Biomedical subjects

H Alm

Publications and source records attributed to H Alm.

At least 19 recordsLinked to original sources

PubMed2007

Ultrastructural analysis reveals striking differences of intercellular contact lengths in bovine embryos produced in vivo, in vitro and by somatic cell nuclear transfer.

Cellular coherence and communication, thus cell-to-cell contact is an indispensable premise to sustain the formation of complex, multi-cellular organisms. We have analyzed intercellular contact lengths in NT-cloned bovine embryos compared to the in vivo or in vitro produced counterparts. Therefore, ultrastructural analysis was carried out by transmission electron microscopy (TEM) at the 8-cell and blastocyst stage of development. To obtain embryos generated in vivo, oviducts of superovulated cows were flushed 3 days after insemination, subsequent to slaughter. Standard in vitro maturation (IVM) and -fertilization (IVF) were utilized to obtain in vitro embryos. Cloned embryos by somatic nuclear transfer were produced by the handmade cloning (HMC) procedure. The points of apposition/focal contact points (CPs) between the blastomeres were of the shortest order in cloned embryos (236 +/- 135 nm) and of highest order in the in vivo produced embryos (2,085 +/- 1,540 nm), although no significant differences regarding the blastomere sizes in the various groups of 8-cell embryos could be established. In summary, the CP lengths in case of in vitro and in vivo 8-cell embryos were, on an average, five or nine times longer, respectively, than in the case of the cloned embryos. These differences of CP lengths vanished in embryos reaching the blastocyst stage of embryonic development in all the three groups of embryos. The observed differences of intercellular contact length at distinct stages of embryonic development could be responsible for differences in intercellular communication between the blastomeres at the beginning of cellular differentiation. These may be one reason for the lower developmental competence of cloned (NT) embryos.

Animals↗

PubMed2006

Selection of developmentally competent oocytes through brilliant cresyl blue stain enhances blastocyst development rate after bovine nuclear transfer.

The aim of the present investigation was to study the effect of oocyte selection on the efficiency of bovine nuclear transfer in terms of increased blastocyst production. For this purpose, prior to in vitro maturation (IVM), oocytes were selected for their developmental competence on the basis of glucose-6-phosphate dehydrogenase (G6PDH) activity indicated by brilliant cresyl blue (BCB) staining. It has been hypothesized that growing oocytes have a higher level of active G6PDH in comparison to the mature oocytes. Compact cumulus oocyte complexes (COCs) were recovered from slaughterhouse-collected bovine ovaries and classified either as control group, which were placed immediately into culture without exposure to BCB stain, or treatment group, which were stained with BCB for 90min before culture. Treated oocytes were then divided into BCB- (colourless cytoplasm, increased G6PDH) and BCB+ (coloured cytoplasm, low G6PDH) based on their ability to metabolize the stain. After IVM, oocytes were subjected to nuclear transfer procedure for the production of cloned embryos which were then cultured for a period of 8 days to determine the blastocyst rate. The BCB+ oocytes yielded a significantly higher blastocyst rate (39%) than the control (21%) or BCB- oocytes (4%). These results show that the staining of bovine cumulus-oocyte complexes with BCB before in vitro maturation could be used to select developmentally competent oocytes for nuclear transfer. In addition, G6PDH activity could prove to be a useful marker for determining the oocyte quality in future.

Analysis of Variance↗

PubMed2006

Influence of Fusarium-toxin contaminated feed on initial quality and meiotic competence of gilt oocytes.

