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Biomedical subjects

T Greve

Publications and source records attributed to T Greve.

At least 109 records · Page 6Linked to original sources
PubMed1992

The freezability of biopsied bovine embryos.

A biopsy of 1-5 blastomeres was taken from 86 bovine compacted morulae, using a technique, that did minimal damage to the zona pellucida. As soon as possible after the micromanipulation the embryos were frozen, without sealing the penetrated zona pellucida. In order to evaluate the viability of the biopsied frozen-thawed embryos, half of them were transferred to synchronized recipients while the remaining half were co-cultured to evaluate the developmental capacity. A control group of 43 intact embryos was subjected to the same procedures. This study revealed a slight, but not significant, decrease in the pregnancy rate of the biopsied embryos after freezing and thawing, although the embryos by co-culture with bovine oviduct epithelial cells revealed normal morphology and developmental rate. It is concluded that the described biopsy technique did not compromise the freezability of 7-day-old bovine embryos.

Journal Article↗
PubMed1992

Use of PMSG antiserum in superovulated cattle?

Two Pregnant Mare Serum Gonadotrophin (PMSG) antisera were tested in 174 dairy cows that were superovulated with PMSG and were then given prostaglandin at 60 hours after PMSG. At 48 hours after injection of prostaglandin, the cows were given either PMSG antiserum (monoclonal (n=56) or polyclonal (n=57)), or saline as control (n=61). Ova (n=1,206) were recovered either nonsurgically or after slaughter. Of these, 757 were evaluated morphologically to be transferable embryos. A proportion of these embryos (n=295 from 52 flushed donors) were transferred to synchronized recipients and the pregnancy results were recorded. The reproductive function of 37 flushed donors was followed for 6 months after superovulation. No significant effect of the PMSG antisera could be demonstrated in any of the parameters studied (i.e., ovulation rate, number of follicles at collection, total yield of ova, fertilization rate, number of transferable embryos, pregnancy results after transfer of embryos, or period required by the donor cows for restitution of reproductive function after superovulation and recovery). It is concluded that use of PMSG antiserum did not improve the embryo yield in terms of the number and quality of transferable embryos or enhance normalization of reproductive function of the donor in the 6-month period after superovulation. Therefore, in an embryo transfer operation, the routine use of PMSG antiserum in a PMSG superovulation regimen in cattle is not recommended.

Journal Article↗
PubMed1992

Coculture of bovine demiembryos prior to freezing.

Pregnancy rates after transfer of frozen/thawed bisected embryos, demiembryos, have until now been very low. In the present study it was attempted to improve the freezability rate of demiembryos by culturing them on a monolayer of bovine oviduct epithelial cells (BOEC) prior to freezing. The cultured frozen/thawed demiembryos showed a lower developmental rate than intact embryos. The pregnancy rate (23%) following transfer was not different from the pregnancy rate after transfer of unfrozen demiembryos (26%). The calving rate (4%), however, was significantly lower than the calving rate after transfer of fresh demiembryos (23%). Although the overall pregnancy rate achieved in this study was low, it can be concluded that a co-culture period upon BOEC prior to freezing does not improve the viability of frozen demiembryos.

Animals↗
PubMed1992

Impact of incubator type on the yield of in vitro produced bovine blastocysts.

Because of suboptimal in vitro production of bovine blastocysts a new incubator model (Mini) was tested against the traditional (Heraeus). The difference between their properties seemed only to be the volume of the incubator space. No difference was noted between the CO2 or the temperature, but the data clearly showed a highly significant increase of the blastocyst rates, 6% versus 51% in the Heraeus and the Mini incubator, respectively, calculated as blastocysts per cleaved embryos. It was concluded that the incubator type or model may be a very important part of the in vitro production of bovine embryos, although we were not able to pin point specific causes for this difference.

Animals↗
PubMed1992

Oviduct epithelial cell co-culture of early porcine embryos.

One- to 16-cell porcine embryos were cultured in either Whittens medium supplemented with bovine serum albumin and fetal calf serum (WM) or in the same medium with porcine oviduct epithelial cell co-culture (WM-Poec). All stages of embryos cultured in WM-POEC had higher cell counts after 144-168 h of development than did embryos in WM. There was however, no significant difference in blastocyst formation rate of embryos cultured in WM-POEC over those cultured in WM. A high proportion of the embryos entering culture at the 1-2-cell were able to pass the 4-cell block stage in both WM and WM-POEC, 81% and 77%, respectively. In both media, most of the 1-2-cell embryos arrested their development at the compacted morula stage and failed to blastulate while embryos initiating culture at the 4- and 8-16-cell embryos formed blastocysts in culture at a rate of 80-90%.

