Search PubMed⌕ Search

Biomedical subjects

T Greve

Publications and source records attributed to T Greve.

At least 91 records · Page 5Linked to original sources
PubMed1995

Impact of Percoll on bovine spermatozoa used for in vitro insemination.

Percoll treatment of bovine frozen/thawed spermatozoa was identified as the cause of low cleavage and blastocyst rates in our in vitro embryo production system. Percoll treatment of spermatozoa yielded very high, stable and repeatable results for many months of cleavage and blastocyst rates of 83 +/- 5% (+/-SD) and 33 +/- 10%, respectively. This was followed by a period with significantly lower cleavage and blastocyst rates of 60 +/- 9% and 14 +/- 5%, respectively. In the last period, the results became even lower, with cleavage and blastocyst rates of 32 +/- 5% and 5 +/- 3%, respectively. These results were significantly different from those of the 2 previous periods. We were able to correlate precisely these changes in outcomes to the introduction of new Percoll batches. When compared with washed spermatazoa and under otherwise identical circumstances, the cleavage and blastocyst rates from spermatozoa treated with Percoll increased significantly from 33 to 73% and from 4 to 26%, respectively. We suggest that this adverse effect of Percoll is not due to Percoll particles per se, but may be ascribed to the effect of unbound polyvinylpyrrolidone (PVP) in the Percoll.

Journal Article↗
PubMed1995

Transcription and localization of growth factor mRNA in the bovine oviduct.

It has become evident that certain growth factors are involved in the regulation of the initial bovine embryogenesis. In the present study, we examined by means of Northern blot and in situ hybridization, the expression and localization in the bovine oviduct of mRNAs encoding for platelet-derived growth factor (PDGF-B), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF-I). Northern blot analysis on oviduct tissue demonstrated transcripts for PDGF-B and bFGF, but not IGF-I mRNAs. Two bands with estimated sizes of 5.0 and 1.5 kb were detected for PDGF-B and two bands with sizes of 7.5 and 4.9 kb for bFGF. In situ hybridization analysis demonstrated localization of PDGF-B mRNA in the lamina epithelialis and tunica muscularis of the oviduct whereas bFGF mRNA was detected in the lamina propria. It is concluded that the lamina epithelialis and lamina propria of the oviduct represent sites of synthesis of PDGF-B and bFGF mRNA, respectively.

Animals↗
PubMed1995

Structural and endocrine aspects of equine oocyte maturation in vivo.

The objectives were to describe the ultrastructure of equine oocytes aspirated from small and preovulatory follicles, and to relate the ultrastructural features to follicle size and follicular fluid steroid concentrations. Mares were examined every second day by transrectal ultrasonography, and follicles measuring > 30 mm were aspirated (in vivo) using a 20-cm-long 12-gauge needle through the flank. Following slaughter, both large and small follicles were aspirated (in vitro) from six mares. The oocytes were isolated under a stereomicroscope and processed for transmission electron microscopy, and the follicular fluid was assayed for progesterone (P4) amd estradiol-17 beta (E2). A total of 29 oocytes (32% recovery rate) were aspirated in vivo, and 15 oocytes were recovered in vitro. According to the stage of nuclear maturation, the oocytes could be divided into the following six categories: 1) the central oocyte nucleus (CON) stage, 2) the peripheral spherical oocyte nucleus (PON-I) stage, 3) the peripheral flattened oocyte nucleus (PON-II) stage, 4) the oocyte nucleus breakdown (ONBD) stage, 5) the metaphase I (M-I) stage, and 6) the metaphase II (M-II) stage. The maturation of the preovulatory follicle was reflected by alterations in the follicular fluid concentrations of steroid hormones. E2 was high in all preovulatory follicles, whereas P4 concentration exhibited a 10-fold increase during follicle maturation, particularly associated with the progression from M-I- to M-II-stage oocytes. The nuclear oocyte maturation included flattening of the spherical oocyte nucleus, followed by increasing undulation of the nuclear envelope, formation of the metaphase plate of the first meiotic division, and, finally, the extrusion of the first polar body and the subsequent formation of the metaphase plate of the second meiotic division. The cytoplasmic oocyte maturation changes comprised breakdown of the intermediate junctions between the cumulus cell projections and the oolemma, enlargement of the perivitelline space, the formation and arrangement of a large number of cortical granules immediately beneath the oolemma, the rearrangement of mitochondria from a predominantly peripheral distribution to a more central or semilunar domain, and the rearrangement of membrane-bound vesicles and lipid droplets from an even distribution to an often semilunar domain, giving the ooplasm a polarized appearance. It is concluded that the final equine oocyte maturation includes a series of well-defined nuclear and cytoplasmic changes that are paralleled by an increase in P4 concentration in the follicular fluid, whereas E2 concentration remains constantly high.