Mycotoxins are contaminants of animal feed that can impair fertility and cause abnormal fetal development in farm animals. The aim of the present study was to investigate the influence of Fusarium-toxin contaminated feed on cumulus morphology and maturation of pig oocytes. Naturally with the Fusarium-toxins deoxynivalenol (DON) and zearalenone (ZON) contaminated wheat was included in feed for gilts at increasing proportions which resulted in increasing dietary concentrations of both toxins (in mgtoxin/kg feed: Group 1 (control), 0.21 and 0.004; Group 2, 3.07 and 0.088; Group 3, 6.1 and 0.235; Group 4, 9.57 and 0.358, for DON and ZON, respectively). Oocytes were recovered from gilt ovaries by follicle aspiration after ovario-hysterectomy. Granulosa cells were analyzed for the expression of the P450(SCC) and 3beta-HSD mRNA by RT-PCR and additionally for P450(SCC) protein by Western blotting. Neither the expression of the P450(SCC) nor of the 3beta-HSD mRNA or the abundance of the P450(SCC) protein was significantly influenced by the mycotoxin application. The distribution of different cumulus cell morphologies was not influenced by group. At the time of recovery, oocytes with compact cumuli in Groups 3 and 4 showed a reduced proportion having immature chromatin in comparison to that for Groups 1 and 2. The proportion of oocytes having degenerated meiotic chromatin was significantly higher in Group 4 than in the other groups. The proportion of oocytes reaching metaphase II in culture was significantly lower in Groups 3 and 4 than in Group 1, and tended to be lower in Group 2 than in Group 1. We conclude that oocyte quality is significantly reduced by feeding of Fusarium-toxins to gilts.

3-Hydroxysteroid Dehydrogenases↗

PubMed2005

Survival and meiotic competence of bovine oocytes originating from early antral ovarian follicles.

The aim of the present study was to examine the growth and survival in culture, and the subsequent meiotic competence, of bovine oocytes recovered from early antral ovarian follicles. Follicles isolated by microdissection of the ovarian slices were sorted into two size groups: (I) 0.2-0.5 mm diameter; and (II) 0.4-0.7 mm diameter. Group I follicles were cultured intact while in Group II, cumulus-oocyte complexes with pieces of parietal granulosa were dissected from the follicles and cultured. Follicles or cumulus-oocyte complexes with parietal granulose were embedded in collagen gel and cultured in TCM 199 supplemented with 3% BSA and 4 mM hypoxanthine for 14 days (Group I) or 7-10 days (Group II). After this, cumulus-oocyte complexes were recovered from the gel. Oocytes that had lost the majority of the cumulus were fixed immediately after recovery. Cumulus-oocyte complexes showing normal morphology were either fixed immediately or were subjected to IVM for an additional 24h, and then were fixed. At the end of the growth culture, 57.6% of the compact COCs in Group I follicles were preserved in the GV configuration, 16.7% had resumed meiosis, and 25.8% were degenerated or did not show detectable chromatin. After IVM, the proportion of oocytes resuming meiosis increased significantly (from 16.7% versus 42.7%; P < 0.05), and 9.1% of all oocytes had reached TI or MII. The isolated cumulus-oocyte complexes in Group II began creating follicle-like structures following 24 h of growth culture (7.1%). The proportion of these structures reached 50.8% on days 2-3, and then gradually decreased due to degeneration. On day 10 only 5.8% of cumulus-oocyte complexes were classified as intact. Of the cumulus intact oocytes recovered from the newly created follicle-like structures at 7-10 days, 54.7% were in the germinal vesicle stage, 31.0% underwent germinal vesicle breakdown, 14.3% were degenerated or the chromatin configuration was not detectable. After 24 h of IVM, 67.6% of oocytes had resumed meiosis, and 21.6% of all oocytes had reached TI and MII. These results show that isolated early follicles and cumulus-oocyte complexes from intact early antral follicles can grow in culture and can develop meiotic competence.

Animals↗

PubMed2005

Endocrine, morphological, and cytological effects of a depot GnRH agonist in bovine.

