Biomedical subjects

T A Kruip

Publications and source records attributed to T A Kruip.

At least 37 records · Page 2Linked to original sources

PubMed1993

Nuclear transfer and electrofusion in bovine in vitro-matured/in vitro-fertilized embryos: effect of media and electrical fusion parameters.

In this study, micromanipulation and electrofusion conditions for the cloning of in vitro-produced bovine embryos (here after termed IVM/IVF embryos) derived from in vitro-matured (IVM) and in vitro-fertilized (IVF) oocytes were established. The effect of DC field strength on the fusion rate was tested in a model system using pronuclear stage embryos in which a cytoplasmic vesicle was removed and reinserted. Efficient fusion (80%) was obtained by applying a pulse of 1.75 kV/cm for 40 microseconds. In vitro development of manipulated pronuclear stage embryos was as efficient as that of unmanipulated control embryos. Different fusion media were compared in the cloning procedure, using IVM oocytes as recipients and blastomeres from day 6 IVM/IVF donor embryos. Zimmermann cell fusion medium reduced the lysis of nuclear transfer embryos compared to F300 (5% vs. 25%). The effects of drugs disrupting the microfilaments and microtubuli were determined. Neither the addition of cytochalasin B (CCB) for 1 hr in the postfusion medium nor incubation of donor blastomeres with nocodazole had a significant effect on the fusion or cleavage rate of the nuclear transfer embryos. Additional experiments demonstrated that there was no difference in developmental potential between nuclear transfer embryos allowed to develop in vitro or in vivo and that the embryos gave a 15% pregnancy rate in recipient cattle.

Animals↗

PubMed1993

Cytogenetic analysis of day-7 bovine embryos by cytophotometric DNA measurements.

A Feulgen staining procedure that stains the DNA of individual fixed nuclei stoichiometrically was used to analyse cytophotometrically the incidence of total ploidy and mixoploidy in 28 day-7 bovine embryos that had been fixed after collection ('non-cultured' embryos). The influence of culture on the incidence of chromosome abnormalities was further studied in another group of 24 embryos ('cultured' embryos) by culturing them for 24 h in Whittingham's medium. Of the total 52 embryos studied, two appeared to be entirely abnormal: one embryo was completely haploid, whereas the other embryo was completely triploid. Individual hyperdiploid nuclei and hypodiploid nuclei were frequently observed in the otherwise diploid embryos. As haploid polar bodies can still be present in morulae and blastocysts (to a maximum of three), only embryos with more than three hypodiploid nuclei were considered as abnormal. Of the 'non-cultured' embryos, 33.3% had one or more hyperdiploid nuclei, whereas 51.9% had more than three hypodiploid nuclei. In this latter group, 35.7% of the embryos also had hyperdiploid nuclei. The results also showed that day-7 bovine embryos that are completely haploid, completely triploid or mixoploid cannot be detected only by examining their morphology. It is concluded that the incidence of, especially, mixoploidy in embryos can be better studied by measuring the DNA content of the individual nuclei of an embryo rather than by analysing chromosomes, as in the latter method only dividing cells can be analysed. The presence of hyperdiploid and hypodiploid nuclei may indicate the frequent occurrence of mitotic segregation failures during mitosis in bovine embryos.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1992

Structural aspects of bovine oocyte maturation in vitro.

Bovine cumulus-oocyte complexes (COCs) were collected from 4-8 mm follicles and graded into four categories on their morphological characteristics. These four categories were matured in vitro and processed for transmission electron microscopy at 24 h after the onset of culture. The morphology of the four groups of oocytes was analysed and compared with that of oocytes that had matured in vivo and were collected 20-23 h after the preovulatory luteinizing hormone peak. After in vivo maturation, oocytes formed a homogeneous group with respect to their morphological characteristics. After in vitro maturation, the oocytes formed a heterogeneous group with respect to their morphology between as well as within the four categories of oocytes. Oocytes from the first three categories showed the same morphology after in vitro maturation. The fourth category showed some specific characteristics: 1) vacuolization, 2) flattening of cumulus cells, and 3) almost complete lack of cortical granules in some category 4 oocytes. These characteristics are interpreted as signs of degeneration. Besides these aspects of degeneration, other deviations from normal development were seen: 1) retraction of cumulus cell process endings from the oocyte without the breaking down of these processes, 2) retardation of some aspects of the cytoplasmic maturation, and 3) incomplete cumulus expansion. It is concluded that oocytes capable of development in vitro show a large morphological variability before the onset of culture. In vitro maturation systems can support normal development, but many oocytes show signs of degeneration and deviant development after in vitro maturation.

