferred by transvaginal endoscopy into the oviducts of temporary recipients and were recovered on Day 7. The transfer of embryos in hyaluronate containing medium ("Hyaluronan"), zygotes stripped of cumulus ("Denuded Zygotes"), embryos embedded in cumulus ("Zygotes with Cumulus"), matured oocytes with capacitated spermatozoa ("GIFT") or embryos embedded in Na alginate ("Alginate") led to increasing recovery rates (13, 30, 56, 63 and 71%, respectively). However, the developmental rate on Day 7 was adversely affected (16, 11, 8, 16 and 8%), whereas the blastocyst rate on Day 8 showed more balanced results (17, 14, 18, 21 and 11%). Our data demonstrate that the structural properties of transferred embryos affect tubal migration and are crucial for subsequent in vivo culture. Embryos enclosed in cumulus cells or alginate synchronize more successfully with the oviductal transport systems than denuded stages or embryos in hyaluronate containing medium.

Alginates↗
PubMed2004

Intrafallopian transfer of gametes and early stage embryos for in vivo culture in cattle.

It may be possible to avoid inadequate in vitro culture conditions by incubating gametes or embryos in the oviducts for a short time. Ideally, an optimized procedure should be devised, combining in vitro and in vivo systems, in order to achieve synchronization in cattle. We transferred gametes as well as embryos in various stages of development and placed them into the oviducts. Embryos were recovered on Day 7 by flushing of oviducts and uterine horns. Blastocyst rates were determined on Day 7 and on Day 8. Experimental designs included transfer of in vitro matured cumulus oocyte complexes into previously inseminated heifers (COCs group), transfer of in vitro matured COCs simultaneously with capacitated spermatozoa (GIFTs group), transfer of four to eight cell stage embryos developed in vitro after IVM/IVF (Cleaved Stages group) and a group of solely in vitro produced embryos (IVP control group). Our results indicate that in vivo culture of IVM/IVF embryos in the homologous bovine oviduct has a positive influence on subsequent pre-implantation development. In addition, we have evidence that in vitro maturation and in vivo fertilization cannot be synchronized.

Animals↗
PubMed2004

The use of cytological examination of vaginal smears (CEVS) in the selection of rabbits for superovulation.

In the present study, we examined two factors associated with the reproduction of rabbit does, cytology of vaginal smears and color of vulva, as potential predictors of the success of superovulation treatment. Vulval color and vaginal smear cytology were assessed in 55 young New Zealand does. Superovulation was then induced by a single administration of eCG (20 IU/kg BW i.m.). Does were artificially inseminated 3 days later, followed by intravenous administration of hCG (120 IU per animal). Does were classified with regard to vulval color (white, rose, red, purple), and the predominant cell type in the vaginal smear (i.e. parabasal, intermediate, superficial, or anuclear). Furthermore, we categorized cells into two groups because we usually observed parabasal and intermediate cells (Group A), and superficial and anuclear cell (Group B) in the same smear. Does were humanely killed 19 h after administration of hCG and the total numbers of corpora lutea (CLs), oocytes, and zygotes (i.e. 1-cell embryos) were determined. The zygotes were assessed by morphological appearance and classified as normal or abnormal. The color of the vulva at the time of eCG treatment did not predict the success of superovulation in terms of the number of CLs, oocytes or zygotes. Does with predominantly superficial cells in vaginal smears yielded significantly fewer CLs and oocytes-zygotes (OZ) compared to does with predominantly parabasal, intermediate, or anuclear cells (P<0.05). Does with predominantly superficial cells in vaginal smears tended to yield fewer normal zygotes (nZ), but this reached significance only when compared to does with predominantly parabasal cells (P<0.05). Does in Group A yielded significantly more (P<0.05) CLs, OZ and nZ compared to does in Group B. Does with predominantly parabasal and intermediate cells in vaginal smears and rose color vulva tended to yield more OZ and nZ (P<0.05). These results suggest that the cytology of vaginal smears may help identify does with a significantly higher likelihood of yielding low numbers of CLs, oocytes, or nZ.

Animals↗
PubMed2003

Nucleolar proteins and ultrastructure in bovine in vivo developed, in vitro produced, and parthenogenetic cleavage-stage embryos.

