Use of cross-species in-situ hybridization (ZOO-FISH) to assess chromosome abnormalities in day-6 in-vivo- or in-vitro-produced sheep embryos

Abstract

Causes of chromosomal differences such as mosaicism between embryos developed in vivo and in vitro may be resolved using animal models to compare embryos generated in vivo with those generated by different production systems. The aims of this study were: (1) to test a ZOO-FISH approach (using bovine painting probes) to detect abnormal chromosome make-up in the sheep embryo model, and (2) to examine the extent of chromosome deviation in sheep embryos derived in vivo and in vitro. Cytogenetic analysis was performed on day 6 in-vivo and in-vitro derived sheep embryos using commercially available bovine chromosome painting probes for sex chromosomes X-Y and autosomes 1-29. A total of 8631 interphase and metaphase nuclei were analyzed from 49 in-vitro-derived and 51 in-vivo-derived embryos. The extent of deviation from normal ovine chromosome make-up was higher (p < 0.05) in in-vitro-produced embryos relative to in-vivo-derived embryos (65.3% vs. 19.6% respectively) mainly due to diploid-polyploid mosaicism. Polyploid cells ranged from 3n to 8 n with tetraploids most predominant among non-diploid cells. The proportions of polyploid cells per mixoploid embryo in in-vitro-produced embryos ranged from 1.4% to 30.3%, in contrast to less than 10% among the in-vivo-derived embryos. It was concluded that in-vitro-derived embryos are vulnerable to ploidy change compared to their in-vivo counterparts. The application of ZOO-FISH to domestic animal embryos is an effective approach to study the chromosome complement of species for which DNA probes are unavailable.

Recent Advances in Reproductive Biotechnologies in Sheep and Goat

“…Co-culture systems in sheep and goat are usually based on including oviductal cells in TCM-199 medium. Semi-defined medium is also used often in small ruminant IVC based on the components of the sheep oviductal fluid (SOF), a medium containing essential and non-essential amino acids supplemented with BSA [48,43]. In our laboratory, sheep embryos cultured in SOF medium supplemented with amino acids and BSA at 38°C in 5% O2, 5% CO2, 90% N2 humidified atmosphere resulted in 19.2% blastocyst rates from total oocytes cultured [43].…”

“…It is obvious that artificial oocyte maturation and culture systems have detrimental effects on embryo development compared to their in vivo counterparts. Recent fluorescent in situ hybridization cytogenetic studies from our laboratory using whole chromosome painting probes have revealed that high incidence of chromosomal abnormalities in sheep blastocysts derived from IVP systems, compared to in vivo derived blastoctsts [43] emphasizing the inherent differences in the two production methods.…”

“…Surrounding cumulus cells are removed by gentle pippetting and washing in fertilization medium and groups of 40-50 oocytes are placed in 4-well dishes in 500 l of synthetic oviductal fluid (SOF) covered by 200 l of mineral oil [31,43]. Motile sperm are obtained by centrifugation of frozen-thawed semen in Percoll gradient (45%/90%) at 500 g for 10 min in room temperature.…”

“…Recent studies have shown that incubation of spermatozoa for a further 1 h period in media supplemented with heat-inactivated estrus serum enhances the capacitation process of spermatozoa (sheep and goat : [41]; deer : [45,46] ). In our laboratory we routinely add heatinactivated sheep serum (2%) to the IVF medium and incubate oocytes with motile spermatozoa for 17 hours resulting acceptable levels of blastocyst development [43]. Although 17 h co-incubation is practiced in IVF, it has been shown that even 4 h co-incubation was enough to obtain acceptable cleavage and blastocyst rates in sheep [41].…”

“…Semi-defined medium is also used often in small ruminant IVC based on the components of the sheep oviductal fluid (SOF), a medium containing essential and non-essential amino acids supplemented with BSA [48,43]. In our laboratory, sheep embryos cultured in SOF medium supplemented with amino acids and BSA at 38°C in 5% O2, 5% CO2, 90% N2 humidified atmosphere resulted in 19.2% blastocyst rates from total oocytes cultured [43]. This rate is confirmed by [38], who showed that SOF culture systems supplemented with BSA and amino acids produce high quality blastocysts in sheep.…”