Animals↗
PubMed1992

Blastomere content of cultured/frozen bovine demiembryos.

The purpose of the study was to evaluate whether a period of co-culture with bovine oviduct epithelial cells (BOEC) could improve the tolerance of bisected bovine embryos to freezing and thawing. Day 6 embryos were bisected and the resulting demiembryos were stained with Hoechst 33342 and cell counts were made by counting intact blastomere nuclei. Of these, 11 were stained as freshly manufactured demiembryos, 25 after co-culture for 24 h with BOEC and 37 stained after 24 h co-culture and freezing and thawing. The staining revealed, that there was no significant difference in cell count of demiembryos that were stained immediately after bisection, compared to those, that were co-cultured for a 24 h period. Also, the co-cultured/frozen/thawed demiembryos had a significant decrease in cell numbers compared to the non-frozen demiembryos. We conclude, that a 24 h period of co-culture with BOEC does not result in appreciable cellular proliferation in demiembryos and therefore instead of improving the survival of frozen/thawed demiembryos by giving them opportunity to multiply their cell number and thus make them more resistant to cell damage, rather compromised the viability of cryopreserved demiembryos.

Animals↗
PubMed1992

[Inhibition of tumor induction by methylnitrosourea in resected urinary bladder].

In the present experiments the dependence of tumour induction upon the different phases of the cell cycle in the proliferating urinary bladder was examined. For stimulation of urothelial proliferation, a one-third resection of the bladder was performed in female Wistar rats. To synchronize the proliferating urothelial cells, hydroxyurea was given. The direct-acting urothelial carcinogen N-methyl-N-nitrosourea (MNU) was administered as a single intravesical dose during different cell cycle phases. The incidence of urothelial bladder tumors was 32.6% in the controls. By comparison, the tumour incidences were 18.9, 9.3, 21.7, 26.3, 25.0 and 30.0%, respectively, when MNU was instilled during the late Gi-, early and late S-, G2+M-, and early and late postmitotic phase. The results obtained from a total of 283 rats clearly document a cell cycle specific inhibition of tumour development in the proliferating urinary bladder particularly when the carcinogen was administered during the early S-phase. MNU has also been shown to produce mesenchymal tumours in the bladder (overall incidence: 4.9%) as well as urothelial tumours in the renal pelvis (3.2%) and ureters (1.4%).

Animals↗
PubMed1991

Sex and development in bovine in-vitro fertilized embryos.

In vitro fertilized bovine embryos were karyotyped at Day 7 and Day 8 post insemination. The results showed that the percentage of males (sex ratio) was dependent on the developmental stage. Among expanded blastocysts (the most advanced embryos), the sex ratio was 100%, followed by 89% for expanding blastocysts, 75% for blastocysts, and 40% for young blastocysts, all analyzed at Day 7. For embryos karyotyped at Day 8, the sex ratio was 20%. The average mitotic index for all in vitro fertilized embryos was 3.5%. These results suggest that the apparent relationship between sex and developmental rate could be used as a method for noninvasive sexing of in vitro fertilized embryos.

Journal Article↗
PubMed1991

In vitro fertilization in cattle: a review.

In vitro maturation and fertilization of cattle oocytes and subsequent in vitro culture of zygotes and embryos is discussed in the context of recent encouraging data. Mass production of embryos produced in this way in the future will have a great impact on animal production and animal breeding plans, for example the so-called MO-ET (multiple ovulation and embryo transfer) plan which aims at establishing nucleus breeding herds.

Animal Husbandry↗
PubMed1991

The effect of endotoxin-contaminated medium on in vitro fertilization and development of bovine oocytes matured in vitro.

The purpose of this study was to determine whether the presence of bacterial endotoxin in the bovine serum albumin (BSA) used to supplement media utilized for sperm preparation and co-culture of bovine sperm and oocytes affects in vitro penetration and embryonic development of oocytes matured in vitro. The chromogenic limulus amoebocyte lysate (LAL) test was used for quantification of the content of endotoxin. The proportion of penetrated ova was significantly greater (P greater than 0.0005) for the endotoxin-contaminated group (89%) versus the non-contaminated group (61%), but this was probably not due to endotoxin contamination. The presence of endotoxin resulted in a high rate of polyspermy (27% versus 4%, respectively; P greater than 0.0005), while the occurrence of parthenogenetic activation was the same for each group (8%). The proportion of total embryos put into culture that developed to the blastocyst stage by day 8 was similar (30% and 26%) for the contaminated and non-contaminated group, respectively. Fifty-three and 69%, respectively, hatched on day 10. These results suggest that endotoxin induces polyspermy, but has no adverse effect on embryonic development.

