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Biomedical subjects

D Rath

Publications and source records attributed to D Rath.

At least 19 recordsLinked to original sources
PubMed2007

Altered mRNA expression patterns in bovine blastocysts after fertilisation in vitro using flow-cytometrically sex-sorted sperm.

Sperm-sexing has been used to produce embryos and offspring of a pre-determined sex in a number of species. However, the fertility of sex-sorted sperm is reduced and the full effects of sperm-sexing remain to be elucidated. The purpose of the present study was to investigate the potential effects of sex-sorted sperm on mRNA expression patterns of developmentally important genes employing in vitro produced bovine embryos. Bovine embryos were produced in vitro with unsorted and sex-sorted sperm and mRNA expression patterns were determined for glucose-3 transporter (Glut-3), glucose-6-phosphate dehydrogenase (G6PD), X-inactive specific transcript (X-ist) and Heat shock protein 70.1 (Hsp) using semi-quantitative endpoint reverse transcriptase-PCR in male and female, day-7 and 8 embryos. The relative abundance (RA) of Glut-3 was higher for day-7 male than female embryos, and day-7 embryos derived from unsorted compared with sex-sorted sperm. The RA of G6PD was higher for embryos derived from unsorted than sex-sorted sperm, and for day-8 female compared with male embryos. The RA of Xist was higher for female than male embryos, and for day-7 female embryos derived from unsorted than sex-sorted sperm. Hsp RA was higher for female compared with male embryos, was similar for day-7 and 8 embryos, and unsorted and sex-sorted sperm derived embryos. These results demonstrate differential expression of developmentally important genes between male and female embryos, and embryos derived from unsorted and sex-sorted sperm.

Animals↗
PubMed2007

Insemination with sex sorted fresh bovine spermatozoa processed in the presence of antioxidative substances.

Flow cytometrically sex sorted spermatozoa are reduced in their fertilizing capacity, particularly when stored either in cooling extender or after freezing in liquid nitrogen. So far, preservation methods for sorted spermatozoa have differed only marginally from procedures used for unsorted semen. In the present study, a TRIS extender was modified to balance major cell damage caused by the sorting process and by liquid storage of the sorted spermatozoa. The new extender, containing a combination of antioxidants (AO) and bovine serum albumin (BSA), significantly increased the lifespan and fertilizing capacity of sex sorted spermatozoa. No significant differences were observed between unsorted controls and sorted samples for motility and status of sperm membranes as tested by fluorescein-isothiocyanat-peanut agglutinin/propidium iodide (FITC-PNA/PI). Acrosome integrity of spermatozoa was significantly better when semen was stored at 15 degrees C for 24 and 48 h in an extender containing AO with or without BSA as compared with controls (p < 0.05). There were no significant differences, in pregnancy rates of heifers inseminated at a natural oestrus, between unsorted controls (16/24, 66.7%) and both sorted groups (AO + BSA: 18/31, 58.1% and AO-BSA: 12/22, 54.5%). Additionally, it was shown for the first time that artificial insemination (AI) with liquid sexed bull spermatozoa stored for 72 h after sorting can result in pregnancy rates similar to AI with non-sorted semen.

Acrosome↗
PubMed2007

Reduction of oxidative stress in bovine spermatozoa during flow cytometric sorting.

