Responses of canine oocytes to in vitro maturation and in vitro fertilization outcome (original) (raw)
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Reproduction, nutrition, development
The objective of this study was to determine an optimum maturation period of canine oocytes for the development in vitro after in vitro fertilization (IVF). Canine oocytes larger than 110 micrometers in diameter, which were collected from ovaries at the follicular phase of the reproductive cycle, were cultured for each time (48, 72 and 96 h) in TCM 199 medium supplemented with 10% canine serum, fertilized, and then cultured in vitro for 8 days. Significantly more oocytes reached metaphase II (MII) in the 72-h culture group than in the 48-h culture group (25.6% vs. 41.0%). The percentages of oocytes that reached MII or beyond after maturation culture did not differ significantly between the 72- and 96-h culture groups, but the percentage of parthenogenetically activated oocytes in the 96-h culture group was significantly higher than that in the 72-h culture group. The percentages of cleaved embryos after IVF were significantly higher in the 48- and 72-h culture groups than in the 96-...
Molecular Reproduction and Development, 2005
Ability of ovarian oocytes from tine domestic dog to complete nuclear maturation in vitro (IVM) varies markedly among donors and generally is 20% or less of all oocytes cultured. To identify the cause(s) underlying these significant variations in meiotic maturation (to metaphase II; Mil), we retrospectively analyzed data from 1,643 oocytes recovered from 90 bitches for which stage of reproduction and season of year were known. Neither stage of reproduction (proestrus/estrus, diestrus, anestrus, or prepuberty) nor season (P>0.05) influenced the ability of oocytes to achieve nuclear maturation in vitro. A second study was conducted to examine the impact of follicular size on meiotic maturation. Populations of large oocytes were recovered from four categories of follicles (ranging from <0.5 to >2 mm in diameter) and cultured in TCM 199 for 48 hr. Follicular size influenced (P<0.05) meiotic competence. Mean percentages of Mil oocytes were 16.9 ±9.2, 26.1 ±7.6, 38.4±9.2, and 79.5± 10.9 for oocytes recovered from <0.5 mm, >0.5-<l mm, 1-2 mm, and >2 mm diameter follicles, respectively. In summary, stage of reproduction and season have no impact on the ability of dog oocytes to achieve nuclear maturation in vitro. However, we demonstrated for the first time that dog oocytes acquire meiotic competency during follicular development. IVM success of selected oocytes from large size follicles (almost 80%) is about 60% higher than measured in most previous studies involving randomly collected oocytes. Mol. Reprod. Dev.
Oocyte biology and challenges in developing in vitro maturation systems in the domestic dog
Animal Reproduction Science, 2007
The oocyte of the domestic dog is unique from that of other mammalian species studied to date. Ovulation occurs either once or twice per year, with the oocyte released at the germinal vesicle stage, and then completing nuclear and cytoplasmic maturation within the oviduct under the influence of rising circulating progesterone. In vivo meiotic maturation of the bitch oocyte is completed within 48-72 h after ovulation, which is longer than 12-36 h required for oocytes from most other mammalian species. Due to these inherently novel traits, in vitro culture systems developed for maturing oocytes of other species have been found inadequate for maturation of dog oocytes. On average, only 15-20% of ovarian oocytes achieve the metaphase II stage after 48-72 h of in vitro culture. Thus far, no offspring have been produced in the dog (or other canids) by transferring embryos derived from in vitro matured oocytes. This review addresses current knowledge about dog reproductive physiology, specifically those factors influencing in vitro developmental competence of the oocyte. This summary lays a foundation for identifying the next steps to understanding the mechanisms regulating meiotic maturation and developmental competence of the dog oocyte. Published by Elsevier B.V.
Effects of oocyte culture density on meiotic competence of canine oocytes
Reproduction, 2002
This study was conducted to determine a suitable ratio of oocytes to medium for in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) collected from bitches at anoestrus and dioestrus and to examine the meiotic competence of COCs cultured singly or in different group sizes. In the first experiment, different numbers of COCs (5, 10, 15 and 20 per drop) were cultured for 72 h in 100 microl drops of maturation medium. The meiotic competence of oocytes from ovaries at anoestrus was affected by the number of COCs incubated, whereas at dioestrus, the incubation number of COCs had no effect. In the second experiment, COCs were cultured singly or in different group sizes for 72 h by suitable oocyte density according to the reproductive cycle of the donor. In the anoestrous group, 1, 5 and 10 COCs were cultured in 10, 50 and 100 microl drops of the medium (10 microl per COC), respectively. In the dioestrous group, 1, 5 and 15 COCs were cultured in 7, 35 and 105 microl drops of the med...
In vitro maturation rates of canine oocytes from anoestrous bitches in simple media
Reproduction Nutrition Development, 2004
The meiotic competence of canine oocytes collected from anoestrous bitch ovaries and cultured for 72 h in different media was studied. The base culture medium was TCM 199 enriched with 10% fetal bovine serum (TCM); the effect of supplementation with EGF (50 ng•mL-1) or ITS (insulin: 10 µg•mL-1 ; transferrin: 5.5 µg•mL-1 ; selenium: 5 µg•mL-1) was also studied. TCM was also compared to a Synthetic Oviductal Fluid (SOF). All the media contained FSH (0.1 UI•mL-1), LH (10 UI•mL-1), 17β-oestradiol (4 µg•mL-1) and kanamycin. Despite the anoestrous stage of the donor bitches, resumption of meiosis occurred in a high proportion of the oocytes, (mean value 77.3%). The number of oocytes showing the 'germinal vesicle breakdown' nuclear stage was not influenced by the type of the culture medium used. ITS had a positive effect on nuclear progression to later stages (from metaphase I to metaphase II); however, this effect was not statistically significant. bitch / anoestrous / oocytes / maturation / in vitro
Reproduction, Fertility and Development
The current in vitro maturation system (IVM) for dog oocytes is inefficient. On the average, only 15% of ovarian oocytes complete nuclear maturation in vitro. For unknown reasons, the ability of oocytes to develop to the metaphase II stage (MII) varies markedly among bitches (Songsasen et al., 2002, Mol. Reprod. Dev. 62, 407–415). The objective of this study was to identify the cause(s) underlying these significant variations in nuclear maturation. Initially, we retrospectively analyzed data obtained during the past 3 years;; 1661 oocytes were obtained from 74 bitches where stage of reproduction for the donor was known based on ovarian morphology. Oocytes were cultured in TCM 199+0.1% polyvinyl alcohol at 38.5°C in 5% CO2 in humidified air under various experimental conditions. Analysis of variance (ANOVA) was performed to compare differences in meiotic competence of oocytes obtained at various reproductive stages and during different seasons. Stage of reproduction did not influence...