In the present study, ribosomal RNA (rRNA) gene activation, monitored through nucleolus development, was studied by autoradiography following (3)H-uridine incubation, transmission electron microscopy, and immunofluorescence confocal laser scanning microscopy of key nucleolar proteins involved in rRNA transcription (topoisomerase I, upstream binding factor, and RNA polymerase I) and processing (fibrillarin, nucleolin, and nucleophosmin) in in vivo developed, in vitro produced, and parthenogenetic bovine embryos. In general, in vivo developed embryos displayed formation of fibrillo-granular nucleoli during the 4th post-fertilization cell cycle. During the previous stages of development, nucleolus precursor bodies (NPBs) were observed. However, on some occasions the initial steps of nucleolus formation were observed already at the 2- and 4-cell stage in cases where such embryos were collected from superovulated animals together with later embryonic stages presenting nucleolar development and autoradiographic labeling. The in vitro produced embryos displayed very synchronous formation of fibrillo-granular nucleoli and autoradiographic labeling during the 4th cell cycle. In vivo developed and in vitro produced embryos displayed allocation of nucleolar proteins to fibrillar and granular compartments of the developing nucleoli during the 4th cell cycle. The parthenogenetic embryos typically displayed formation of fibrillo- granular nucleoli during the 5th cell cycle and autoradiographic labeling was not observed until the morula stage. Moreover, the 1-, 2-, and 4-cell parthenogenetic embryos practically lacked NPBs. On the other hand, parthenogenetic embryos displayed allocation of nucleoar proteins to nuclear entities during the 4th cell cycle. In conclusion, both in vivo developed and in vitro produced bovine embryos displayed activation of transcription and nucleolar development during the 4th cell cycle. However, in vivo developed embryos flushed together with later developmental stages displayed premature activation of these processes. Parthenogenetic bovine embryos, on the other hand, displayed a delayed activation.

Animals↗
PubMed2002

Expression and characterization of functional recombinant bovine follicle-stimulating hormone (boFSHalpha/beta) produced in the milk of transgenic rabbits.

Bovine follicle-stimulating hormone (boFSH) is a heterodimeric glycoprotein that belongs to the pituitary gonadotropins. Bioactive FSH is composed of alpha and beta subunits which require extensive N-glycosylation and sialylation. The mammary gland of transgenic livestock is an attractive source for the synthesis of post-translationally modified proteins. Two mammary gland-specific gene constructs with the cDNA for the boFSH alpha (boFSHalpha) and beta (boFSHbeta) subunits controlled by bovine alpha-s1 casein regulatory sequences were co-microinjected into fertilized rabbit oocytes. Two FSHalpha/FSHbeta double transgenic rabbit lines were established. The transgene expression was strictly lactation and mammary gland specific. Protein analysis revealed the presence of the boFSH heterodimer in the milk of transgenic rabbits showing a molecular weight similar to that of purified pituitary gland derived boFSH (boFSH-P). Subunit specific antibodies detected both polypeptides with the expected molecular sizes. Biochemical characterization demonstrated the expected isoelectric points of the recombinant boFSH. The presence of the post-translationally added terminal sialic acid residues was indicated by wheat germ agglutinin (WGA) lectin Western blotting. The biological activity of the recombinant mammary gland produced boFSH was determined using a FSH-dependent reporter cell line. The bioactivity of the recombinant boFSH was comparable to that of purified boFSH-P.

Animals↗
PubMed2002

Measurement of cortisol metabolites in faeces of ruminants.

Twenty-one metabolites were detected in faecal samples collected after infusion of (14C)cortisol into the jugular vein of sheep. Using high-performance liquid chromatography/radiometric analysis plus mass spectrometry. One group of metabolites had molecular weights of between 302 and 308, and another group of 350, which indicates that the substances have a C19O3 or a C21O4 structure. Therefore, an enzyme immunoassay against 5beta-androstane-3alpha-ol-11,17-dione-17-CMO:BSA was established. Faecal samples were collected from 10 cows immediately after transport and then during a course in which non-invasive diagnostic procedures were being taught (course 1). For comparison, faeces were sampled from another 5 cows that were being used for teaching invasive procedures (course 2). Six cows from a university farm served as controls. In the animals used in course 1, the highest concentrations of cortisol metabolites were measured immediately after transport to the university (median value: 2.2 micromol/kg faeces). During the first 5 days at the university, the concentrations decreased to 0.52 micromol/kg (median) and remained at this level during the rest of the course. The median concentration in the samples that were taken during coursc 2 (collected about 2 months after transport) was 0.48 micromol/kg. There was no significant difference in the excretion of cortisol metabolites between these cows and the controls. We conclude from these data that, using the enzyme immunoassay against 5beta-androstane-3alpha-ol-11,17-dione-17-CMO, we were able to detect transport/novel environment stress but not the potential disturbance that cows experience during diagnostic procedures.

Animals↗
PubMed2001

Fertilization and embryo recovery rates in superovulated chios ewes after laparoscopic intrauterine insemination.