Animals↗
PubMed1995

Bovine oocyte diameter in relation to maturational competence and transcriptional activity.

The aims of the present series of experiments were to establish a possible relationship between bovine oocyte diameter and follicle size, investigate the developmental ability of oocytes of different diameter groups, and investigate the relationship between oocyte diameter and RNA transcriptional activity of the oocyte. Follicles were recovered from slaughterhouse ovaries by mechanical dissection, measured, and assigned to one of the following size categories: > or = 4 mm, < 3-4 mm, < 2-3 mm, < 1-2 mm, and < 1 mm. Oocytes were liberated from the follicles and their diameters recorded. The correlation coefficient between oocyte diameter and follicle size was 0.32 (P < 0.0001). Oocytes were recovered by aspiration and/or slicing of slaughterhouse ovaries and divided into four groups ( < 100 microns, 100- < 110 microns, 110- < 120 microns, and > 120 microns) based on diameter. Oocytes were processed through standard procedures for in vitro maturation and stained in order to assess nuclear development. Rates of in vitro development to metaphase II were 21.2%, 42.3%, 75.9%, and 80.7%, respectively, for the four groups. On a separate occasion immature oocytes from the above diameter groups were cultured in the presence of 3H-uridine for 45 min and scored for degree of RNA synthesis as indicated by the presence of autoradiographic labeling. Oocytes < 110 microns showed a greater degree of 3H-uridine incorporation than those > or = 110 microns, suggesting that they were involved in RNA synthesis and therefore still in the growth phase.

Animals↗
PubMed1995

Development of in vitro matured and fertilized bovine embryos, cultured from days 1-5 post insemination in either Menezo-B2 medium or in HECM-6 medium.

Bovine embryos were produced by in vitro maturation and fertilization of abattoir oocytes. The embryos were randomly allocated either to coculture with bovine oviduct cells in Menezo-B2 medium (control group), or to culture in the defined HECM-6 medium. At Day 5 after insemination the HECM-6 embryos were transferred to Menezo-B2 medium with (HECM-B2/BOEC) or without (HECM-B2) oviduct cells for further culture. The proportion of cleaved embryos and blastocysts, the morphology and the speed of development were compared for the control and HECM groups. Significantly more HECM-6 embryos than control embryos cleaved (88 +/- 3% vs 76 +/- 5% (+/- SD)). Significantly fewer blastocysts developed in the HECM-B2 than in the control group (28 +/- 2% vs 35 +/- 3%), in addition the speed of development was delayed and the morphology was impaired. In the HECM-B2/BOEC group no differences in neither morphology, blastocyst rates (31 +/- 8%) nor speed of development could be demonstrated, when compared with the control group. A portion of the control and HECM-B2 embryos were vitrified at Days 7-8, but no differences were noted in survival or morphology at 48 and 72 h post thawing. It can be concluded, that the defined medium HECM-6 can support bovine embryonic development through the 8-16 cell in vitro block stage without the use of coculture in a reliable way. In our system it was however necessary to transfer the embryos at Day 5 to coculture in Menezo-B2 medium to ensure optimal continuation of development.

Journal Article↗
PubMed1995

Factors affecting the developmental stage of embryos recovered on day 7 from superovulated dairy cattle.

The objective of this retrospective study was to evaluate the factors influencing developmental stage of bovine embryos recovered from superovulated dairy cattle 7 d after estrus. From 217 superovulated dairy cows and heifers, 2,211 eggs were recovered, of which 1,495 were classified as transferable embryos based on morphological evaluation of developmental stage and quality. From the evaluated embryos, 1,429 were non-surgically transferred to recipients to produce 623 calves. The transferable embryos were classified into five developmental stages and four quality grades. The least-developed transferable embryos tended to be classified into poorer quality grades. A multifactorial statistical model was used to analyze whether the following factors were associated with the developmental stage and quality grade of the embryos: donor breed, parity, gonadotropin preparation, embryo sex, insemination bull, embryologist (the person evaluating the embryo), year, and season of recovery. Among these factors, only the embryologist and the donor animal accounted for significant variation in embryo development. It was concluded that the developmental stage of embryos recovered at d 7 from superovulated cattle, when evaluated by simple morphological criteria, was correlated with the embryo's quality and was affected by the donor animal but in this study not by the embryo sex, donor breed and parity, gonadotropin preparation, and insemination bull used. The embryo's quality grading was influenced by the embryologist. Consequently, sexing of an embryo recovered from superovulated cattle is not possible by simple morphological evaluation of the embryo's developmental stage.

