Vitrification of biopsied mouse embryos.

Solid surface vitrification (SSV) was compared with in-straw vitrification for cryopreservation of biopsied mouse embryos. Eight-cell stage embryos were zona drilled and one blastomere was removed. Developed morulae or blastocysts were vitrified in microdrop (35% EG + 5% PVP + 0.4 M trehalose) or in straw (7.0 M EG + 0.5 M sucrose). Following recovery, embryos were cultivated in vitro or transferred into recipients. Cryopreservation had an effect not only on the survival of biopsied embryos but also on their subsequent development in vitro. Cryosurvival of biopsied morulae vitrified in straw was significantly inferior to SSV. The post-warm development of biopsied and non-biopsied morulae was delayed on Day 3.5 and 4.5 in both vitrification groups. A delay in development was observed on Day 5.5 among vitrified non-biopsied blastocysts. The percentage of pups born from biopsied morulae or blastocysts following cryopreservation did not differ from that of the control. No significant differences could be detected between methods within and between embryonic stages in terms of birth rate. The birth rate of biopsied embryos vitrified in straw was significantly lower compared to the non-biopsied embryos. The novel cryopreservation protocol of SSV proved to be effective for cryopreservation of morula- and blastocyst-stage biopsied embryos.

Chilling sensitivity of carp (Cyprinus carpio) embryos at different developmental stages in the presence or absence of cryoprotectants: work in progress.

The unsolved problem of cryopreservation of the yolk-rich teleost embryos may be related, in part, to their sensitivity to chilling and cryoprotective agents. The aim of this study was to gain data on the sensitivity of carp embryos to low temperatures at different developmental stages and on the possible protective and toxic effects of cryoprotectants. A total of 86,400 morulae, half-epiboly and heartbeat-stage embryos was selected and then placed in water or in 1 M methanol, dimethyl sulfoxide (Me2SO), glycerol or 0.1 M sucrose solution at 0, 4 or 24 degrees C for 5 min or 1 h. Following these treatments, the embryos were held in a 24 degrees C water bath until the evaluation of hatching rates. In every developmental stage a significant decrease of hatching rates following exposure to 4 or 0 degree C was detected. Sensitivity to chilling changed significantly with development (heartbeat < morula < half-epiboly). Half-epiboly stage embryos were less sensitive to a short period of exposure to cryoprotectants than morula and heartbeat stages. A 1-h exposure to cryoprotectants revealed a stage dependent sensitivity. Toxicity increased in the order of methanol < Me2SO < glycerol in morula and half-epiboly stages, and methanol < glycerol < Me2SO in the heartbeat stage. The results show morulae are partially protected against chilling in Me2SO and sucrose, half-epiboly in Me2SO, sucrose and methanol, and heartbeat-stage in methanol and glycerol. The results further suggest that carp embryos are sensitive to chilling and that toxicity and protective effects against chilling of cryoprotectants are stage-dependent. The finding on the low chilling sensitivity of heartbeat-stage embryos and the protective effect of certain cryoprotectants may be useful in designing cryopreservation protocols.

Comparison of milk production of the progeny of BLAD-carrier and healthy Holstein bulls in Hungary.

'Bovine Leukocyte Adhesion Deficiency' (BLAD) is a recessive monofactorial, lethal inheritable defect occurring in Holstein-Friesian cattle and often passed on by well-known top bulls. The aim of this study was to find a relationship between the BLAD genotype of bulls, their genetic evaluation for milk and their daughters' milk production. BLAD-carrier and healthy bulls were compared on the basis of their breeding value published in November 1997. The first 100 bulls ranked according to the Total Production Index (TPI) were used, including nine BLAD carriers with 2,835 daughters and 77 healthy sires with 21,950 female progenies. For 14 bulls the BLAD genotype was not indicated. The healthy animals significantly outperformed the BLAD carriers, which result contradicts our earlier findings (Dohy et al., 1996; Jánosa and Dohy, 1997). In a BLAD elimination programme, the identification of BLAD carriers and properly planned mating are of great importance in order to avoid 'inter se' mating of BLAD-carrier top animals which can be of significant influence in Holstein breeding.