Forty superovulated dairy ewes of the Greek Chios breed were used in an experiment to evaluate the efficiency of laparoscopic intrauterine insemination on fertilization and embryo recovery rates as well as embryo quality. Estrus was synchronized by intravaginal progestagen impregnated sponges and superovulation was induced by administration of 8.8 mg o-FSH i.m. following a standard 8 dose protocol. A small volume (0.3 mL) of diluted fresh ram semen was deposited in each uterine horn 24 to 28 h after onset of the estrus by a laparoscopic technique. The animals were allocated randomly into two groups (Group A and B) of 20 animals each. In Group A, embryos were recovered 18 to 24 h after the intrauterine insemination and in Group B on Day 6. The average number of corpora lutea was 12.8 +/- 1.2 and 11.5 +/- 1.1 (+/- SEM); the overall embryo recovery was 66.4% and 57% and the percentage of recovered fertilized ova was 81% and 82.8% in Groups A and B, respectively. More fertilized ova were collected per ewe from Group A (P < or = 0.1). Results indicated that in Chios breed, superovulation using homologous FSH combined with laparoscopic AI leads to good ovarian response with satisfactory results in fertilization, embryo recovery and quality of embryos. This could lead to improved and more efficient methods for obtaining large numbers of high quality oocytes and embryos for embryo transfer programs which could contribute to genetic improvement and increase of the population size.

Animals↗
PubMed2001

Collection of tubal stage bovine embryos by means of endoscopy. A technique report.

Here we describe the development and optimization of endoscopy-mediated transvaginal access for collecting ova and embryos from the bovine oviduct. The novel technique was developed in three experimental setups: In Experiment 1 embryos were collected unilaterally from nonstimulated heifers. We flushed the oviducts of superovulated heifers unilaterally (Experiment 2) and bilaterally (Experiment 3). In Experiment 1 the oviducts of 18 heifers were successfully cannulated, which resulted in the collection of twelve 1-cell to 8-cell embryos and one empty zona. Unilateral flushing of 13 animals (Experiment 2) resulted in 84 ova with 6.3 +/- 3.2 observed ovulation sites. Bilateral flushing of 25 animals (Experiment 3) resulted in 293 ova plus 10 empty zonae from 11.8 +/- 5.4 ovulation sites. Given our experience from these studies we optimized the technical equipment by improving the flushing metal catheter (Experiment 4). The novel catheter hermetically sealed the lumen of the ampulla at the moment, the medium was flushed through the oviduct. This resulted in a visible flow of medium via oviducts toward the embryo filter connected to an embryo flushing catheter that was fixed in the uterine horns. Our endoscopy-guided method is minimally invasive and facilitates the flushing of tubal stage embryos.

Animals↗
PubMed2001

Ribosomal RNA gene expression and chromosome aberrations in bovine oocytes and preimplantation embryos.

This review focuses on the key features of development of the bovine oocyte and embryo, with comparisons of the developmental characteristics of embryos produced in vivo and in vitro. The oocyte is transcriptionally quiescent in the primordial and primary follicle. In the secondary follicle transcription is initiated in the oocyte and a ribosome-synthesizing nucleolus is established in this cell. Transcription and nucleolar activity are enhanced in the tertiary follicle during oocyte growth. When the oocyte reaches approximately 110 microm in diameter, corresponding to a follicle of about 3 mm in diameter, transcription ceases and the nucleolus is inactivated, forming a dense spherical remnant. During the final phase of follicular dominance this remnant becomes vacuolated and, in conjunction with resumption of meiosis, it disperses. The rRNA genes are apparently re-activated during the four-cell stage, that is, the third cell cycle after fertilization, but a nucleolus is not formed. During the subsequent cell cycle, that is, during the eight-cell stage, ribosome-synthesizing nucleoli are again established. Bovine embryos produced in vitro apparently display the same pattern of nucleolus development as that in embryos developed in vivo. Examination of the ploidy of embryonic cells using fluorescence in situ hybridization has revealed that the production of bovine embryos in vitro is associated with increased chromosome aberrations in the embryos. Blastocysts produced in vitro display a significantly higher rate of mixoploidy, that is, when the embryo consists of both normal diploid and abnormal polyploid cells, than that in embryos developed in vivo. The rate of mixoploidy among embryos produced in vitro increases with increasing developmental stage. Moreover, after fertilization in vitro, initially there is a high rate of 'true' polyploidy, that is, when all cells of the embryos are polyploid. However, the polyploid embryos are eliminated before they cleave beyond the eight-cell stage, the stage at which major activation of the embryonic genome occurs in cattle.

