In Vitro Culture Conditions Using Chemically Defined Media for In Vitro Matured and Intracytoplasmically Inseminated Porcine Oocytes

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Abstract. The aim of this study was to investigate the effects of the presence and the numbers of corpora lutea (CL) in porcine ovaries on in vitro oocyte maturation and embryonic development following intracytoplasmic sperm injection (ICSI). At oocyte collection, the ovaries of non-delivered and delivered pigs were classified into four groups by CL presence. The effect of the number of CL was also investigated following re-division of the non-delivered groups into four groups. In addition, the progesterone (P4) concentrations in follicular fluid (FF) of all the groups were measured to confirm the relationship between the presence and numbers of CL. Throughout the present study, the oocytes recovered from the CL-holding ovaries showed high (P<0.05) oocyte maturation rates, blastocyst rates and P4 concentrations in FF. Furthermore, in the non-delivered groups, the blastocyst rates and P4 concentrations in FF seemed to coincide with the CL numbers in each ovary. From these findings, we concluded that the presence and number of CL in the ovary can be used as an indicator for estimation of the developmental competence of porcine oocytes. Additionally, the present study suggests that P4 in FF influences in vitro oocyte maturation and embryonic development in porcine in vitro production. Key words: Corpus luteum, Embryonic development after ICSI, Oocyte maturation, Pig, Progesterone (J. Reprod. Dev. 55: [655][656][657][658][659][660] 2009) o date, in vitro production (IVP) of embryos has been attempted in many species including pigs [1][2][3][4][5][6][7][8][9][10][11], cattle [12][13][14][15] and sheep [16,17]. In these trials, the oocytes used for IVP were obtained from animals in various stages of growth, e.g., sexual maturity. The usage of younger oocyte donors enables reduction of the generation interval and rapid expansion of a valuable genotype. In many cases, however, the oocytes obtained from prepubertal animals have had a lower developmental competence than those of postpubertal or aged animals [1,6,8,[14][15][16][17][18]. The low quality of oocytes recovered from prepubertal animals is the principal problem for IVP.In IVP of porcine embryos, prepubertal pigs without corpora lutea (CL) in their ovaries are commonly utilized as oocyte donors. The prepubertal individuals are characterized by the fact that they have not completed maturation or ovulated yet. Ovulation produces morphological and physiological changes in the ovaries. CL are formed after ovulation and secrete progesterone (P4), which seems to be transferred to follicular fluid (FF), leading to a change of the hormonal balance in the FF. Therefore, the preovulatory oocytes of prepubertal pigs without CL are exposed in FF with a lower P4 concentration than those of adult pigs [1].Supplementation of the culture media with P4 accelerates the meiosis resumption in porcine oocytes [19]. Thus, P4 might be a key molecule in oocyte maturation and subsequent development. Pigs are multi-ovulatory animals. Therefore, it is likely that under the presence of a...

Section: Discussionmentioning

confidence: 99%

Section: Intracytoplasmic Sperm Injection (Icsi)mentioning

confidence: 99%

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Effects of the Presence and the Numbers of Corpora Lutea in Non-Delivered and Delivered Pigs on In Vitro Oocyte Maturation and Embryonic Development

Yoshizawa

Watanabe

Fukui

2009

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“…In Experiment 1, immature oocytes recovered from prepubertal whales (n=4) were cultured for two different IVM culture durations (30 or 40 h) using the WOW method (0.5 mm in diameter) according to previous reports [7,10,11]. After IVM for 30 or 40 h, the nuclear status of the oocytes was determined by whole-mount preparation.…”

Section: Experimental Designmentioning

confidence: 99%

Attempt at Intracytoplasmic Sperm Injection of In Vitro Matured Oocytes in Common Minke Whales (Balaenoptera acutorostrata) Captured During the Kushiro Coast Survey