The goal of the study was to investigate the effect of antioxidant supplementation on the quality of frozen-thawed flow cytometrically sorted bull spermatozoa. Twelve ejaculates from two Holstein Friesian bulls were sorted according to the Beltsville Sperm Sexing Technology. Each ejaculate was divided into three parts and processed as (i) unsorted controls, (ii) according to a standard sorting protocol and (iii) in the presence of different antioxidants (S-AO). Cooling and freezing of the samples were performed in the same way for all three groups, except that antioxidants were added to the TRIS-egg-yolk freezing extender for those semen samples that were already sorted in the presence of antioxidants. The semen quality in frozen-thawed samples was determined by morphology analysis immediately after thawing, motility estimation in a thermo-resistance test after 0, 6, 12 and 24 h incubation at 37 degrees C and Fluorescein isothiocyanate conjugated PNA/propidium iodide (FITC-PNA/PI) staining after 0, 12 and 24 h of incubation at 37 degrees C. There was a significantly higher (p < 0.05) percentage of motile spermatozoa in S-AO samples in comparison to unsorted frozen-thawed control at 0, 6 and 24 h after thawing and compared with normally sorted samples at all times after thawing. The percentage of damaged acrosomes was significantly lower (p < 0.05) in S-AO samples than in the unsorted controls (20.8 +/- 6.9% vs 30.3 +/- 12.0%). The percentage of morphologically abnormal spermatozoa in this group was significantly lower (p < 0.05) than in the unsorted controls and normally sorted samples (25.8 +/- 5.2%, 36.0 +/- 12.5% and 35.1 +/- 7.4%, respectively). Analysis of frozen-thawed spermatozoa with FITC/PI revealed no significant difference in membrane integrity at 0 and 12 h after sorting, but after 24 h of incubation the S-AO samples had a significantly higher (p < 0.001) percentage of spermatozoa with intact membranes in comparison to unsorted controls and normally sorted semen (40.7 +/- 6.3%, 7.8 +/- 4.7% and 7.4 +/- 4.6%, respectively). The percentage of acrosome-reacted spermatozoa was significantly lower (p < 0.05) in the S-AO samples than in the unsorted controls (14.1 +/- 7.5%, 23.4 +/- 5.4% and 28.8 +/- 6.3% vs 25.9 +/- 14.4%, 38.5 +/- 16.7% and 79.8 +/- 4.1%, for 0, 12 and 24 h after thawing, respectively) and in comparison to normally sorted semen 24 h after thawing (67.3 +/- 10.0%). This study demonstrates the highly protective effects of antioxidants on the quality of flow cytometrically sorted frozen-thawed bull spermatozoa.

Acrosome↗
PubMed2006

Utilization of frozen-thawed epididymal ram semen to preserve genetic diversity in Scrapie susceptible sheep breeds.

The European Union has introduced transmissible spongiform encephalopathy (TSE) resistance breeding programmes for several sheep breeds to cope with the genetic susceptibility to Scrapie infections. Due to the different allele frequencies among breeds, strong selection for ARR alleles is associated with a loss of genetic diversity in small populations and in larger populations with unfavourable ARR allele frequencies. To ensure maintenance of genetic diversity, an adhoc cryopreservation programme was initiated employing epididymal sperm from 109 rams representing 16 different breeds within one breeding season. Epididymal semen was chosen for this adhoc programme because time consuming training of rams for ejaculated semen collection via an artificial vagina was not possible. Prior to freezing, average sperm motility was 79.7% and acrosome integrity was 93.7%. After freezing, these levels were decreased to 60.5 and 72.8%, respectively. An insemination trial using frozen-thawed epididymal semen resulted in a lambing rate of 87.5%. Results show that this semen preservation method is robust and efficient and associated with high fertility. It may also be useful for other animal species.

Acrosome↗
PubMed2006

Non-surgical deep intra-uterine transfer of in vitro produced porcine embryos derived from sex-sorted frozen-thawed boar sperm.

Embryos and offspring of a pre-determined sex have been produced in pigs using AI and IVF with unfrozen sperm, and after surgical insemination with sex-sorted frozen-thawed sperm. The aims of this study were to demonstrate that sex-sorted frozen-thawed boar sperm could be incorporated into pig IVF for the production of embryos of a pre-determined sex and that these embryos could be successfully non-surgically transferred. Oocytes were matured in vitro, fertilised with either unsorted or sex-sorted frozen-thawed sperm and cultured until the eight-cell stage. These embryos were then transferred to recipients (n = 7) non-surgically (n = 70 embryos per sow). Oocyte cleavage was similar between sex-sorted (1538/5044; 30.5%) and unsorted (216/756; 28.6%) frozen-thawed sperm, and PCR sex-determination of the embryos confirmed that they were of the predicted sex (n = 16). Delayed return to oestrus (>23 days) was observed in five recipient sows (71.4%). Fetal sacs were observed by transcutaneous ultrasound on Day 18 in one of these sows. Pre-sexed porcine IVP embryos can be successfully produced using sex-sorted frozen-thawed boar sperm, and these embryos are capable of initiating pregnancies when transferred to recipients. However, further refinement of porcine ET protocols are required to enable development to term.

Animals↗
PubMed2006

Structural, biochemical and functional aspects of sperm-oocyte interactions in pigs.

