Use of Low-Salt Culture Medium for in Vitro Maturation of Porcine Oocytes is Associated with Elevated Oocyte Glutathione Levels and Enhanced Male Pronuclear Formation after in Vitro Fertilization1

The aim of this study was to evaluate the effect of progesterone supplementation and stage of oestrous cycle on in vitro maturation (IVM) of canine oocytes. Oocytes were cultured in medium supplemented with 0, 2000, 4000 or 8000 ng progesterone ml(-1) (Expt 1; n=274 oocytes) or 0, 20, 200 or 2000 ng progesterone ml(-1) (Expt 2; n=789 oocytes). In Expt 3, oocytes (n=1202) were cultured in a bi-phasic system of meiotic arrest followed by IVM, both in the presence of 0, 20, 200 or 2000 ng progesterone ml(-1). Rates of meiotic resumption for Expt 1 ranged from 40.0% to 58.5%; there were no significant differences among groups. In Expt 2, rate of meiotic resumption was significantly lower in the 2000 ng progesterone ml(-1) treatment (35.5%) compared with the 200 ng progesterone ml(-1) treatment (54.0%; P<0.05). There were no significant differences in rates of maturation to metaphase II among treatments in Expt 1 (1.8-8.6%) or Expt 2 (8.4-14.7%); however, oocytes collected from ovaries of bitches in oestrus and dioestrus had higher rates of maturation to metaphase II than did oocytes from bitches at pro-oestrus or anoestrus (P<0.01). In Expt 3, no differences were observed in rates of maturation among treatment groups. Rates of maturation to metaphase II of oocytes from bitches in dioestrus were significantly higher than those from bitches in pro-oestrus (P<0.01). These results indicate that supplementation of culture medium with progesterone either during maturation or during meiotic arrest before maturation does not increase the rate of IVM of canine oocytes. However, stage of oestrous cycle is a key factor in the selection criteria for meiotically competent canine oocytes for use in in vitro experiments.

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316EFFECTS OF ESTRADIOL-17² AND PROGESTERONE SUPPLEMENT ON THE RESUMPTION OF MEIOSIS OF CANINE OOCYTES MATURED IN VITRO

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab316 ◽

2004 ◽

Vol 16 (2) ◽

pp. 277

Author(s):

M.K. Kim ◽

Y.H. Fibrianto ◽

H.J. Oh ◽

G. Jang ◽

K.S. Lee ◽

...

Keyword(s):

Oocyte Maturation ◽

In Vitro Maturation ◽

Culture Medium ◽

Linear Models ◽

Uv Light ◽

Germinal Vesicle ◽

Domestic Animal ◽

Estradiol 17Β

In the bitch, oocytes are ovulated at the germinal vesicle (GV) stage and mature in the isthmus of the oviduct around 3 days after ovulation, it is not known what elements trigger the release of this meiotic arrest. Canine IVM has shown limited success with maturation rates, usually around 20% (MII) (Farstad W, 2000 Anim. Reprod. Sci. 60–61, 375–387). Estrogen and progesterone are suggested to play a significant role in causing oocyte resumption of meiosis and progression to MII stage. The purpose of this study was to investigate the role of estradiol-17β (E2) and progesterone (P4) during in vitro maturation of canine oocytes in serum-free tissue culture medium (TCM)-199. Canine oocytes collected from bitches were categorized into three groups based on estrous stages, follicular, luteal, or anestrus, at routine ovariohystrectomy. Oocytes were cultured in vitro in TCM-199 supplemented with E2, P4 or E2+P4 according to experimental design at 39°C in 5% CO2 and O2. After 72h of maturation culture, oocytes were denuded, fixed in a 3.7% paraformaldehyde solution for 10min, stained with Hoechst 33342 in glycerol, and observed under the UV light. Three groups of oocytes were cultured in TCM-199 supplemented with different concentrations (0, 0.1, 1.0 or 2.0μgmL−1) of E2 (Experiment 1, n=898, replications: 5) or P4 (0, 0.5, 1.0 or 2.0μgmL−1, Experiment 2, n=734, replications: 5). Multiple comparisons were implemented using Generalized Linear Models in the SAS 8.12 program. The rates of oocyte maturation to MII stage were higher (P<0.05) in follicular stage oocytes cultured with 2μgmL−1 E2 (17.9%) compared to other supplement groups (0 to 7.6%). No differences (P<0.05) in rate of MII stage oocytes among P4 supplement groups were observed. In Experiment 3, to investigate the combined effects of E2 and P4 on in vitro maturation, three groups of oocytes were cultured in TCM-199 supplemented with 2μgmL−1 E2 and various concentration of P4 (0, 0.5, 1.0 or 2.0μgmL−1, Experiment 3, n=1613, replications: 5). The rate of oocyte maturation to MII stage (11.5%) was higher (P<0.05) in follicular stage oocytes cultured with 2μgmL−1 E2+2.0μgmL−1 P4 supplement compared to other supplement groups (0 to 6.4%). In conclusion, the present study demonstrated that E2 supplement in the culture medium increased maturation of canine oocyte to MII stage and that supplement of P4 alone did not promote oocyte maturation. However, P4 supplemented with E2 further promoted oocyte maturation in the follicular stage compared to E2 supplement alone, indicating that P4 acts synergistically with E2 on canine oocyte maturation in the presence of E2. From our results, we conclude that canine oocytes are exposed to high levels of P4 during maturation due to the preovulatory luteinization of canine follicles which gives rise to high intrafollicular as well as intratubal P4 concentrations-this is very different from the situation in oocytes from other domestic animal species. This study was supported by Biogreen 21-1000520030100000.