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PubMed1991

A new method for bovine embryo production: a potential alternative to superovulation.

Eight cows were used to study the feasibility of transvaginal ultrasound-guided puncture of follicles as a method for the collection of immature oocytes for embryo production in vitro. In six trials at intervals of seven days, 104 oocytes were collected. After in vitro maturation and fertilisation the 104 oocytes were transferred to the oviducts of sheep. Six days later, 75 oocytes were recovered by flushing the oviducts. Twenty-four per cent of the recovered oocytes/embryos had developed into transferable and viable morulae and, or, blastocysts. The data show that this non-surgical and repeated collection of immature oocytes can be used successfully for the in vitro production of bovine embryos. The procedure may produce yields of embryos comparable to those obtainable by conventional superovulation procedures.

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PubMed1991

The effects of alpha-amanitin and cycloheximide on nuclear progression, protein synthesis, and phosphorylation during bovine oocyte maturation in vitro.

Bovine cumulus oocyte complexes were cultured for various periods and either denuded and orcein stained or radiolabeled with 35S-methionine or 32P-orthophosphate. Specific inhibitors were added to the culture medium to investigate mRNA and protein synthesis requirements for both nuclear and cytoplasmic changes during maturation in vitro. Inhibition of mRNA synthesis by alpha-amanitin during the first 2 h of culture prevented the phosphorylation of some specific proteins preceding GVBD and decreased the occurrence of GVBD from 97% to 27%. In addition, in oocytes that had undergone GVBD, only part of the changes in protein synthesis after GVBD were observed. Addition of alpha-amanitin after 3 h of culture had no effect on meiotic maturation. When cumulus oocyte complexes were cultured in the presence of cycloheximide, the phosphorylation of specific proteins was also blocked and only 5% of the oocytes underwent GVBD. Addition of cycloheximide after 4, 6, or 8 h of culture resulted in an increasing percentage of GVBD, but the oocytes became arrested in metaphase I. When cycloheximide was added from 12 h of culture onwards, nuclear progression to metaphase II was increasingly restored. It is concluded that after the onset of culture, both mRNA and protein synthesis are necessary for the phosphorylation of specific proteins and for GVBD. Furthermore, transcription during the first hours of culture is needed for the synthesis of new proteins after GVBD.

Amanitins↗

PubMed1991

Heterologous cell contacts and metabolic coupling in bovine cumulus oocyte complexes.

The integrity of the cumulus cell processes were studied in four categories of bovine cumulus oocyte complexes (COCs) selected on their morphological characteristics. Three different types of cumulus cell process endings (CCPEs) were identified, one penetrating the cortex, another not penetrating the cortex, and a third form was intermediate and more rare in appearance. The process endings that penetrated the cortex frequently made gap junctions with the oolemma. The division of the three types of CCPEs over the four different COC categories was specific for three of the four categories. The first-category COC predominantly possessed the penetrating CCPE, the fourth-category COC possessed predominantly the nonpenetrating CCPE, and the second and third categories had both types of CCPEs. The metabolic coupling of the cumulus-oocyte contacts was assessed by means of incorporation of 3H-choline into the oocyte. The majority of category 4 COCs transferred low levels of choline into the oocyte while the majority of the oocytes of the other three categories transferred high levels of choline into the oocyte. Category 4 includes a smaller proportion of oocytes capable of cleaving after fertilization than the other three categories. This reduced developmental capacity is probably due to the loss of metabolic coupling before the onset of culture.

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PubMed1991

Protein synthesis and phosphorylation patterns of bovine oocytes maturing in vivo.

To investigate protein synthesis and phosphorylation during bovine oocyte maturation in vivo, oocytes were collected at consecutive times after the preovulatory luteinizing hormone (LH) peak. Therefore, heifers treated for superovulation were ovariectomized between 3 and 20 h after the maximum of the LH peak. Subsequently, cumulus-enclosed oocytes, selected from nonatretic follicles greater than 10 mm, were radiolabeled with 35S-methionine or 32P-orthophosphate for 3 h and individually prepared for gel electrophoresis. Changes in the protein synthesis patterns were observed coinciding with germinal vesicle breakdown (GVBD). No changes were detected during the ensuing maturation period or coinciding with the extrusion of the first polar body. In addition, the protein phosphorylation patterns exhibited striking differences around GVBD. In particular, a phosphoprotein band of 19 kDa and the two heavily phosphorylated proteins with apparent molecular weights between 50 and 60 kDa were present in patterns of oocytes in the germinal vesicle stage. The results are discussed in relation to previous data obtained during maturation in vitro.