Polyspermic fertilization is still a major issue in porcine IVF systems. New information is available to characterize the zona pellucida (ZP) at different developmental stages by scanning electron microscopy (SEM) and by confocal microscopy to show the distribution of ZP glycoproteins. SEM images indicated no differences between in vivo and in vitro matured oocytes; however a change in the surface structure between immature and matured oocytes, as well as between mature oocytes and preimplantation embryos was obvious. In addition, spermatozoa were more tightly fixed in the ZP of in vivo produced compared to the ZP of in vitro produced embryos. The ZP undergoes biochemical changes during maturation prior to fertilization. The acidity of the ZP increases during maturation as indicated by a shift of 1.3 pl units for ZPB/ZPC and 0.8 pl units for ZPA in 2D gel electrophoresis, which is based on increasing sulfation of the oligosaccharides during maturation. Mass spectrometry in combination with in-gel deglycosylation allowed the mapping of new glycosylation sites. Functionality of the ZP also depends on its maturation status. Induction of the acrosome reaction was delayed when capacitated spermatozoa were exposed to immature oocytes.

Animals↗
PubMed2005

Effect of oviductal and cumulus cells on zona pellucida and cortical granules of porcine oocytes fertilized in vitro with epididymal spermatozoa.

The objective of this study was to evaluate the effects of porcine oviductal epithelial cell (POEC) monolayers and cumulus cells on the zona pellucida (ZP) and cortical granules (CG) of in vitro matured porcine oocytes. Denuded and cumulus-enclosed oocytes were exposed to POEC before or during in vitro fertilization (IVF). The functional effects of the co-culture system were the tested on the ZP resistance, measured by the time necessary to dissolve the ZP with 0.1% pronase, and the distribution and density of the cortical granules. CG density in the equator and cortex of each oocyte was evaluated by confocal microscopy after staining with fluorescein isothiocyanate-labelled peanut agglutinin (FITC-PNA). Both variables were assessed immediately after an in vitro maturation period (IVM group), 3 and 6h after culture with or without (Control) oviductal cells (Experiment 1) and 3h after insemination with frozen-thawed epididymal spermatozoa in the presence or absence (Control) of oviductal cells (Experiment 2). The time to dissolve the ZP of oocytes from IVM group was 440.4 +/- 61.7 s and no difference was observed among groups in Experiment 1. In contrast, the density of CG was affected; oocytes pre-incubated for 6h had a higher density than those pre-incubated for 3 h (P <0.001). Oocytes fertilized in vitro in the presence of POEC (Experiment 2) had a similar ZP digestion time as control oocytes 3 h after insemination. The presence of POEC during IVF as well as the presence of cumulus cells had no effect on the density and distribution of CG. However, a significant decrease in the density of CG was observed in the fertilized oocytes compared to in vitro matured oocytes (P <0.001). It is concluded that under the conditions employed the oviductal and cumulus cells in the perifertilization period had no effect on ZP hardening and CG density. However, an increase in CG density was observed when oocytes were maintained in culture. In addition, no hardening of ZP was observed after IVF, and denuded and cumulus-enclosed oocytes showed similar cortical reactions after insemination with epididymal spermatozoa regardless of the presence of POEC.

Animals↗
PubMed2005

First established pregnancies in Mediterranean Italian buffaloes (Bubalus bubalis) following deposition of sexed spermatozoa near the utero tubal junction.

At the time of AI following Ovsynch protocol, a total of 51 buffaloes were randomly divided in a first group (n = 30) subjected to conventional AI into the uterine body with 20 million non-sex sorted frozen-thawed spermatozoa, while a second group (n = 21) was inseminated near the utero-tubal junction (UTJ) ipsilateral to the ovary carrying the preovulatory follicle with 2.5 million live (4 million total) sex-sorted frozen-thawed spermatozoa. The semen used for flowcytometric sorting was collected and processed on a farm in Italy, and then shipped to a laboratory in Germany. Eleven buffaloes were inseminated with X-chromosome bearing spermatozoa and 10 with Y-chromosome bearing spermatozoa. Conception rates after conventional and UTJ inseminations were 43.3% (n = 13) and 42.8% (n = 9) respectively (p = 0.97). Eight of the nine foetuses obtained after insemination with sexed spermatozoa corresponded to the sex as predicted by the cell sorting procedure (five male and four female foetuses by ultrasound vs six male and three female foetuses by cell sorting). In conclusion, for the first time buffalo semen has been successfully subjected to procedures for flowcytometric sperm sorting and freezing. Low doses of sexed spermatozoa have been deposited near the UTJ giving conception rates similar to those of conventional AI with full dose.