Animals↗
PubMed1999

Species-specific alternative splicing of transgenic RNA in the mammary glands of pigs, rabbits, and mice.

Gene-farming techniques provide an effective tool for the production of recombinant proteins in livestock. Transgenes consisting of genomic DNA sequences are as a rule more efficiently expressed than those in which the product of interest is encoded by a cDNA. However, the processing of pre-mRNA from genomic constructs may yield unexpected messenger RNAs and subsequently protein variants. We describe the appearance of different alternative mRNA splice patterns of a gene construct in which a mutant human growth hormone (hGH-N) gene is transcriptionally controlled by 2.5 kb of mouse whey acidic protein (WAP2) regulatory sequences in the mammary gland of different livestock species. Compared to the transcription products in transgenic mice harboring the same gene construct and to cell transfection experiments, expression analysis in transgenic pigs and rabbits revealed different mRNA splice patterns with regard to the proportion of the processed transcripts. Apart from already-known physiological mRNA splice products, previously undescribed processed hGH transcripts were observed in these species. Sequence analysis of the transgenes suggests that the species-specific hGH mRNA patterns may be caused by species- and tissue-specific differences in trans-acting splice factors.

Alternative Splicing↗
PubMed1999

Human nerve growth factor beta (hNGF-beta): mammary gland specific expression and production in transgenic rabbits.

Transgenic rabbits carrying gene constructs encoding human nerve growth factor beta (hNGF-beta) cDNA were generated. Expression of hNGF-beta mRNA was restricted to the mammary gland of lactating rabbits. Western Blot analysis revealed a polypeptide of 13.2 kDa in the milk of transgenic animals. hNGF-beta was purified from the milk by a two-step chromatographic procedure. Electrospray mass spectroscopy analysis of purified hNGF-beta depicted a molecular weight of 13,261 Da per subunit. The biological activity of the hNGF-beta was tested using PC12W2 cells and cultures of dorsal root ganglion neurons from chicken embryos. Crude defatted milk from transgenic animals and purified hNGF-beta demonstrated full biological activity when compared to commercial recombinant hNGF-beta.

Animals↗
PubMed1999

The 2.3 kb smooth muscle myosin heavy chain promoter directs gene expression into the vascular system of transgenic mice and rabbits.

BACKGROUND: Smooth muscle cells (SMC) are a preferential target for gene therapeutic approaches in atherosclerosis and restenosis. However, the undesirable expression of putative therapeutic genes in tissues other than the vascular wall is a considerable safety limitation for clinical trials, thus requiring the identification of a smooth-muscle-specific promoter sequence. Since the 2.3 kb rabbit Smooth Muscle Myosin Heavy Chain (SMHC) promoter was shown to be transcriptionally active in primary vascular but not visceral or other non-SMC in vitro, this fragment was chosen for in vivo analysis. METHODS AND RESULTS: Transgenic mice and rabbits were established expressing a luciferase reporter gene under control of the 2.3 kb rabbit SMHC promoter. In contrast to the endogenous expression pattern of the SMHC gene both species revealed light emission predominantly in the arterial system including coronary arteries. Low activities were measured in large veins and the gastrointestinal system. In situ hybridization of murine embryos using a luciferase riboprobe confirmed reporter gene expression in large arteries with no detectable mRNA in the viscera. Unlike adult animals, ectopic luciferase activities were found in ventricular myocardium during murine development ceasing 1 week post partum. CONCLUSIONS: In two animal species, the 2.3 kb SMHC promoter appeared to be effective in discriminating between the pathways regulating vascular and visceral smooth muscle gene expression. The vascular-specific expression profile of the 2.3 kb SMHC promoter suggests that the 2.3 kb SMHC promoter contains the regulatory elements necessary for selective gene targeting into vascular SMC of large arteries including coronary arteries in vivo.

Animals↗
PubMed1999

Repeated endoscopic ovum pick-up in sheep.