Live Births from Domestic Dog (Canis familiaris) Embryos Produced by In Vitro Fertilization
Development of assisted reproductive technologies (ART) in the dog has resisted progress for decades, due to their unique reproductive physiology. This lack of progress is remarkable given the critical role ART could play in conserving endangered canid species or eradicating heritable disease through gene-editing technologies-an approach that would also advance the dog as a biomedical model. Over 350 heritable disorders/traits in dogs are homologous with human conditions, almost twice the number of any other species. Here we report the first live births from in vitro fertilized embryos in the dog. Adding to the practical significance, these embryos had also been cryopreserved. Changes in handling of both gametes enabled this progress. The medium previously used to capacitate sperm excluded magnesium because it delayed spontaneous acrosome exocytosis. We found that magnesium significantly enhanced sperm hyperactivation and ability to undergo physiologically-induced acrosome exocytosis, two functions essential to fertilize an egg. Unlike other mammals, dogs ovulate a primary oocyte, which reaches metaphase II on Days 4-5 after the luteinizing hormone (LH) surge. We found that only on Day 6 are oocytes consistently able to be fertilized. In vitro fertilization of Day 6 oocytes with sperm capacitated in medium supplemented with magnesium resulted in high rates of embryo development (78.8%, n = 146). Intra-oviductal transfer of nineteen cryopreserved, in vitro fertilization (IVF)-derived embryos resulted in seven live, healthy puppies. Development of IVF enables modern genetic approaches to be applied more efficiently in dogs, and for gamete rescue to conserve endangered canid species.
Influence of season and parity on the recovery of in vivo canine oocytes by flushing fallopian tubes
Animal Reproduction Science, 2007
Assisted reproductive technology (ART) in dogs largely depends on the in vivo matured oocytes due to lack of a suitable in vitro maturation system. The present study evaluated the technique of flushing fallopian tubes to collect in vivo matured canine oocytes by laparotomy, and determined the effects of seasons, and parity of donor bitches on the success of oocyte retrieval. Oocytes were retrieved from anesthetized bitches by laparotomy. About 7 ml of TCM-199 supplemented with HEPES was used to flush each individual fallopian tube. Oocytes were categorized as good, fair, poor, immature or aged based on the nuclear stage, cumulus cell layers, color and homogeneity of ooplasm. Oocytes categorized as being good or fair were considered usable, while poor, aged or immature oocytes were considered unusable for ART. A significantly higher number of oocytes per bitch were retrieved during the spring (11.2) compared to the winter (7.9). The oocyte recovery rates were 89.4, 92.2, 89.7 and 89.3% for spring, summer, autumn, and winter, respectively. The highest percentage of usable oocytes (74.7%) was retrieved during autumn (P > 0.05). The number of oocytes was influenced by the parity of the donor bitch. Significantly more oocytes were collected from the multiparous bitches (10.3) compared to nulliparous bitches (7.7). The percentage of usable oocytes was more in multipara (71.5%) compared to nullipara (64.7%) (P > 0.05). Collection of in vivo produced oocytes by laparotomy represents a potential source of matured oocytes for ART in dogs.
The canine oocyte: uncommon features of in vivo and in vitro maturation
The biology of the canine oocyte is unusual compared with that of other mammalian females. The present paper reviews both in vivo and in vitro specificities of canine oocytes. Final follicular growth in the bitch is characterised by an early appearance of LH binding sites in the granulosa, a high proportion of polyovular follicles and a preovulatory luteinisation, starting at the time of the LH surge. Through follicular fluid, preovulatory oocytes are thus exposed to high levels of progesterone, as high as 1000-fold plasma concentrations. The composition of the follicular fluid is affected by the size of the female. The more specific aspect of oocyte biology in the bitch is ovulation: oocytes are expelled immature, at the Prophase I stage. Ovulatory follicles are 6-8 mm in diameter, releasing oocytes from 110 mm, with dark cytoplasm. Resumption of meiosis occurs from 48 h postovulation, MII stages appearing 48-54 h after ovulation. The mechanisms controlling such a late meiotic resumption are still unknown. Granulosa cells seem to play a central role as in other mammalian species, but not with cAMP as the principal mediator. The importance of a transient reactivation of oocyte transcription a few hours before meiotic resumption is to be explored. These specific features may contribute to the low efficiency of IVM. Only 10-20% oocytes reach the metaphase stage and suffer from a poor cytoplasmic maturation. Moreover, in vitro culture of canine oocytes is associated with a high proportion of degeneration. To date, IVM of the oocytes is the main limiting factor for the development of assisted reproductive techniques in the canine. A better knowledge of the basic physiology of folliculogenesis and the molecular mechanisms controlling oocyte meiosis resumption in this species may allow us to overcome this obstacle.