Animals↗
PubMed2005

Susceptibility of in vivo- and in vitro-produced porcine embryos to classical swine fever virus.

The objective of this study was to investigate the susceptibility of in vivo- and in vitro-produced (IVP) porcine embryos to classical swine fever virus (CSFV). IVP zona pellucida (ZP)-intact porcine embryos (n = 721) were co-cultured with CSFV for 120 h. After washing according to the International Embryo Transfer Society guidelines (without trypsin) and transferring embryos to CSFV-susceptible porcine kidney cells (PK15 cell line), no virus was isolated. However, when 88 IVP ZP-intact porcine embryos were co-cultured with CSFV for only 48 h before being transferred to PK15 cells, virus was isolated in three of six replicates. Similarly, 603 in vivo-produced porcine embryos were co-cultured with CSFV for 120 h. Subsequently, CSFV was isolated in eight of 50 groups (16%) and the ability of these to form a blastocyst was significantly reduced when compared with the control group (68.2 +/- 19.9% vs 81.9 +/- 9.7%; p < or = 0.001). In contrast, the development of CSFV-exposed IVP porcine embryos was not affected when compared with control embryos (19.1 +/- 10.8% vs 18.9 +/- 10.6%; p > or = 0.05). After removal of the ZP of IVP embryos and subsequent co-culture with CSFV, the virus was isolated from all groups of embryos. These data suggest that virus replication had occurred in the embryonic cells. In conclusion, data indicate that in vivo- and in vitro-produced ZP-intact porcine embryos differ in their susceptibility to CSFV. Hatched or micro-manipulated embryos may increase the risk of transmission of CSFV by embryo transfer, which has to be confirmed by in vivo tests under isolation conditions.

Animals↗
PubMed2004

Production of piglets with sexed semen employing a non-surgical insemination technique.

The aim of the present study was to ascertain whether multiparous sows could successfully be inseminated with sexed semen non-surgically. Spermatozoa were stained with Hoechst 33342 and separated flowcytometrically in X- and Y-chromosome bearing sperm populations employing the Beltsville Sperm Sexing Technology (BSST). After weaning, estrus was induced in sows with PMSG and hCG. Animals were inseminated once per estrus non-surgically with a specially designed catheter into the tip of the uterine horn, employing 50x10(6) of either sexed or non-sexed spermatozoa diluted in 2 ml Androhep. Pregnant sows were allowed to go to term. Mean pregnancy rate from inseminations with unsexed spermatozoa was 54.5% whereas inseminations with sexed spermatozoa resulted in 33.3% pregnant sows. All but one piglet born after insemination with sexed semen were of the predicted sex. The sex of those piglets born after inseminations with non-sexed spermatozoa was 61.1% for male and 38.9% for female sex. It is concluded that non-surgically inseminations with flowcytometrically sexed spermatozoa can be conducted successfully.

Animals↗
PubMed2004

Nucleolar ultrastructure and protein allocation in in vitro produced porcine embryos.