Animals↗
PubMed1995

Follicular development and ovulation in sows: effect of hCG and GnRH treatment.

Follicular growth, chronology of ovulation and embryo morphology were compared in sows ovulating spontaneously and sows, in which the ovulation was attempted induced by hCG or GnRH. Indwelling catheters were placed on day 1 (weaning = day 0) in the ear veins of 18 sows, which were then randomly divided into 3 groups: a control group (N = 6), a group (N = 6) given 750 iu hCG (Physex) im 76h after weaning (hCG group) and a group (N = 6) given 500 micrograms GnRH (Fertagyl) im 76h (N = 3) or 100h after weaning (N = 3) (GnRH group). Follicular diameter and time of ovulation were monitored by ultrasonography every 4h from day 3 until ovulation or development of cysts by means of a sector scanner fitted with a 5.0/7.5 MHz multiangle probe. Heat detection was performed every 8h from day 3 until ovulation. On day 13, the sows were slaughtered, the number of corpora luteae (CL) was counted, and embryos were flushed from the uteri. The control group showed clear heat symptoms, and on day 3, the follicles were typically 3-7 mm and grew up to 7-10 mm over 2 days, where they remained for approximately 24h until ovulation took place 41h +/- 9h after first sign of standing heat. The hCG group exhibited no signs of heat, and the follicles only reached 5-8 mm in diameter at time of ovulation, which occurred 40h +/- 1h after hCG-injection. The GnRH group exhibited inconsistent signs of heat, and the follicles reached a maximum size of 7-12 mm in diameter where they remained for more than 24h. Only 2 sows in this group ovulated within 84-92h after the GnRH injection, and development of bursa cysts and cystic follicles was a common finding. The average number of CL was 18.2 +/- 5.7 per sow (N = 16, range: 3-27) with no significant difference between the groups. Total embryo recovery was 79 +/- 13% with no significant difference between groups. The embryo diversity calculated as standard deviation of the maximum diameter was higher in the hCG group as compared with the control group.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Oocyte morphology in dominant and subordinate follicles.

The structure of oocytes aspirated from the dominant and its subordinate follicles was investigated from the achievement of follicular dominance to ovulation. Ovulation was induced in 18 heifers and 5 cows by injection of cloprostenol at days 8-14 (day 0 = day of ovulation), and follicular development was monitored by ultrasonography. The animals were slaughtered at days 3-11, but animals slaughtered on days 8-11 were given a second injection of cloprostenol at day 7 to allow ovulation of the dominant follicle of the first follicular wave. Oocytes were aspirated from the dominant (largest) and two largest subordinate follicles and processed for transmission electron microscopy, whereas the follicular fluids were analyzed for concentrations of estradiol-17 beta (E2) and progesterone (P4). Dominant follicular growth was associated with increase in the concentration of E2 and P4 in the follicular fluid, which was E2-dominated. From days 3-7, the dominant oocytes had pronounced junctional contacts with the cumulus cells and a nonundulating nuclear envelope but showed an increase in the number of lipid droplets and a decrease in the size of Golgi complexes, the size of cortical granule clusters, and the number of microvilli stacks. After cloprostenol injection on day 7, but before the anticipated LH surge, the dominant oocytes showed a reduced oocyte-cumulus contact, vacuolization of the nucleolus, undulation of the nuclear envelope, and dispersal of the mitochondrial clusters. The morphological alterations occurring in the dominant oocytes before the anticipated LH surge are suggested to be a prerequisite for the oocyte to achieve the competence to undergo final maturation. Subordinate follicles ceased growing at about days 3-4 and their follicular fluid had low E2:P4 ratio or was P4-dominated. Subordinate oocytes displayed degenerative features in their cumulus investment and nuclear activation and maturation especially after day 5. The structural changes associated with oocyte degeneration showed similarities with the processes seen before and during final maturation of the dominant oocytes.

Animals↗
PubMed1994

Ultrastructural features of secretory cells in the bovine oviduct epithelium.