The present study was conducted to assess effects of the gonadotropin-releasing hormone agonist (GnRHa) triptorelin in dairy heifers. The peptide was released from a commercial 4-week depot formulation (Decapeptyl Depot) administered at animals' estrus (day 0). First experiment (EXP I, n=5), which was aimed to explore the availability of peptide, detected a maximum of triptorelin concentration between day 2 and 5 after depot injection, and the peptide remained detectable by RIA in peripheral blood for about 3 weeks. In further experiments, the peptide release was terminated on day 9 (EXP II, n=16) or day 21 (EXP III, n=47). Treatment effects were studied on follicular development, the characteristics of cumulus-oocyte complexes (COCs) (EXP II; EXP IIIa) and secretions of LH and progesterone (EXP IIIb). Results showed that the occurrence of the pre-ovulatory LH surge was more uniform in treated heifers than that in controls. The duration of ovulation periods was similar amongst the heifers of EXP II, but more compact amongst those of EXP III each compared with the respective controls. Post-ovulatory, the number of LH pulses was significantly reduced by treatment, whereas both basal LH and progesterone concentrations were elevated on a few days. Follicular growth was reduced only by the prolonged influence of the GnRHa. There were increased proportions of both degenerated COCs and immature oocytes from small follicles (<3mm in diameter), and meiotic configuration and quality of oocytes isolated from follicles 3-5mm were changed after the prolonged, 21-day treatment. These results indicate that a continuous influence of a GnRHa over more than 1 week may increasingly impair the development of bovine follicles and oocytes. This may have some significance for the development of novel GnRH-based techniques in regulating the reproductive function in cattle.

Animals↗

PubMed2005

Bovine blastocyst development rate in vitro is influenced by selection of oocytes by brillant cresyl blue staining before IVM as indicator for glucose-6-phosphate dehydrogenase activity.

The aim of this present study was to increase the efficiency of blastocyst production from cows after in vitro maturation/fertilization (IVM/IVF) by oocyte selection before maturation. Oocytes were selected on the basis of brillant cresyl blue (BCB) staining, used to indicate glucose-6-phosphate dehydrogenase (G6PDH) activity. To re-valuate the hypothesis that growing oocytes are expected to have a high level of active G6PDH, while mature oocytes have low G6PDH activity, cumulus oocyte complexes (COCs) were recovered from slaughterhouse ovaries by slicing the surface of the ovary. Only oocytes with a compact cumulus investment were used. Oocytes were placed into three groups: (1) control--placed immediately into culture; (2) holding control--COCs kept in PBS containing 0.4% BSA for 90 min before placement into culture; and (3) treatment--incubation with BCB for 90 min before culture. Treated oocytes were then divided into BCB- (colorless cytoplasm, increased G6PDH) and BCB+ (colored cytoplasm, low G6PDH) on their ability to metabolize the stain. Activity of G6PDH was determined via measurement of NADP reduction induced by G6P as substrate oxidized by G6PDH in the cytosol of control, BCB- and BCB+ groups; G6PDH activity was significant higher in BCB- COCs than in control and BCB+ COCs. After IVM, oocytes were fertilized in vitro. Embryos were cultured to day 8. The rate of maturation to metaphase II was significantly higher for control and BCB+ oocytes than for BCB- oocytes. The BCB+ oocytes yielded a significantly higher proportion of blastocysts (34.1%) than did control or holding control oocytes (18.3 and 19.2%); and both controls and BCB+ oocytes had significantly higher blastocyst development than did BCB- oocytes (3.9%). These results show that the staining of bovine cumulus oocyte complexes with BCB before in vitro maturation may be used to select developmentally competent oocytes for IVF. In addition, G6PDH activity may be useful as a marker for oocyte quality in future studies on factors affecting developmental competence.

Animals↗

PubMed2004

Mitochondrial aggregation patterns and activity in porcine oocytes and apoptosis in surrounding cumulus cells depends on the stage of pre-ovulatory maturation.