The nucleolus formation was studied as an indirect marker of the ribosomal RNA (rRNA) genes activation in porcine embryos following oocyte maturation, fertilization, and culture in vitro. Nucleologenesis was assessed by transmission electron microscopy (TEM), light microscopical autoradiography following 20 min of 3H-uridine incubation, and immunocytochemical localization of key nucleolar proteins involved in rRNA transcription (upstream binding factor (UBF), topoisomerase I, and RNA polymerase I) and processing (fibrillarin, nucleophosmin, nucleolin) by confocal laser scanning microscopy. During the first four post-fertilization cell cycles, TEM revealed spherical nucleolus precursor bodies (NPBs), consisting of densely packed fibrils, as the most prominent intra-nuclear entities of the blastomeres. Fibrillo-granular nucleoli were observed in some blastomeres in a single embryo during the 5th cell cycle, i.e., the tentative 16-cell stage, where formation of fibrillar centres (FC), a dense fibrillar component, and a granular component on the surface of the NPBs was seen. In this embryo, autoradiographic labeling was detected over the nucleoplasm and in particular over the nucleoli. Fibrillarin was immunocytochemically localized in the presumptive NPBs of the pronuclei. This protein was again localized to the presumptive NPBs together with nucleolin from late during the 3rd cell cycle, i.e., the four-cell stage in some embryos. UBF, RNA polymerase I, and nucleophosmin were localized to the presumptive NPBs in a proportion of the embryos at the 4th cell cycle, i.e., the tentative eight-cell stage and onwards. Toposiomerase I was not localized to intra-nuclear entities even during the 5th post-fertilization cell cycle. Moreover, a considerable proportion of the blastomere nuclei apparently did not show localization of other nucleolar proteins. In conclusion, porcine embryos produced in vitro display a substantial delay in or even lack of the development of functional nucleoli.

Animals↗
PubMed2003

Effects of oviductal and cumulus cells on in vitro fertilization and embryo development of porcine oocytes fertilized with epididymal spermatozoa.

This study was designed to evaluate the effects of adding porcine oviductal epithelial cell (POEC) monolayers before or during the fertilization of denuded or cumulus-enclosed oocytes, in terms of fertilization results and subsequent embryo development. The variables determined were: penetration rate, mean number of spermatozoa per oocyte, male pronucleus formation rate, monospermy rate, cleavage rate after 48 h of fertilization, blastocyst rate, and mean number of nuclei per blastocyst. We used cumulus-free and cumulus-enclosed oocytes preincubated or fertilized in the presence of POEC, once the purity in epithelial cells of these cultures had been assessed. All the experiments involved the use of frozen-thawed epididymal spermatozoa to avoid replicate variability. The POEC cultures prepared showed a high proportion of epithelial cells (over 95%). Preincubation of oocytes with POEC before fertilization showed no effects on the fertilization variables determined. In contrast, during IVF under our experimental conditions, these cells attached to the cumulus cells and their interaction had a significant effect on some of the fertilization variables analyzed. The presence of POEC and cumulus cells during IVF increased oocyte penetrability. Moreover, in the absence of POEC, cumulus cells resulted in a reduced monospermy rate. On subsequent embryo culture, a lower cleavage and blastocyst formation rate were recorded when the oocytes had been preincubated with POEC before IVF.

Animals↗
PubMed2003

Advances in biotechnology: new tools in future pig production for agriculture and biomedicine.

Biotechnology in livestock comprises an arsenal of reproductive biotechniques and molecular genetics. While molecular genetics are poorly developed in swine, reproductive techniques are more advanced and applied under field conditions. This review describes three selected examples of our own research to illustrate the implication of biotechnology in future pig reproduction. Sperm sexing technology is now available and can be used to generate piglets of the desired sex by IVF and ICSI. First studies also indicate satisfactory success rates following intrauterine insemination with sexed spermatozoa. Cloning technique and production of transgenic pigs require information about the regulation and time course of gene expression during in vitro production and pre-implantation development. Information on gene expression is scare in porcine embryos. With the exception of transcripts for the oestrogen receptor gene, no mRNA's from the activated porcine genome have been identified. Recent development of cDNA arrays might help to identify a larger amount of genes in single embryos. Remarkable progress has been made in organ transplantation technology. As the demand for human organs is increasing rapidly, the pig might serve as donor of xenotranplants, provided the transmission of zoonoses from the donor animal to the human recipient is prevented, donor organ anatomy and function are compatible, and immunological rejections (HAR, VAR MAC) can be overcome. The most promising strategy is the synthesis of human complement regulatory proteins in the pig. Transgenic pigs have been generated for hDAF or hCD 46 and their hearts have been transplanted into non human primates for up to 90 days. HCD 59 driven by CMV promotor provides significant protection against HAR at least under in vitro and in vivo conditions. Current studies indicate a temporary use in patients within the next 3-5 years. As the microinjection technology to produce transgenic offspring is time consuming and very expensive, nuclear transfer technology provides a possibility for multiplication without going through the germ line with recombination effects. A functional nuclear transfer system will be crucial for xenotransplantation as it is anticipated that the expression of several transgenes will be required.

Agriculture↗