Fukui

Iwayama

Matsuoka

et al. 2007

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Abstract. The present study was conducted during the Kushiro Coast Survey in an attempt to produce common minke whale embryos. In Experiment 1, we attempted to determine the appropriate culture duration (30 or 40 h) for in vitro maturation (IVM) of immature oocytes using the Well of the Well method. In Experiment 2, and intracytoplasmic sperm injection (ICSI) was applied to matured oocytes from prepubertal and adult common minke whales after IVM culture (40 or 48 h), and then their embryonic development was assessed. In Experiment 1, the maturation rate of oocytes cultured for 40 h (30.4%) was significantly higher than that of oocytes cultured for 30 h (6.8%; P<0.01). In Experiment 2, a total of 35 and 46 immature oocytes derived from adult (n=2) and prepubertal (n=6) minke whales, respectively, were cultured for 40 or 48 h. The maturation rate in the oocytes from the adult whales (34.2%) tended to be higher than that of the oocytes from the prepubertal whales (19.6%), but there was no significant difference. Following ICSI, 3 out of the 10 inseminated and cultured oocytes from the adult whales cleaved (2-, 8-, and 16-cell stages); all of these oocytes had been matured for 40 in culture. However, these oocytes did not develop to further stages. Only one of the 6 oocytes derived from the prebuertal whales, IVM cultured for 40 h and inseminated, developed to the 4-cell stage. The present results indicate that a 40 h IVM culture produces significantly higher rates of in vitro maturation than a 30 h IVM culture for common minke whale oocytes. Following ICSI, some oocytes cleaved to the 16-cell stage, but no further development was observed.

“…These results indicate the importance of adding follicular fluid (FF) collected from pregnant females to IVM medium for whale immature oocytes. It has recently been suggested that chemically defined culture media can be used for IVM and IVC in order to clarify the effectiveness of various additives in the media, such as hormones and growth factors [6][7][8][9]. However, FF has been widely demonstrated to have a positive effect on IVM culture of various mammalian oocytes, and it has been shown that addition of FF during IVM enhances the male pronuclei formation of oocytes after IVF and subseq u e n t e m b r y o n i c d e v e l o p m e n t [ 1 0 - 1 4 ] .…”

mentioning

confidence: 99%

Follicle Size-Dependent Changes in Follicular Fluid Components and Oocyte Diameter in Antarctic Minke Whales (Balaenoptera bonaerensis)

Nagai

Mogoe²,

Ishikawa³

et al. 2007

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Abstract. The concentrations of various components of follicular fluid were compared among three groups of follicles (small, <5 mm; medium: 5-10 mm; large, >10 mm) with a control that consisted of the components of umbilical serum using seven pregnant Antarctic minke whales. Follicular oocytes recovered from the follicles were also used for measurement of oocyte diameter after removing the cumulus cells. The mean diameter of the ooplasm in the oocytes from the large follicles (143.2 µm) was significantly greater than those from the small (127.1 µm) and medium (131.7 µm) follicles, although there were no significant differences in diameter of the whole oocyte and thickness of the zona pellucida among the three follicular sizes. The osmolarity of the follicular fluid from the small follicles (363.3 mOsmol) was significantly lower than that of the medium follicles (388.9 mOsmol) and tended to be lower than that of large (381.9 mOsmol) follicles, respectively, both of which were similar to that of the umbilical serum (379.5 mOsmol). There was no significant difference in the concentrations of all components of the follicular fluid between the medium and large follicles. As compared with the values of the umbilical serum, the total-protein, glucose, albumin and chlorine concentrations of the follicular fluid from the medium and large follicles were significantly higher, and the total cholesterol and calcium concentrations were significantly lower. The concentrations of lactic acid (85.3-136.0 mg/dl) of the follicular fluid from the three groups of follicles were significantly lower than that of the umbilical serum (360.0 mg/dl). Among the follicles, the follicular fluid from the small follicles (136.0 mg/dl) contained a significantly higher concentration of lactic acid than that from the large follicles (85.3 mg/dl). The progesterone concentrations were not significantly different among the fluid from the three group of follicles and the umbilical serum: however, the estradiol 17-β concentrations of the follicular fluid increased with the size of the follicle (14.3 and 34.6 ng/ml for small and large follicles, respectively). These results offer new information concerning whale reproductive physiology, especially for improvement of in vitro oocyte maturation and related technologies in whales.