In this study, we evaluated the distribution and oxidative activity of mitochondria in ex vivo pre-ovulatory porcine oocytes using the fluorescence probe MitoTracker CMTM Ros Orange. Cumulus-oocyte complexes (COCs) were classified according to cumulus morphology and time from hCG administration. The meiotic configuration of the oocytes and the degree of apoptosis in the surrounding cumulus cells were also evaluated. Estrus was synchronized in 45 crossbred Landrace gilts by feeding altrenogest for 15 days and administering 1000 IU PMSG on Day 16. The LH peak was simulated by treatment with 500 IU hCG, given 80 h after PMSG. Endoscopic oocyte recovery was carried out 2 h before or 10, 22, or 34 h after hCG administration. Altogether 454 COCs were aspirated from follicles with a diameter of more than 5 mm. Cumulus morphology in the majority of COCs recovered 2 h before and 10 h after hCG was compact (60.4 and 52.7%, respectively; P<0.05). At 22 h after hCG, COC morphology changed significantly from 10 h dramatically: 74% of COCs had an expanded cumulus (P<0.01). At 34 h after hCG, 100% of recovered COCs had an expanded cumulus. The percentage of oocytes with a mature meiotic configuration differed among COC morphologies and increased as the interval after hCG administration increased (P<0.05). The type of mitochondrial distribution in the oocytes (n=336) changed from homogeneous to heterogeneous as the interval after hCG administration increased (P<0.01) and was associated with the cumulus morphology. Representative mitochondrial distributions were found as follows: -2 h: fine homogeneous in compact and dispersed COCs; 10 h: granulated homogeneous in compact and dispersed COCs; 22 h: granulated homogeneous in expanded COCs; and 34 h: granulated heterogeneous and clustered heterogeneous in expanded COCs (P<0.01). The oxidative activity of mitochondria measured by fluorescence intensity (Em: 570 nm) per oocyte after Mitotracker CMTM Ros Orange labeling increased in the oocyte as the post-hCG interval increased (P<0.01) and depended on the type of mitochondrial distribution. Lowest oxidative activity of mitochondria was found in oocytes with fine homogeneous distribution (253.1+/-9.4 microA). The oxidative activity increased (334.4+/-10.3 microA) in oocytes with granulated homogeneous distribution of mitochondria, and reached highest level in oocytes with granulated heterogeneous (400.9+/-13.0 microA) and clustered heterogeneous distributions (492.8+/-13.9 microA) (P<0.01). Mitochondrial activity in oocytes coincided with apoptosis in surrounding cumulus cells which increased in a time-dependent manner during pre-ovulatory maturation in vivo (P<0.01). These results indicate that there is a relationship between meiotic progression, cumulus expansion and mitochondrial redistribution and their oxidative activity during final pre-ovulatory maturation in pig oocytes. It appears that increased levels of mitochondrial activities in oocytes are correlated to increased levels of apoptosis in surrounding cumulus cells, in which mitochondria may play a role.

Animals↗

PubMed2004

[Prolactin-binding activity of bovine granulosa cells on luteinization and exposure to somatotropin in vitro].

The influence of luteinization and bovine somatotropin (ST, 5-50 ng/ml) during cultivation of bovine granulosa cells on their ability to bind [125I]-labeled bovine prolactin (PRL) was studied. On the second day of cultivation in serumfree medium, granulosa cells from immature antral follicles underwent spontaneous luteinization, in both the absence and presence of ST. The level of [125I]-PRL specific binding to cells increased after two days of cultivation, with a negative correlation being revealed between estradiol production by the cells and their PRL-binding activity. At the same time, the addition of ST to the culture medium had no effect on the level of [125I]-PRL specific binding to native and luteinizing granulosa cells. The findings suggest a stimulatory influence of the luteal differentiation process on the PRL-binding activity of bovine granulosa cells, this influence is independent of the action of ST.

Animals↗

PubMed2004

[The influence of somatotropin, prolactin and insulin on granulosa cells from bovine atretic follicles].

An influence of somatotropin, prolactin and insulin on destructive processes in bovine granulosa cells from small antral follicles following atresia in vivo was studied in vitro. As compared to control, the addition of the studied hormones to serum-free suspension system was shown to result in increase in number of cells without signs of chromosome degeneration after 24 and 48 hrs of incubation. The revealed inhibitory action of somatotropin, prolactin and insulin on chromatin degeneration in granulosa cells was not due to the hormonal influence on proliferative activity of the cells. The stimulatory action of insulin on the viability and estrogen-secretory activity of granulosa cells cultured for 1 day was also found. At the same time, somatotropin and prolactin did not affect the estradiol and progesterone production by the cells. The data obtained suggest that the inhibitory action of somatotropin and prolactin on destructive processes in cultured granulosa cells is not related to the hormonal regulation of the steroidogenic activity of the cells, whereas the similar action of insulin may be partially due to its stimulatory influence on the estradiol secretion.

