Cytogenetic analysis of caprine 2- to 4-cell embryos produced in vitro

Abstract

Prepubertal goat in vitro matured/in vitro fertilised oocytes produce only a small percentage of blastocysts. The present study examines the incidence of chromosomal anomalies in 2- to 4-cell embryos in vitro produced (IVP) from prepubertal oocytes fertilised with the semen of two males. Cumulus-oocyte complexes were obtained by slicing ovaries from slaughtered prepubertal goats. Oocytes were matured in TCM199 supplemented with 20% heat inactivated Donor Bovine Serum (DBS), 10 μg/ml FSH + 10 μg/ml LH + 1 μg/ml 17β-oestradiol for 27 h at 38.5 °C in 5% CO2 in air. IVM oocytes were inseminated with the sperm from two males prepared using the swim-up and heparin-capacitation procedures. At 24 h post-insemination (hpi) the oocytes were transferred to 100 μl drops of SOF medium for a further 24 h. At 17 hpi a sample of oocytes was stained with lacmoid to evaluate the nuclear stage after fertilisation. The cleavage rate was determined at 24, 36 and 48 hpi and chromosome slides were prepared according to the gradual-fixation technique and stained with Leishman. A total of 1070 2- to 4-cell embryos from prepubertal goat oocytes were studied, but it was only possible to analyse 241 cytogenetically. Of these, 40% exhibited a normal diploid chromosome complement, 59% were haploid and 1% were triploid. There were significant differences between the two males in sperm oocyte penetration and oocyte cleavage but no differences were found in chromosomal anomalies. In conclusion, the low number of embryos karyotyped and the high number of haploid embryos found in this study suggested a high incidence of abnormal fertilised embryos and deficient cytoplasmic maturation of the oocyte which inhibits sperm head decondensation.

Developmental competence of prepubertal goat oocytes selected with brilliant cresyl blue and matured with cysteamine supplementation

“…In prepubertal goat oocytes, the high incidence of asynchronous fertilisation (oocytes with a feminine pronucleus and intact sperm head) could be due to the low intracytoplasmic GSH levels of these oocytes. Previous studies with prepubertal goats have shown that the asynchronous fertilised oocytes develop to haploid embryos [11]. In our study the percentage of cleaved embryos in the control group (42.3%) was higher than the percentage of normally fertilised (2PN) oocytes (22.2%).…”

“…However, blastocyst development rates were still lower (4% in BCB+ oocytes) than those obtained with oocytes from adult goats [5][6][7][8][9]. Prepubertal goat IVM-IVF-oocytes show a low incidence of male pronuclear (MPN) formation [10] and a high percentage of haploid embryos (59% of 2-4 cell embryos; [11]). The transformation of the fertilising sperm head into the MPN has been shown to be positively related to intracellular glutathione levels (GSH; [12]).…”

Novel Scientific Methods and Technology in the Reproductive Medicine

“…King [27] suggested that approximately one quarter of the abnormalities (haploidy, polyploidy, mixoploidy) may be attributed to errors in meiosis with the remaining three quarters occurring around the time of fertilization and early embryonic development. Similar results have been reported for pig [35], horse [36], goat [37], sheep [38] and humans [39].…”

In vitromaturation and fertilization of prepubertal and pubertal black Bengal goat oocytes

“…Moreover, there are reports that oocytes from prepubertal goats show higher percentages of polyspermic fertilization and lack of sperm head decondensation up to the male pronucleus stage [36], low percentage of blastocysts formation [19] and production of high percentages of haploid embryos [51]. The sperm separation method has a great influence on IVF, cleavage and embryo development.…”