S. Andries

Effect of cumulus cell coculture and oxygen tension on the in vitro developmental competence of bovine zygotes cultured singly

The customary practice in bovine in vitro embryo production (IVP) is to handle oocytes and embryos in groups; although there are several reasons for establishing an IVP system for individual embryos that allows for following a single oocyte from retrieval through development to the blastocyst stage. To date, reports of individual IVP are inconsistent, and in most cases, resulted in unsatisfactory blastocyst rates. The objective of this study was to develop an efficient system for routine in vitro culture of individual bovine embryos. Single culture of zygotes in 2 different culture volumes (20 and 500microL) yielded less than 3% blastocysts in experiment 1. In an attempt to improve these results, cumulus cells were added to the culture medium in experiment 2, after which blastocyst rates increased from 2.9 to 21.8% (P<0.05). The third experiment revealed that an atmospheric oxygen tension, which is commonly used with somatic cell coculture, was not beneficial during individual embryo-cumulus cell coculture, because it resulted in lower blastocyst rates (Odds ratio 0.57, P<0.001) and in lower blastocyst cell numbers (P<0.05), when compared to culture in 5% oxygen. Grouped vs. single culture and reduced oxygen tension did not have a significant effect on cleavage and hatching rates. In experiment 4, three different cumulus cell coculture conditions during individual culture were tested and compared with the cleavage, blastocyst and hatching rates, and cell number of group culture (73.2%, 36.4%, 66.7% and, 155.1+/-7.26, respectively). The outcome variables after individual embryo culture on a 5-day-old cumulus cell monolayer (74.1%, 38.2%, 71.9% and 133.4+/-9.16, respectively), and single culture in the presence of added cumulus cells (69.9%, 31.9%, 66.7% and 137.3+/-8.01, respectively) were not significantly different from those obtained after group culture (P<0.05). Though, individual culture in a cumulus cell conditioned medium significantly reduced both the cleavage (59.0%) and blastocyst rates (6.3%). These results demonstrate that single culture of bovine zygotes can be fully sustained by coculture with cumulus cells in a low oxygen environment; implementation of these findings in our IVP system produced blastocysts comparable in quantity and quality to those obtained by group culture. These results were consistently achieved after acquiring experience and expertise in the handling of single zygotes.

Follicle survival and growth to antral stages in short-term murine ovarian cortical transplants after Cryologic solid surface vitrification or slow-rate freezing

This study was designed to asses murine preantral follicle survival and growth, after cryopreservation of ovarian tissue by two different methodologies, solid-surface vitrification by the Cryologic vitrification method (CVM) and slow-rate freezing (SRF). Cryotreated tissue was stored in liquid nitrogen for 24h, and upon warming follicle viability was assessed by live/dead fluorescent probes, and by 7-day autotransplantation of both cryotreated tissue types to the left and right kidney capsule of the donor animals (n=16). The live/dead assay immediately upon tissue warming did not allow a distinction to be made in terms of follicle viability between the CVM and SRF cryoprocedure. In grafted tissue, follicular survival and growth was assessed by conventional histological examination and proliferating cell nuclear antigen immunohistochemistry. In each experimental group (control, CVM and SRF), follicles were classified according to developmental stage, and a comparison of the proportions of follicle stages between the three groups was executed by statistical analysis of variance. The fraction of primordial follicles in CVM and SRF grafts significantly decreased as compared to control tissue, whereas intermediary and primary follicles significantly increased. The proportion of secondary and antral follicles after SRF was significantly larger than after CVM, but did not differ significantly between CVM and control tissue. The observed massive follicle activation is a typical transplantation effect, but testifies to the survival of cryopreserved follicles. In both types of cryotreated tissue, growing follicles, including antral stage, were present in grafts from all recipient animals. The significantly more abundant further developed stages in SRF treated tissue, however, suggest that CVM treated tissue may have suffered a growth disadvantage. To our knowledge, this is the first time that the CVM technique has been utilized to vitrify preantral follicles.

Acute poisoning with amphetamines (MDEA) and heroin: antagonistic effects between the two drugs

A case of oral ingestion of large doses of both the amphetamine-derivative 3,4-methylene dioxyethamphetamine (MDEA) and heroin is reported. Despite high serum levels of both drugs, the patient did not present with the classic signs and symptoms normally seen during intoxication with these drugs. The patient recovered after symptomatic treatment. The possibility that opposite pharmacological properties of the two drugs prevented the patients death is discussed.