Comparison of different treatments for parthenogenetic activation of bovine oocytes matured in vitro.

In vitro matured bovine oocytes were treated by three parthenogenetic activation methods. Basic treatment with Ca-ionophore and cytochalasin D was combined with cycloheximide (Group 2), electric impulse (Group 3), and cycloheximide and electric pulse (Group 1) treatments, respectively. Survival and the in vitro development of parthenogenetic embryos to hatched blastocyst stage were compared. Rates of the first cleavage and morula development significantly differed among the treatment groups. Further development rates of the treated embryos up to blastocyst stage did not differ. The best results were obtained by the combination of cycloheximide and electric treatments (Group 1; 14% blastocyst, 7% hatched blastocyst). The results suggest that the combined treatment for oocyte activation is the most efficient and should be applied for cloning procedures.

Preimplantation genetic diagnosis in cattle: a review.

Preimplantation Genetic Diagnosis (PGD) is reviewed and novel fields where it may be applied are investigated. Technical advances of PGD in cattle embryos have already enabled its integration as a part of the MOET (Multiple Ovulation Embryo Transfer) breeding system. PGD for well-defined selection targets can enhance cattle breeding and embryo trade. It allows embryo selection according to their sex, and it may be used to breed special cow lines, or top bulls, by selecting embryos for valuable production traits using Marker Assisted Selection (MAS). A good allelic profile and/or the insertion of a transgene can be detected by PGD. This review article presents the technical requirements for PGD, and shows that this biotechnological method has great economic potential.

A simplified biopsy method for precompacted mouse embryos: a technical report.

This article presents a new, simple and rapid embryo biopsy method. The blastomere for genetic analysis can be separated from a precompacted mouse embryo after a partial zona digestion with the use of a holding pipette. For the micromanipulation only two microcapillaries and micromanipulators are needed. The development of the biopsied embryos was studied during in vitro culture and in utero following embryo transfer. There was no significant difference between the treated and the control groups in the ratio of embryos that developed to the blastocyst stage, although the biopsied embryos were delayed in their development because they contained significantly fewer cells compared to the control ones at the same stage. Although there was no difference in the ratio of implantation, the development of the biopsied embryos in utero was also delayed 12-24 hours on the 9th day of pregnancy. No difference in development was visible from the 13th day of pregnancy. Statistically, no differences were found in the developmental ratio (number of developed fetuses/transferred embryos) of the control and treated embryos during gastrulation (9th day of pregnancy), at the beginning of organogenesis (13th day of pregnancy) and before birth (19th day of pregnancy). The embryo biopsy method presented here can be a new and useful tool for preimplantation genetic diagnosis.

Effect of swim up and Percoll treatment on viability and acrosome integrity of frozen-thawed bull spermatozoa.

The aim of this study was to investigate the effect of the swim up and Percoll methods to select frozen-thawed bull spermatozoa with high quality membrane and acrosomal integrity and final concentration. Semen samples from six Holstein-Friesian bulls were examined. The whole experiment was repeated three times. Before and after both treatments, spermatozoa were subjected to a double-staining method and evaluated by brightfield light microscope using 40x dry, or 100x oil immersion objectives. The concentration of spermatozoa evaluated by haemocytometer was 8.8 x 10(7)/ml after thawing, and the percentage of live cells with intact acrosome was 45.8%. Both treatments significantly increased the proportion of live spermatozoa compared with no treatment, and the use of Percoll gradient resulted in a significantly higher percentage of living cells with an intact acrosome (88.2%) than the swim up method (69.4%). The concentration of spermatozoa after Percoll separation (9.3 x 10(6)/ml) was higher than that after the swim up method (5.8 x 10(6)/ml). These results indicate that spermatozoa with a higher viability and acrosome integrity can be obtained by Percoll separation than by the swim up method. Therefore the use of Percoll-treated spermatozoa in IVF systems can be more expedient.