Animals↗

PubMed2003

Changes in cumulus-oocyte complexes of pregnant and non-pregnant camels (Camelus dromedarius) during maturation in vitro.

The aim of the present study was to examine the cumulus morphology and the oocyte chromatin quality of camel cumulus-oocyte complexes (COCs) at the time of recovery, and to monitor changes in oocyte chromatin configuration and apoptosis in cumulus cells from camel COCs during in vitro maturation (IVM) (0, 12, 24, 32, 36, 42, and 48 p.IVM) depending on pregnancy of donors. A total of 1023 COCs were isolated from sliced ovaries after slaughtering of 47 pregnant and 43 non-pregnant camels in an abattoir. The mean number of COCs per donor was 10.3 in pregnant and 12.5 in non-pregnant donors. The cumulus morphology of COCs was independent of the type of donor and was divided in COCs with compact (26.9 and 28%), dispersed (39.3 and 46%), corona radiata cumulus investment (27.9 and 21.7%) and without cumulus (6 and 4.2%), respectively for pregnant and non-pregnant donors. The highest proportion of COCs exhibited dispersed cumulus (P<0.05). Oocytes with meiotic stages of diplotene >50% were found only in compact (55 and 56.5%) and in dispersed COCs (58.4 and 60%), respectively for pregnant and non-pregnant donors. During IVM (0-48h) the first significant onset of specific meiotic stages were different in oocytes from pregnant donors: metaphase 1 (24-32h), metaphase 2 (36-42h), versus oocytes from non-pregnant donors: metaphase 1 (24h), metaphase 2 (32-48h) (P<0.05). The level of apoptotic cells in cumuli of matured COCs increased during IVM and was higher in matured COCs from non-pregnant donors for each time point during IVM (P<0.01). Camel oocytes meiosis during IVM is accompanied by a drastic increase of apoptosis in the surrounding cumulus cells 0-32 and 0-24h during IVM, respectively for pregnant and non-pregnant donors. The oocytes of pregnant camels require 36h of maturation to reach levels of >50% metaphase 2 stage in comparison to oocytes from non-pregnant donors where 32h are sufficient. The earlier onset of apoptosis in the COCs derived from non-pregnant donors possibly determines the faster progression of the oocytes through the final stages of meiosis.

Animals↗

PubMed2002

The influence of the mycotoxins deoxynivalenol and zearalenol on in vitro maturation of pig oocytes and in vitro culture of pig zygotes.

The aim of the present study was to investigate the influence of specific toxins on in vitro maturation and embryo culture. alpha- and beta-zearalenol were tested at increasing levels from 3.75 to 90 microM and deoxynivalenol from 0.94 to 7.5 microM in order to evaluate the effect on in vitro maturation rate of porcine cumulus-oocyte complexes. Furthermore, the influence of alpha-zearalenol (3.75-30 microM) was appraised on the developmental competence of in vivo-derived zygotes during 5 days of in vitro culture. All three substances affected maturation and degeneration rates in a dose-dependent manner, but to different extents. Significant differences were obtained at a concentration of 7.5 microM alpha-zearalenol and higher. beta-zearalenol negatively affected the process of oocyte development beginning at a concentration of 30.0 microM (P<0.05). Deoxynivalenol had significant influence on oocyte maturation at a concentration of 1.88 microM (31.4 vs 79.3% for control). Differences in embryonic development in vitro were observed at a concentration of 15 microM alpha-zearalenol (P<0.05). These data demonstrate a negative effect of alpha-zearalenol on embryonic development of zygotes, and a compound-specific, dose-dependent negative effect of the three substances on meiotic progression of porcine oocytes.

Animals↗

PubMed2001

Effect of sperm cryopreservation and treatment with calcium ionophore or heparin on in vitro fertilization of horse oocytes.