The non-ciliated (NC) cells of the bovine oviduct epithelium, have been shown to release embryotrophic substances to the oviduct lumen. The aim of the present study was to investigate the ultrastructure, focusing on aspects of the secretory machinery, of NC cells in different segments of the oviduct during and after transoviduct migration of zygotes and embryos. Dairy heifers (n = 8) were superovulated with an ECG/cloprostenol regimen, and the time of ovulation was estimated by ultrasound scanning. Samples from the infundibulum, ampulla, isthmus and uterotubal-junction of the oviduct were surgically collected from animals at 19-96 h and 7 1/2-8 1/2 days after ovulation and processed for transmission electron microscopy, following standard procedures. The NC cells contained characteristic membrane-bound secretory granules composed of a lamellar cortex encaging an amorphous medulla. The two components could still be recognized during extrusion of the granule content into the oviduct lumen by exocytosis. During granulogenesis, small maturing granules without the lamellar structure were observed, but distinct condensing vacuoles were absent. An abundance of granules was found in the early versus the late group. In both groups the uterotubual junction was almost free of granules. This segment, on the contrary, was characterized by the presence of primary and secondary lysosome-like bodies. In the early group the intracellular location of the granules varied between oviduct segments.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Acrosomal status in fresh and frozen/thawed stallion spermatozoa evaluated by scanning electron microscopy.

Semen from stallions with equal fertility at natural services, but yielding semen with either satisfactory or poor fertilizing capability after freezing and thawing, was processed for scanning electron microscopy before and after freezing and thawing. In all fresh semen samples the following three categories of acrosomal defect were noticed: (1) minor fenestrations of the plasma membrane (PM) and outer acrosomal membrane (OAM), (2) complete vesiculation and loss of PM and OAM and (3) lack of a large circular part of PM and OAM. The frequency of these defects ranged from 15% to 27%. All frozen and thawed samples displayed defects of categories 1-3 at similar frequencies as the fresh ones. However, additional defects categorized as: (4) major fenestrations of PM and OAM and (5) complete vesiculation of PM and OAM without loss of the vesicles were noted upon freezing and thawing. The total frequency of defects categorized as 4 and 5 ranged from 8% to 21%, and they seemed to be more frequent in stallions with poor fertility after freezing and thawing although the difference was not significant. Moreover, a particular defect categorized as (6) loosening of the whole acrosomal cap was found exclusively in stallions yielding semen with poor freezability.

Acrosome↗
PubMed1994

Follicular development and embryo recovery following 3 versus 8 FSH injections in heifers.

Ovarian follicular dynamics and embryo yield were studied during 2 different FSH regimens for superovulation of cattle. Twenty heifers were given intramuscular injections of FSH (total of 35 mg NIH) either once daily for 3 days (Group 3x1) or twice daily for 4 days (Group 4x2). At 72 h after the first FSH injection, each animal was injected with 0.75 mg cloprostenol. Inseminations were performed at 12 h and 24 h after the onset of heat. Transrectal ultrasonography was performed on the day of the first FSH injection, the day of cloprostenol injection, the day of insemination and finally on the day of embryo recovery (day 6 or 7 after heat). The numbers of small (2-4 mm), medium (5-9 mm) and large (> 10 mm) size follicles were recorded. The total number of corpora lutea, eggs and transferable embryos were recorded on the day of embryo recovery. No differences were found between the 2 groups in either of the parameters studied (p > 0.05). It can be concluded that treatment with this FSH preparation once daily for 3 days gives a folliculogenic and superovulatory response similar to a treatment regimen where it is given twice daily for 4 days.

Animals↗
PubMed1993

Follicular dynamics prior to and during superovulation in heifers.

The present ultrasonographic study examined the relationship between certain follicular parameters and the superovulatory response in gonadotropin-stimulated heifers. Thirty heifers received a total of 35 mg FSH twice daily for 4 d and 0.75 mg cloprostenol were given to induce luteolysis and estrus at 72 h after the initial FSH injection. Transrectal ultrasonography was performed once daily from 1 or 2 d before the initial FSH injection and until the day of estrus. The number of small (2 to 4 mm), medium (5 to 9 mm), and large (>/=10 mm) size follicles as well as the diameter of the large follicles were recorded. Embryos were recovered non-surgically 6 or 7 d after estrus, and the number of corpora lutea was determined by palpation per rectum. Heifers with >2 or </=2 corpora lutea were classified as responders or nonresponders, respectively. All follicular categories were affected by treatment with FSH in the responders (P<0.0001), while in the nonresponders only small follicles and the total number of follicles showed a change after treatment (P<0.05). All follicular categories were different between the 2 groups (P<0.005). There was no effect of corpus luteum location (ipsilateral vs contralateral) on any of the follicular categories (P>0.05). The number of large follicles and the sum of medium and large follicles were positively correlated (r=0.43 and r=0.54, respectively; P<0.05) with the number of corpora lutea palpated on the day of embryo recovery (6 to 7 d after estrus). In conclusion, there was an effect of the day relative to initiation of FSH treatment on all follicular categories in heifers responding positively to superovulation, and there was no effect of side (left or right ovary) or of corpus luteum diameter (ipsilateral or contralateral).