Endoscopy is an effective and minimally invasive technique which offers the possibility of repeated ovum pick-up (OPU). In this study 4 different treatment programs (Groups A, B, C and D) for repeated endoscopic OPU in sheep were investigated. The number of follicles and oocytes, quality of cumulus-oocyte-complexes (COCs), and detectable effects on fertility of the donor ewes were compared. Each group consisted of 5 East Friesian Milksheep. In Group A, follicles were punctured twice a week, in Group B once a week, and in Group C once a week followed by administration of 1500 IU PMSG 48 h prior to OPU. In Group D follicles were punctured and the sheep stimulated with 1500 IU PMSG 48 h prior to OPU once every 2 weeks. The PMSG-stimulated sheep received anti-PMSG immediately after OPU. Over a period of 10 weeks 216 OPU-sessions were performed. A total of 1978 follicles was punctured, and 1098 oocytes were recovered, for a collection rate of 55.5%. In the Groups A, B, C and D an average of 6.8, 8.6, 12.2 and 14.9 follicles per animal and session was aspirated, and an average of 3.8, 4.9, 7.0 and 7.6 COCs per animal per session was recovered, respectively. No significant differences between groups were observed in the collection rates (51.1 to 57.1%) or in the quality of the COCs, and 65 to 70% of the COCs were suitable for in vitro production of ovine embryos. Seven sheep developed small adhesions between the ovary and infundibulum. After the study 15 ewes became pregnant following natural mating with the same fertile ram (5 from Group A, 1 from Group B, 4 from Group C and 5 from Group D). In conclusion, OPU once a week in PMSG/anti-PMSG treated ewes was found to be the most effective treatment program for oocyte collection.

Animals↗
PubMed1998

Tubal transfer of bovine embryos: a simple endoscopic method reducing long-term exposure of in vitro produced embryos.

Although numerous trials had shown the need to define a procedure to get free access to the bovine oviduct, there was no adequate report of a technique which was accepted for the routine transfer of early tubal-stage embryos. We have now report an endoscopically mediated transvaginal method for transferring embryos into the oviduct. The in vitro produced embryos were loaded into a curved glass capillary tube which was connected to a perfusor tube plus 1-mL syringe. The capillary tube was directly inserted via the infundibulum into the ampulla. After first having checked the ovaries for the presence of a corpus luteum the embryos were deposited under visual guidance in about 20 to 50 microL medium. Twenty-four Simmental and Brown Swiss heifers received 26 embryos and 9 animals became pregnant, of which 7 recipients delivered 8 live calves. With practice, the time used for endoscopic transfer was reduced to less than 10 min. The results demonstrate that the described technique is suitable for practical application. Especially for the early transfer of IVP-derived embryos this technique might be advantageous. In conclusion, this method is also of great potential interest for the recovery of tubal-stage embryos and for the in vivo culture of embryos followed by conventional flushing at Day 7.

Animals↗
PubMed1998

Laparoscopic recovery of pronuclear-stage goat embryos.

The oviducts of 16 Saanen does, superovulated with follicle-stimulating hormone (FSH) and synchronised with prostaglandin F2 alpha were flushed 75 to 86 hours after the injection of prostaglandin. The mean (sd) ovulation rate was 13.7 (3.9). The flushings were directed orthograde through a flexible intravenous catheter, which was introduced into the oviduct via the infundibulum. The flushing medium was recovered by a balloon-catheter, which was placed in the uterine lumen near the uterotubal junction. Five does were flushed unilaterally either because they had one blocked oviduct or because they had ovulated on only one ovary. The overall embryo recovery rate was 72 per cent. Nine weeks later 11 of the donor ewes were examined laparoscopically and no adhesions of the reproductive organs were observed. Eight of these does were synchronised with progestagen-vaginal sponges, superovulated with FSH and their oviducts were flushed again. Their mean ovulation rate was 16.0 (4.3) and 86 per cent of the embryos were recovered. The optimal time to obtain pronuclear stage embryos was 75 to 78 hours after the injection of prostaglandin. All the embryos recovered within this period were at the pronuclear stage whereas 28 per cent of those recovered one to six hours later were at the two- or four-cell stage.

Animals↗
PubMed1998

Stable production of human insulin-like growth factor 1 (IGF-1) in the milk of hemi- and homozygous transgenic rabbits over several generations.

One transgenic rabbit line was generated carrying a fusion gene consisting of the cDNA for human IGF-1 fused to a mammary gland specific expression cassette derived from bovine alpha-S1-casein sequences. Transgene expression was shown to be strictly tissue and lactation period specific. The transgenic rabbit line was bred for six generations. All transgenic animals showed stable production of biologically active IGF-1 over the generations and no apparent effect on the physiological or reproductive performance was observed. The absence of adverse effects on homozygous transgenic rabbits suggested the absence of insertional mutagenesis. Eight hemizygous transgenic offspring analysed produced on average 363 +/- 12 micrograms/ml (ranging from 223 +/- 61 to 484 +/- 39 micrograms/ml) mature human IGF-1 in their milk, whereas three homozygous animals produced on average 543 +/- 41 micrograms/ml (ranging from 360 +/- 15 to 678 +/- 80 micrograms/ml). Homozygous hulGF-1 females clearly showed a significantly increased production performance of the recombinant protein.

Animals↗