Little information is available on methods of sperm capacitation for IVF in the horse. In this study, we summarized results of several independent trials that compared acrosome reaction, hyperactivation and chromatin integrity of fresh or cryopreserved stallion spermatozoa after treatment with heparin or with calcium ionophore. We also examined the influence of spermatozoa storage (fresh vs. cryopreserved), capacitation treatment, oocyte maturation time and cumulus morphology on the penetration rate and fertilization rate. We recovered cumulus-oocyte-complexes (COCs) from ovaries by ultrasound guided follicle aspiration or by scraping of follicles from ovaries obtained at a slaughterhouse. Upon recovery, we evaluated the cumulus morphology, and the COCs were matured in vitro for 18 to 24 or 26 to 40 h. Fresh semen and cryopreserved semen were treated either with heparin (200 microg/mL) or calcium ionophore (7.14 microM). Overall, 28.4% (99/349) of the oocytes were penetrated, and 12.9% (45/349) were fertilized. Fresh spermatozoa treated with calcium ionophore showed a higher penetration rate than cryopreserved spermatozoa (36.0 vs. 0%). Fresh and heparin-treated spermatozoa showed a penetration rate of 29.1%, and the same treatment for cryopreserved spermatozoa showed a penetration rate of 33.7%; none of these differences was significant (P>0.05). Fertilization rates after the calcium and heparin treatment followed the same trend and also showed no significant differences. Prolonged maturation period resulted in higher penetration (P<0.05) and fertilization rates in compact (26 to 40 h: 37.7 and 13.1% vs. 18 to 24 h: 13.1 and 2.8%) and in tendency in expanded COCs (26 to 40 h: 40.0 and 30.3% vs. 18 to 24 h: 29.4 and 13.5%). In oocytes with only a few cumulus cells, the rates tended to be higher after the shorter incubation (18 to 24 h: 33.5 and 18.8% vs. 26 to 40 h: 17.2 and 6.5%). We observed hyperactivation more frequently in fresh than in cryopreserved semen after different treatments (43.2, 39.1 and 35.4% for heparin, calcium ionophore and control vs. 15.7, 10.8 and 5.7%, respectively). We observed significant changes in the acrosome reaction of fresh spermatozoa after heparin treatment (62.6 vs. 48.2%, P<0.05), as well as in cryopreserved spermatozoa after calcium ionophore treatment (31.7 vs. 17.6%, P<0.05). The chromatin integrity was significantly reduced after heparin treatment of fresh spermatozoa, in comparison to control and calcium ionophore (81.0 vs. 87.3 and 86.6, P<0.02). We also observed a similar reduction of chromatin quality after heparin treatment in cryopreserved spermatozoa, but the difference was significant only between heparin and calcium ionophore treatment [77.4 vs. 86.4 (P<0.02) and 84.9]. The results in the this retrospective study show that capacitating fresh spermatozoa with calcium ionophore, or using heparin in cryopreserved spermatozoa, results in higher penetration and fertilization rates of in vitro matured horse oocytes. A prolonged maturation time of 26 to 40 h is necessary for compact cumulus oocyte complexes to achieve the fertilization capacity. Further investigation is needed to show the developmental capacity of these fertilized oocytes.

Acrosome Reaction↗

PubMed2001

Platelet-activating factor (PAF)-like activity, localization of PAF receptor (PAF-R) and PAF-acetylhydrolase (PAF-AH) activity in bovine endometrium at different stages of the estrous cycle and early pregnancy.

PAF-like activity in the endometrium increased from days 2-4 to day 12 and day 20 in both cyclic and pregnant cows. There was an increase in platelet aggregation induced by PAF-like activity in the endometrium of pregnant animals on day 20 as compared to cyclic animals at the same point in time. Two major bands of PAF-R protein at 67 kDa and 97 kDa were detected by Western blot analysis. PAF-R was localized mainly in luminal and glandular epithelium of the endometrium, but the staining was markedly increased in the endometrium of pregnant cows on day 20 compared to cyclic animals on the same day. The purified PAF-AH from the endometrium is similar to in plasma. In cyclic cattle, no changes in PAF-AH activity of endometrium were observed, whereas a decrease in enzyme activity occurred in pregnant cows on day 20 as compared to cyclic animals on the same day. We suggest that the bovine endometrium produces PAF-like activity, expresses the PAF-R and possesses a PAF-AH activity which varies during pregnancy.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed2000

Nuclear configuration of bovine oocytes derived from fresh and in vitro-cultured preantral and early antral ovarian follicles.