Animals↗
PubMed1991

Bisection of bovine morulae and blastocysts from superovulated Danish dairy cows.

Sixtyfour compacted morulae and blastocysts were bisected with a microscalpel. The majority of the demi-embryos (n = 122) were reinserted into separate zona pellucidae (ZP) before non-surgical transfer to 113 synchronized recipients, as singles (n = 98) (DE-S) or in pairs (n = 30) (DE-P). Thirty non-manipulated embryos (E) were transferred during the same period and served as controls. Pregnancies were diagnosed by rectal palpation 4-7 weeks after transfer. The pregnancy rates for DE-S, DE-P and E were 32%, 53% and 40%, respectively (P greater than 0.05). A substantial number of abortions were recorded between 50 and 250 days of pregnancy among the recipients with DE-S. The fetal survival rate for DE-S was reduced to 21% and significantly lower (p less than 0.05) than the survival rates of DE-P (43%) and E (40%). The quality of DE and the presence of ZP did not significantly influence the results. No conclusive reasons for the fetal loss could be found but different possibilities are discussed.

Animals↗
PubMed1990

Preovulatory plasma estradiol-17beta concentrations and ovulation rates in PMSG/anti-PMSG treated heifers.

Possibilities for early characterization of the superovulatory response were studied in 41 PMSG/PG-treated dairy heifers, of which 21 received an additional treatment of PMSG-antiserum. Plasma was obtained at 33, 36, 41, 47 and 51 h after PG for hormone analyses. After slaughter at 6 or 7 d after insemination, the number of follicles and corpora lutea (CL) were recorded, and ova were recovered for morphological evaluation. Significant correlations were demonstrated between plasma concentrations of estradiol-17beta (E2) at 33, 36 and 41 h after PG and the ovulation rate (number of CL). Each of these correlations was equal to the one found by using the peak concentration of E2 achieved during the preovulatory E2 surge. In heifers with preovulatory E2 surges, as determined with the blood sampling scheme used, both the ovarian response (number of CL and follicles) and the quality of ova recovered (number of transferable embryos) was clearly better compared to heifers without this surge. None of the parameters studied was affected significantly by treatment with PMSG-antiserum. It is concluded that plasma E2 determinations at fixed times in relation to prostaglandin treatment can be used to characterize the superovulatory response in donor cattle in terms of the ovulation rate and the quality of ova recovered. No evidence was found in favor of using PMSG-antiserum for improving either the superovulatory response or such characterization.

Journal Article↗
PubMed1989

A comparative ultrastructural study of in vivo versus in vitro fertilization of bovine oocytes.

Heifers were superovulated by PMSG or FSH, and oestrus was induced by prostaglandin. One group of animals was ovariectomized 19-26 h after the LH peak, the content of preovulatory follicles aspirated, and the oocytes processed for in vitro fertilization. Another group was inseminated and ova were collected from the oviducts for study of in vivo fertilization. All ova were examined ultrastructurally. The developmental rate following in vitro fertilization was delayed compared to fertilization in vivo. A high proportion of the in vitro fertilized ova showed polyspermic penetration of the zona pellucida, and supernumerary spermatozoa were found in the ooplasm of some ova. In vivo fertilization was associated with release and subsequent dispersal of the cortical granule content in the perivitelline space. In contrast to this the released granule content of the in vitro fertilized ova remained undispersed close to the oolemma. This feature may account for the high incidence of polyspermic penetration of the zona pellucida. In addition, the study provided an ultrastructural visualization of the initial contact between the equatorial segment of the spermatozoon and the microvilli of the oocyte, and the subsequent internalization of the sperm head.

Acrosome↗
PubMed1989

A comparative study of attachment of human, bovine and mouse blastocysts to uterine epithelial monolayer.