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PubMed1991

Characteristics of bovine estrous cycles during repeated transvaginal, ultrasound-guided puncturing of follicles for ovum pick-up.

Repeated transvaginal ultrasound guided puncturing of visible follicles was performed for ovum pick-up (OPU) during Periods A and B, each of which lasted 3 mo. During Period A, 10 cows (A) were used in the study. Period B commenced 1 mo after Period A and two groups of animals were used. The first group (B1) consisted of 9 of 10 cows from Group A. The second experimental group of animals in Period B consisted of 11 cows (B2) which had not been submitted to previous puncture. During the study, all visible follicles larger than 3 mm were punctured and aspirated three times, on Day 3 or 4, Day 9 or 10 and Day 15 or 16 of the estrous cycle. The mean estrous cycle length (+/- SEM) after repeated follicle puncture did not differ among the three groups and was 22.3 +/- 0.4, 22.5 +/- 0.4 and 22.1 +/- 0.3 d for groups A, B1 and B2, respectively. The mean total number (+/- SEM) of punctured follicles per estrous cycle in Group A (13.1 +/- 0.5) was significantly larger than in Groups B1 (11.2 +/- 0.4) and B2 (11.6 +/- 0.4). The largest number of follicles punctured for ovum pick-up in all three groups was always on Day 3 or 4 of the estrous cycle: 4.9 +/- 0.3 follicles; the mean (+/- SEM) number of punctured follicles on Day 9 or 10 and Day 15 or 16 was significantly (P<0.05) lower: 3.4 +/- 0.2 and 3.9 +/- 0.2, respectively. In Period A, primarily 3- to 5-mm follicles were punctured per estrous cycle, while 6- to 10-mm follicles were predominantly punctured in Period B (P<0.05). Recovery rate of oocytes on Day 3 or 4, Day 9 or 10 and Day 15 or 16 were 53, 50 and 52%, respectively. Most oocytes (P<0.05) were aspirated from follicles smaller than 10 mm.

Journal Article↗

PubMed1991

Transvaginal ultrasound guided follicular aspiration of bovine oocytes.

A transvaginal ultrasound guided follicular aspiration technique was developed for the repeated collection of bovine oocytes from natural cycling cows. In addition, the feasibility of using this method for collecting immature oocytes for in vitro embryo production was also evaluated. Puncturing of visible follicles for ovum pick-up was performed in 21 cows over a three month period. All visible follicles larger than 3 mm were punctured and aspirated three times during the estrous cycle on Day 3 or 4, Day 9 or 10 and Day 15 or 16. The mean (+/- SEM) estrous cycle length after repeated follicle puncture was 22.2 +/- 0.3 days. The mean total number of punctured follicles per estrous cycle was 12.6 +/- 0.3. The largest (P<0.05) number of follicles punctured (5.1 +/- 0.3) for ovum pick-up was on Day 3 or 4 of the estrous cycle. The overall recovery rate of 541 punctured follicles was 55%. Most oocytes (P<0.05) were aspirated from follicles smaller than 10 mm. Following in vitro maturation and fertilization (IVM/IVF), 104 oocytes were transferred to sheep oviducts. Six days later, 75 ova/embryos were recovered, after flushing the oviduct of the sheep, of which 24% developed into transferable morulae and blastocysts. In this study, a reliable nonsurgical, follicular aspiration procedure was used for the repeated collection of immature oocytes which could be used successfully for in vitro production of embryos. This procedure offers a competitive alternative to conventional superovulation/embryo collection procedures.

Journal Article↗

PubMed1991

Morphology of preovulatory bovine follicles as related to oocyte maturation.

Thirty-three preovulatory bovine oocytes and their follicles were collected during the period of final maturation in normally cyclic cows. Cell density of the membrana granulosa, mitotic index of the membrana granulosa, and the occurrence of eosinophilic granulocytes around the basal membrane as well as the maturational stage of the oocyte were determined. Cell density decreased during the period of final maturation. Mitotic indices also decreased after an initial high level in the first hours of the final maturation. Eosinophilic granulocytes were only seen during the last hours of final maturation. The maturational stages of the oocytes were related to distinct maturational stages of the follicular wall as determined by morphological characteristics. We propose a scoring system for the maturity of the follicular wall based on cell density, presence of mitotic figures and the presence of eosinophilic granulocytes outside the vascular compartment.

Journal Article↗

PubMed1991

Follicular and oocyte maturation in cows treated for superovulation.