The aim of this experiment was to characterize the growth and nuclear configuration of oocytes isolated from late preantral and early antral bovine ovarian follicles immediately after recovery and after the in vitro culture. Individual follicles were isolated by microdissection from slices of the ovarian cortex. Follicles were sorted by diameter into 175 to 224, 225 to 274 and 275 to 325 microm-size classes. The follicles selected for in vitro culture were placed singly into 40 microL droplets of medium (TCM 199 enriched with FCS, insulin, transferrin, sodium selenite, sodium pyruvate, 1-glutamine, hypoxanthine, FSH and estradiol-17beta) and cultured for 6, 8, 11, 14 or 17 d. The sizes of follicles and oocytes were related to the duration of culture and gradually increased as culture duration was prolonged. The analysis of the relationship between mean diameters of oocytes at the time of recovery and after the in vitro culture, has shown significant differences after culture lasting 8 d (76.9+/-9.9 vs. 86.1+/-11.1 microm; P < 0.05), 11 d (77.0+/-9.9 vs. 91.9+/-17.5 microm; P < 0.01), 14 d (80.0+/-9.5 vs. 97.9+/-16.5 microm; P < 0.01) and 17 d (82.6+/-6.6 vs. 97.2+/-11.5 microm; P< 0.01). No statistical differences were shown among oocytes in the 5 pre-culture groups (79.5+/-8.8; 76.9+/-9.9; 77.1+/-9.9; 80.1+/-9.5 and 82.6+/-6.6 microm). Meiotic arrest was preserved in 71.9% of oocytes in our culture system up to 14 d. Frequency of the germinal vesicle (GV) stage did not significantly differ among oocytes evaluated "fresh" or cultured for 6, 8, 11 or 14 d. No relationship was observed between the size class of follicles and the frequency of the GV-stage. Prolonging the culture period to 17 d drastically decreased the percentage of oocytes in the GV-stage (18.7%) and increased the percentage of oocytes having premature initiation of meiosis (GVBD; 46.3%) and degeneration (25.0%). These results suggest that out of all culture periods used in our experiment, Day 14 was found to be the longest culture time allowing for both oocyte growth and maintenance of nuclear configuration at the GV-stage.

Animals↗

PubMed1999

Effects of prolactin on intracellular stored calcium in the course of bovine oocyte maturation in vitro.

At present there are divergent opinions as to the role of prolactin (PRL) in the mechanisms of meiotic regulation in mammals. We investigated the effects of bovine PRL (bPRL) on bovine oocyte maturation in different culture systems and varying levels of intracellular stored calcium ([Ca2+]is) in the oocytes. Cumulus-oocyte complexes (COC) were incubated in TCM 199 containing either 10% fetal calf serum (FCS) in the absence (System 1) or presence (System 2) of FSH and estradiol, or 6 mg/mL bovine serum albumin (BSA) in the presence of FSH and estradiol (System 3). Levels of [Ca2+]is in oocytes were determined by using the fluorophore chlortetracycline. The addition of 50 ng/mL bPRL to different culture media increased the percentage of oocytes at telophase I and metaphase II stages (Systems 1 and 2) and/or decreased the percentage of oocytes with degenerated chromosomes (Systems 1 and 3). Compared with the control, lower levels of [Ca2+]is were observed in oocytes cultured for 2.5 h in those systems in which bPRL decreased the rate of oocytes with degenerated chromosomes (1.27+/-0.11 vs. 1.67+/-0.09 arbitrary units (AU) in System 1, P<0.001 and 1.27+/-0.12 vs. 1.52+/-0.04 AU in System 3, P<0.001). These findings show that the effects of bPRL on bovine oocyte maturation depend on the composition of the culture system and that the decline in the rate of oocytes with degenerated chromosomes in response to bPRL may be the result of the decrease in [Ca2+ ]is levels at early stages of oocyte maturation.

Animals↗

PubMed1998

Morphology of porcine cumulus-oocyte-complexes depends on the stage of preovulatory maturation.