The in-vitro attachment of human, bovine and murine blastocysts to monolayer cultures of uterine epithelium were studied by transmission electron microscopy. The human trophoblastic cells intrude between uterine epithelial cells forming a multilayer during attachment in vitro, thus resembling the intrusive type of penetration observed in vivo. The bovine trophoblastic outgrowth resembled an epitheliochorial attachment as the trophoblast formed an attachment plate on top of the endometrial cells without penetration. In the murine attachment study, the trophoblast cells immediately displaced the uterine cells and formed contact with the culture vessel.

Animals↗
PubMed1989

Sex determination of bovine embryos based on embryonic cleavage rates.

Bovine embryos (n = 64) collected at day-6 from 10 superovulated and inseminated donors were evaluated upon recovery and again after incubation for approx. 12 hrs. According to cleavage rate they could be divided into a fast, an intermediate and a slow developing group, respectively. This grouping was correlated to embryonic sex determined by karyotyping (n = 29). From the fast developing group we found 7 males and 2 females (male ratios 78%), from the intermediate group 3 males and 8 females (male ratio 27%) and from the slow group 1 male and 8 females (male ratio 11%). From this preliminary study it is concluded that the rate of embryonic development may be used as a sex discriminator.

Animals↗
PubMed1989

Follicular correlates with in-vitro fertilization in cattle.

The follicular microenvironment may affect the quality and the fertilizability of an oocyte in vivo as well as in vitro. Determination of the concentrations of certain follicular fluid compounds upon aspiration could therefore be used as important clinical measures to predict the developmental ability of an oocyte submitted to in-vitro fertilization. In cattle gonadotrophin stimulation (superovulation) frequently leads to perturbed follicular steroidogenesis and oocyte maturation, and fertilization rates in vivo as well as in vitro may be reduced. These abnormal changes are monitored in the peripheral profiles of LH, progesterone and oestradiol-17 beta during the periovulatory period, and measurements of these hormones may be used to discriminate between good and poor oocyte donors. Concurrent measurements of progesterone and oestradiol-17 beta in the aspirated follicular fluids and determination of the cytogenetic features of the oocytes will reflect these deviations but, because the variation between follicles within an animal is so large, it is not possible to rely on single hormone determinations; if attempted they should be interpreted with caution. Presently, no non-invasive method such as steroid measurement and flow cytometry accurately reflects oocyte quality and function in cattle and it is seriously questioned whether such clinically applicable methods are valid. It is suggested that evaluation of oocyte quality is done on three levels: (1) the animal (peripheral hormones), (2) the ovary (stimulation, premature ovulation, follicular appearance); and (3) the follicle (cumulus oophorus complex, steroids).

Animals↗
PubMed1989

Ultrastructural aspects of oocyte maturation and fertilization in cattle.

The preovulatory surge of LH triggers follicular and oocyte maturation in cattle. Oocyte maturation includes disruption of the gap junctions between cumulus-cell projections and oocyte and the breakdown of the envelope of the oocyte nucleus within 12 h after the LH peak; at approximately 15 h metaphase of the first meiotic division occurs and spatial rearrangements of mitochondria and vesicles are seen in the ooplasm; at approximately 19 h the first polar body is abstricted and the second metaphase appears; and at 21-22 h the cortical granules migrate to solitary positions along the oolemma, the Golgi compartment decreases, and the smooth endoplasmic reticulum (SER) transforms. Ovulations occur in unstimulated and superovulated cattle at approximately 24 h and 24-33 h, respectively. The acrosome reaction, which is preceded by swelling of and appearance of small vesicles in the acrosome, is completed on the surface of the zona pellucida. During the subsequent gamete fusion the microvilli of the ovum contact the equatorial segment of the sperm head, and the acrosomal region is subsequently internalized into the ooplasm surrounded by a vesicle. Within the following 2-3 h the formation of the maternal and paternal pronucleus is initiated, the cortical granules are released, conspicuous Golgi complexes develop, and the SER is transformed; at 5-7 h the pronuclei enlarge, and arrays of annulate lamellae develop. Subsequently, the pronuclei migrate close together; at approximately 20 h the envelopes of the pronuclei are broken down and synkaryosis is seen; and at approximately 24 h the 2-cell stage emerges. Artificial control of oocyte maturation and fertilization in cattle may lead to deviation in these processes. Superovulation may affect oocyte maturation adversely, and in-vitro fertilization may lead to increased frequencies of polyspermy due to deviations in cortical granule release and dispersal. Knowledge about these basal processes of oocyte maturation and fertilization are fundamental in the context of egg manipulation (oocyte enucleation at cloning, gene injection into pronuclei at specific stages etc.) in cattle.

Animals↗