Nuclear Maturation Of Canine Oocytes Research Articles

Effects of meiosis-inhibiting agents and equine chorionic gonadotropin on nuclear maturation of canine oocytes.

Experiments were conducted to determine the effects of meiosis-inhibiting-agents and gonadotropins on nuclear maturation of canine oocytes. The culture medium was TCM199 + 10 ng/ml epidermal growth factor supplemented with 25 microM beta-mercaptoethanol, 0.25 mM pyruvate, and 1.0 mM L-glutamine (Basal TCM). Initially, oocytes were cultured in Basal TCM alone or in Basal TCM + dibutylryl cyclic adenosine monophosphate (0.5, 1, 5, or 10 mM dbcAMP) for 24 hr. Dibutylryl cAMP inhibited resumption of meiosis in a dose-dependent manner; 60% of oocytes remained at the germinal vesicle (GV) stage after being cultured for 24 hr in 5 mM dbcAMP. The meiosis-inhibitory effect of dbcAMP appeared to be reversible, as the oocytes resumed meiosis and completed nuclear maturation after being cultured for an additional 48 hr in its absence. Oocytes were then cultured in Basal TCM alone or in Basal TCM + roscovitine (12.5, 25, or 50 microM) for 24 hr. Although approximately 60% of oocytes cultured in 25 microM roscovitine remained at the GV stage, this percentage was not significantly different from the 48% that also remained at the GV stage when cultured in its absence. Oocytes were cultured in Basal TCM + 25 microM roscovitine for 17 hr, exposed briefly to equine chorionic gonadotropin (eCG), and then cultured in Basal TCM for 48 hr. Short exposure of oocytes to eCG was beneficial, as it significantly increased the proportion of oocytes developing beyond germinal vesicle breakdown (P < 0.05) with approximately 20-30% of these were metaphase I (MI) oocytes. Study of the kinetics of nuclear maturation demonstrated that large numbers of oocytes remained at MI even after being cultured for 52 hr following brief exposure to eCG. This study showed that in vitro maturation of canine oocytes can be somewhat improved by short exposure of oocytes to eCG. However, further studies are still required to derive effective methods to mature canine oocytes in vitro.

Nuclear maturation of canine oocytes cultured in protein-free media.

The objective of this study was to determine the ability of canine oocytes to complete nuclear maturation in a protein-free medium. Oocytes obtained from ovaries of bitches aged 6 months to 2 years were cultured either in TCM199 or CMRL1066 medium without protein supplementation in 5% or 20% O(2). Sixteen of 121 (13%) oocytes cultured in TCM199 reached metaphase II, but only 1 of 135 oocytes cultured in CMRL1066 did so (P < 0.05). Oxygen concentration did not affect nuclear maturation. An additional 103 oocytes were cultured in TCM199 for 48 hr, inseminated with chilled ejaculated spermatozoa, fixed in 1:3 acetic acid-ethanol and then stained with aceto-orcein; 34% of these oocytes were penetrated by spermatozoa. To determine developmental competence of oocytes cultured in a protein-free medium, 85 oocytes were cultured in TCM 199 for 48 hr, inseminated and then cultured; 7 early stage embryos were produced. The effects of growth hormone, beta-mercaptoethanol (betaME), luteinizing hormone (LH) and energy substrates, alone or in combination, on nuclear maturation of oocytes cultured in a protein-free medium were also determined. Growth hormone enhanced cumulus expansion, but did not improve nuclear maturation. beta-mercaptoethanol had no effect on nuclear maturation. However, percentages of MII oocytes significantly decreased when the oocytes were cultured for 48 hr in the medium containing LH or a high concentration of glucose (P < 0.05). In conclusion, canine oocytes are able to complete nuclear maturation in a protein-free medium. The specific type of medium and other supplements significantly influence the meiotic maturation of canine oocytes.

The effect of oocyte size and bitch age upon oocyte nuclear maturation in vitro

In vitro maturation in the bitch has yet to be fully investigated, and perfection of the technique is essential for future gamete salvage programs in endangered canine species. For optimal success with these techniques, knowledge of the individual animal and of oocyte effects upon maturational competence would be useful. Two factors were therefore studied using an aceto-orcein staining technique, which has been shown to be effective for monitoring nuclear maturation of canine oocytes following oocyte culture in medium supplemented with bovine serum albumin (BSA). Oocytes of different sizes were cultured in vitro and their nuclear maturation monitored. It was shown that the selection of oocytes which had acquired meiotic competence through adequate intrafollicular growth was important for in vitro maturation. In vitro maturation of oocytes from bitches aged 1 to 6 yr, and from those 7 yr and older was then compared, and it was found that oocytes from young bitches had a greater potential to mature than those collected from the older animals.

Effect on development of chilling in vitro-produced bovine embryos at various cleavage stages

In vitro embryo production in the domestic bitch can provide valuable insights for conservation of endangered canids. In the present study, canine oocytes underwent in vitro maturation (IVM) in simple or complex media, with production of in vitro matured and fertilized (IVM/IVF) canine embryos. Cumulus–oocyte complexes (COCs) were harvested from ovaries by slicing and subjected to IVM in four media (SOF, TCM 199, Ham-F10, and DMEM/F12). After culture for 48 h, oocytes were stained and examined for nuclear maturation. There were no significant differences in the mean (±S.D.) percentage of nuclear maturation (metaphase II) of oocytes cultured in SOF (18.6 ± 7.6%), TCM 199 (18.3 ± 4.5%), Ham-F10 (13.9 ± 8.2%), or DMEM/F12 (11.9 ± 4.2%). For assessment of embryo development, oocytes were matured for 48 h in synthetic oviductal fluid (SOF), fertilized with frozen-thawed sperm, and presumptive zygotes were cultured for 7 d, either in SOF or as co-cultures with BRL cells in TCM 199. Percentages of IVM/IVF oocytes that developed to the 2-cell, 3–4-cell, and 5–7-cell stages were higher (P < 0.05) following culture in SOF versus BRL cell co-cultures (33.6 ± 1.2% vs 13.7 ± 1.2%, 24.7 ± 0.5% vs 8.7 ± 1.1%, and 15.1 ± 2.2% vs 4.3 ± 1.3%, respectively). However, none of the embryos developed beyond the 8–16-cell stage. In conclusion, simple or complex media successfully induced resumption of meiosis and nuclear maturation of canine oocytes. Furthermore, SOF supported in vitro development of IVM/IVF canine embryos to the 8–16-cell stage.