The aim of this investigation was to determine the relationship between the morphology of the cumulus-oocyte-complexes (COCs) and the meiotic configuration of oocytes as an LH peak mimicked by hCG. Estrus was synchronized in a total of 29 crossbred Landrace gilts by feeding Regumate for 15 d and administering 1000 IU PMSG. The LH peak was simulated by treatment with 500 IU hCG at 80 h after PMSG. Endoscopic oocyte recovery was carried out 2 h before and 10, 22 and 34 h after hCG. Only macroscopically healthy follicles with a diameter of more than 5 mm were punctured. Altogether, 410 follicles from 57 ovaries were punctured and 251 COCs were aspirated. Oocyte recovery rate increased from 48.5% (P < 0.01) of the early, not yet preovulatory follicles (2 h before hCG) to 80.8% of late preovulatory follicles (34 h after hCG). Cumulus morphology in COCs recovered 2 h before and 10 h after hCG was heterogeneous, with most (72.9 to 57.4%; P < 0.01) showing a compact or slightly expanded cumulus. Starting at about 22 h after hCG, COC morphology changed dramatically (86.7% of COCs with expanded cumulus; P < 0.01), and 34 h after hCG, 98.3% of the COCs had only an expanded cumulus. The percentage of oocytes with a mature meiotic configuration increased (11.2; 7.1; 41.4 and 70.2%, respectively, n = 238 oocytes; P < 0.01) as the interval post hCG increased (-2, 10, 22, 34 h, respectively). Meiotic configuration was related to COC morphology: compact COCs--88.9% diplotene, expanded COCs--53.8% metaphase II (M-II), and denuded oocytes--69.2% degenerated chromatin. These results indicate that there is a relationship between oocyte recovery rate, COC morphology, and meiotic configuration and preovulatory follicle maturation after the application of hCG.

Animals↗

PubMed1997

Comparison of different methods for the recovery of horse oocytes.

The object of this study was to compare 4 different methods of oocyte recovery from mares; 1) transvaginal follicle aspiration in vivo; 2) follicle aspiration in vitro; 3) oocyte recovery by isolation of follicles in vitro and 4) follicle scraping in vitro. Oocyte recovery was highest after follicle scraping (71.1%) and follicle isolation and rupture (61.3%). Follicle aspiration in vitro and in vivo yielded oocytes on 31.2% and 19.3% of occasions, respectively. The output of different types of cumulus-oocyte-complexes was different among the methods; the portion of compact cumulus-oocyte-complexes was significantly higher with follicle scraping (50.7%) and follicle isolation (44.5%) than with aspiration in vivo (31.9%) and in vitro (23.7%). The recovery rate of oocytes from small follicles (<15 mm) was significantly higher than from larger follicles (P<0.05) using transvaginal follicle aspiration. The proportion of oocytes that were degenerate (exhibited shrunken, dense or visibly damaged ooplasm) ranged from 1.2% after follicle scraping, to 17.2% after aspiration in vivo. These results indicate that, for the recovery of horse oocytes in vitro, follicle scraping and follicle isolation give the highest recoveries of cumulus-intact oocytes.

Animals↗

PubMed1997

Neuropsychological aspects of driving after brain lesion: simulator study and on-road driving.

Twenty-nine patients with brain lesion and 29 matched controls participated in the study. The patients were socially well recovered with a high rate of employment. Compared with the controls, they performed significantly worse on a neuropsychological test battery, especially on executive and cognitive functions. Patients drove as well as controls in predictable situations in the advanced simulator used. In unpredictable situations, they demonstrated longer reaction times and safety margins, as well as difficulties in allocating processing resources to a secondary task. The patients showed significantly less attention, worse traffic behavior, and less risk awareness when driving in real traffic. Forty-one percent of the patients did not pass the driving test. The neuropsychological test battery was factor analyzed into four factors: executive capacity, cognitive capacity, automatic attentional capacity, and simple perceptual-motor capacity. The second factor was the mast significant with a simultaneous capacity test predicting driving performance with 78% confidence.

Journal Article↗