The maturational stage of oocytes and their follicles was assessed at 24/26 h after the preovulatory luteinizing hormone (LH) peak by means of morphological criteria. Follicles were obtained from cows treated for superovulation (PMSG/PG) with additional anti-PMSG to neutralize the residual PMSG. Follicular fluid was also recovered and analyzed for progesterone and estradiol levels. Seventy-two percent of the oocytes were at the Metaphase II (M(II)) stage of meiosis, whereas only 28% of the follicular walls were at the proper maturational stage; assessed on morphological characteristics, 78% of the follicles were progesterone-dominated. Earlier maturational stages of oocytes and follicles were also present, including those that are restricted to periods shortly after the LH peak in the normally cyclic cow. It is concluded that upon treatment for superovulation not all oocytes and follicles mature synchronously, and that not all oocytes mature in harmony with their follicles.

Journal Article↗

PubMed1990

[Biotechnology or the revelation of reproduction in farm animals].

The increased knowledge of endocrinology, cytobiology and embryology has also made stock farmers familiar with biotechnology. The genetic potency of both male and female animals may be utilised to better advantage today. However, the normal reproductive capacity, present in the form of large numbers of sex cells in female animals, is not utilised to an optimum extent. In spite of all recent developments so far, ninety-nine per cent of all ova are still lost. Since 1987, it has become possible to obtain immature ova from the living animal and to let them mature, fertilize and develop into embryos capable of transplantation outside the body. It may be expected that, in the long run, the number of embryos per excellent animal will show an enormous increase when this method is used. In addition, in vitro maturing of ova may be used in cloning embryos. This requires a large number of mature egg cells which act as recipients of the nuclei of embryonic cells, after the nuclei have been removed from these ova. One nucleus of an embryonic cell in each denucleated mature egg cell. These embryonic cells may be derived from an embryo which has disintegrated into individual cells or from an embryonic cell culture (embryonic stem cells). The only condition is that these cells have not yet become differentiated and have retained their omnipotent character.

Animals↗

PubMed1990

Detection of corpora lutea and follicles in cows: a comparison of transvaginal ultrasonography and rectal palpation.

The ovaries of 59 pluriparous cows of unknown reproductive history were palpated, scanned and dissected on the day of slaughter to compare the accuracy of rectal palpation and transvaginal ultrasonography with a 5 MHz linear array for the detection of corpora lutea and follicles. The rectal palpation was first carried out to judge the presence of follicles of more than 5 mm diameter, and corpora lutea which were classified as young (days 1 to 4), mid-cycle (days 5 to 16) or old (days 17 to 21) according to morphological criteria. The cows were then examined for follicles and corpora lutea by ultrasonography and the corpora lutea were again classified directly as young, mid-cycle or old according to their appearance. The cows were then slaughtered, their ovaries dissected, and the follicles over 5 mm in diameter were counted and the corpora lutea were classified in the above mentioned age categories. For the detection of a mid-cycle corpus luteum the sensitivity and predictive value of rectal palpation were, respectively, 83.3 per cent and 73.2 per cent and for ultrasonography the sensitivity and predictive value were 80.6 per cent and 85.3 per cent, respectively. However, both techniques were inaccurate for the detection of young and old corpora lutea. For detecting follicles ultrasonography was a significantly better method than rectal palpation. Ultrasonography detected 95 per cent of follicles larger than 10 mm whereas rectal palpation detected only 71 per cent of these follicles. Both techniques failed with follicles 5 to 10 mm in diameter; only 21.5 per cent were detected by rectal palpation and 34.3 per cent by ultrasonography.

Animals↗

PubMed1990

Analysis of protein synthesis in morphologically classified bovine follicular oocytes before and after maturation in vitro.

Bovine cumulus oocyte complexes (COCs) were isolated from antral ovarian follicles (4-8 mm). Immature COCs were classified into four categories, based on the homogeneity and clearness of the ooplasm and the transparency and compactness of the cumulus investment. In this study, the incorporation of TCA-precipitable 35S-methionine and the protein synthesis patterns of oocytes of these four categories were examined. Before maturation in vitro, similar incorporation rates and identical protein synthesis patterns were observed between oocytes of categories 1-3. Immature oocytes of category 4 showed reduced incorporation rates and exhibited aberrant protein synthesis patterns. After maturation in vitro, the patterns of category 4 oocytes were identical with the patterns of those in categories 1-3. The incorporation of 35S-methionine into in vitro matured oocytes was lower (P less than .001) in all categories. Based on these results, it is concluded that the initial classification of oocytes into four categories can be reduced to two categories.

Animals↗

PubMed1990

Changes in protein synthesis and phosphorylation patterns during bovine oocyte maturation in vitro.