Journal Article↗
PubMed1993

Character of preovulatory follicles and oocytes after different superovulatory treatments in heifers.

The relationship between the opaqueness of the surface of bovine preovulatory follicles, degree of expansion of oocyte-cumulus investment, presence of perivitelline space, and abstriction of the first polar body was examined in heifers treated with PMSG (Group I), FSH-P (Group II), and FSH-P/GnRH (Group III). Follicles greater than 8 mm in diameter were inspected by laparoscopy 65 hours after treatment with cloprostenol in all groups and were classified as clear or opaque based on their surface appearance. Subsequently, oocytes were recovered from the follicles and characterized. The proportion of clear exceeded that of opaque follicles in all groups. The oocyte recovery rate was highest for clear follicles in Group I and II, while in Group III the rates were identical for clear and opaque follicles. The proportion of oocytes with expanded cumulus investment was highest in Group III. In Group II and I decreased proportions of expansion was seen especially among oocytes from opaque follicles. The proportion of oocytes with perivitelline space was highest in Group II and III. Again, this effect was most pronounced among oocytes from "opaque" follicles. The proportion of oocytes with a polar body was highest in Group III followed by Group II while only few oocytes in Group I displayed a polar body. It is concluded that treatment with FSH-P and especially in combination with GnRH reduced the incidence of follicular atresia as measured by opaqueness and improved oocyte quality as indicated by cumulus expansion, formation of the perivitelline space, and abstriction of the first polar body.

Journal Article↗
PubMed1993

Pronucleus formation in bovine oocytes activated by a single electric pulse.

Bovine oocytes matured in vitro were stimulated by a single pulse of direct current (DC), then cultured for 0.5-6 h and evaluated by light (LM) and transmission electron microscopy (TEM). At the light microscopic level, the beginning of oocyte chromatin decondensation was first observed 3 h post-activation (14%). A well-developed pronucleus with evenly granulated nucleoplasm surrounded by nuclear membrane was found in 12, 61 and 81% oocytes at 4, 5 and 6 h post-activation, respectively. The TEM evaluation revealed that nuclear membrane vesicules were first visible at 0.5 h post-activation and became even more prominent at 1 h. Based on these observations, it is concluded that a nuclear membrane starts to form immediately after oocyte activation, while a well-developed pronucleus appears at 4-6 h.

Animals↗
PubMed1993

RNA synthesis in porcine blastomere nuclei introduced into in vitro matured ooplasm.

The objective was to investigate the RNA synthesis in porcine blastomere nuclei upon transplantation into in vitro matured enucleated oocytes. Nuclei from 2- to 8-cell porcine embryos were introduced into the ooplasm of in vitro matured and enucleated porcine oocytes by electrofusion, and the resultant reconstructed embryos were cultured in vitro. Before fusion or at different intervals after this event embryos were incubated with [3H]-uridine, fixed, and histologically processed for autoradiography in order to detect RNA synthesis. About two thirds of the embryos were considered to depict normal development. All blastomeres displayed pronounced RNA synthesis before fusion, at 3 and 9 h after fusion the synthesis decreased or ceased, and at 24-49 h some embryos resumed synthesis at the 1- to 2-cell stage.

Animals↗
PubMed1992

Morphological development and sex of bovine in vitro-fertilized embryos.

Bovine in vitro-fertilized embryos at the blastocyst stage were collected at days 7, 8, and 10 postinsemination and sex was determined via the polymerase chain reaction (PCR) to compare the embryonic development with the sex of the embryos. The percentages of males (sex ratio) after division of the embryos into three developmental groups were 68%, 48%, and 35% in the fast, intermediate, and slow groups, respectively (P = 0.014). The percentages of males on days 7, 8, and 10 were 60%, 40% and 33%, respectively (P = 0.043). The average sex ratio for the whole material was 50%. It is thus concluded that male bovine preimplantation embryos develop faster than female embryos.

Animals↗