Sequential protein synthesis and protein phosphorylation patterns were generated by radiolabelling bovine cumulus-oocyte complexes after various periods of culture with [35S]methionine and [32P]orthophosphate respectively. The radiolabelled oocytes were assessed for their nuclear status and used individually for gel electrophoresis. Marked changes in the protein synthesis patterns were observed exclusively after germinal vesicle breakdown (GVBD), whereas oocytes which remained in the germinal vesicle stage showed a consistent protein synthesis pattern. The changes were observed after 8 and 16 h or culture, shortly after GVBD and before first polar body extrusion. From 3 h of culture, dominant phosphoprotein bands with apparent molecular weights of 24,000 and two between 50,000 and 60,000 were observed. The latter bands displayed slight molecular weight changes, which were not closely time related. After GVBD, the phosphoprotein band with Mr 19,000 was no longer observed. This study demonstrates that specific changes in protein synthesis and protein phosphorylation are programmed during bovine oocyte maturation.

Animals↗

PubMed1989

Morphology of immature bovine oocytes.

Bovine cumulus oocyte complexes (COCs) as used for in vitro maturation and fertilization can be classified into different categories by light microscopical inspection. We have distinguished four categories based on compactness and transparency of the cumulus investment and homogeneity and transparency of the ooplasm. The four categories were studied for their morphological characteristics at the ultrastructural level and for their developing capacity in an in vitro maturation system. In categories 1 and 2 oocytes, organelles were evenly distributed. In categories 3 and 4, oocytes organelles were clustered and the distribution of the organelles mimicked the characteristics of oocytes during final maturation. Cumulus cell process endings penetrated the cortex of the oocyte or were located superficial to the cortex of the oocyte. In category 1 oocytes, most of the process endings penetrated the cortex. In category 4 oocytes, most of the process endings did not penetrate. In categories 2 and 3 oocytes, both forms of process endings did occur. After in vitro maturation, only category 4 oocytes showed a decreased developing capacity. Categories 1-3 oocytes showed equal developing capacity in an in vitro maturation system.

Animals↗

PubMed1989

Intrinsic and extrinsic factors influencing steroid production in vitro by bovine follicles.

Nonatretic bovine follicles were cultured on grids in a conventional static system, in roller tubes or in a continuous flux system. Culture medium from the static and roller tube system was replaced after 24 h of culture, while in the continuous flux system, only a small sample was taken aseptically at that time. Steroid concentrations in these samples were estimated by radioimmunoassay (RIA) and related to intrinsic factors like follicle size, day of cycle and micromorphological appearance at the end of culture and to extrinsic factors like the culture system, O(2)-concentration applied in the gasphase and time of culture. Bovine nonatretic follicles from 2 to 8 mm showed the same amount of estradiol-17beta (E2) production as pmol mg protein(-1) 24 h. Follicles over 8 mm had a significantly higher E2 production. Follicles from the follicular phase of the cycle produced more E2 than follicles from the luteal phase, independent of follicular size and culture system. Degeneration of follicles during culture resulted, independent of the culture system, in a decline of E2-production, and in an increase of P4-production; whereas the T-production initially (primary atresia) rose but subsequently (secondary and tertiary atresia) declined. The difference between the culture systems were reflected by quantitative differences in the production of the steroids measured. The most striking difference between the continuous flux system on one hand and the static and rolling tube system on the other is the predominant E2 production in the former by every follicle. It is thought that this difference might be caused by a better 02 supply in the continuous flux system. This hypothesis is tested in the static culture system. The more 02 the more E2 production. The increase in culture time resulted in an increase of E2 and P4, whereas the testosterone production was not significantly decreased.

Journal Article↗

PubMed1988

[The role of prolactin during the estrus cycle of cattle].

Suppression of the prolactin level in the peripheral blood by chronic treatment with bromocryptin throughout the oestrous cycle of the cow, had no effect on: 1) LH concentrations during the cycle, 2) progesterone secretion of the corpus luteum, 3) duration of the early luteal phase, the luteal phase and the period of luteal regression, 4) oestradiol secretion during the follicular phase, 5) number and quality of follicles greater than 2 mm, 6) number of binding sites on granulosa cells and thecal tissue of the preovulatory follicle and 7) the concentration of oestradiol, androstenedione and progesterone in the follicular fluid of the preovulatory follicle. Follicles from bromocryptin treated and control heifers did not possess specific binding sites for prolactin. It is concluded that prolactin is not involved in the regulation of the oestrous cycle in cattle.

Animals↗