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148 Effect of concentration of methionine in maturation and culture medium on cleavage rate of oocytes from alpaca (Lama pacos)

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab148 ◽

2019 ◽

Vol 31 (1) ◽

pp. 199

Author(s):

M. L. Uchuari ◽

M. Artica ◽

J. C. Villanueva ◽

W. F. Huanca ◽

W. Huanca

Keyword(s):

In Vitro Maturation ◽

Culture Medium ◽

Egg Yolk ◽

Cumulus Cells ◽

Cleavage Rate ◽

Sodium Pyruvate ◽

Maturation Medium ◽

Thermos Flask ◽

Lama Pacos

Maturation time of oocytes from alpacas is around 38 to 40h (Huanca et al. 2009) that would induce an increase in reactive oxygen species during in vitro maturation and IVF and cause cytotoxic damage to gametes. The objective of this study was to determine the optimal concentration of methionine during in vitro maturation on cleavage rate of alpacas oocytes following IVF. Cumulus-oocyte complexes were collected from slaughterhouse ovaries and transported in a thermos flask containing a saline solution 0.9% and antibiotic, antimycotic at 35°C. Cumulus-oocyte complexes were aspirated from follicles >2mm and evaluated with a stereomicroscope for selection. Only cumulus-oocyte complexes with a homogeneous cytoplasm and with 2 or more layers of cumulus cells were selected to be cultured in maturation medium TCM-199 supplemented with 10% FCS (v:v) plus 0.5μg mL−1 FSH, 10μg mL−1 hCG, 0.2mM sodium pyruvate, 50μg mL−1gentamycin and 1μg mL−1 oestradiol under mineral oil by 38h. Testes of mature males were collected from a slaughterhouse and transported to the laboratory. Caudal epididymide was isolated, and fluid, rich in spermatozoa, was aspirated in syringes containing 2mL of Tris-fructose-egg yolk extender. Motile spermatozoa were obtained by centrifugation at 700×g in a Percoll discontinuous gradient (22.5: 45.0%) for 10min. The supernatant was removed by aspiration, and the pellet was resuspended in TL stock and centrifuged again at 700×g for 5min. Spermatozoa and oocytes were co-incubated by 18h at 39°C with 5% CO2. Presumptive zygotes were culture in KSOMaa medium and evaluated at 72h. The treatments include 0, 14 and 21 μM of methionine in maturation and culture medium. Data were analysed by ANOVA, and results are presented in Table 1. The results suggest that addition of methionine in maturation and culture medium improve the cleavage rate in oocytes from alpacas. Table 1.Cleavage rate (%) following in vitro maturation at different concentrations of methionine Proyect 405-PNICP-PIAP-2014, INNOVATE-PERU, is acknowledged.

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Effect of LH and Estradiol-17β Supplementation at Different Time Points on In vitro Development of Preantral Follicles in Sheep

Indian Journal of Animal Research ◽

10.18805/ijar.b-4119 ◽

2020 ◽

Author(s):

L.S.S. Varaprasad Reddy ◽

B.R. Naik ◽

A.V.N. Sivakumar ◽

B. Punyakumari ◽

J. Suresh

Keyword(s):

In Vitro Maturation ◽

Culture Medium ◽

Follicular Development ◽

Standard Medium ◽

In Vitro Development ◽

Preantral Follicles ◽

Time Points ◽

Vitro Development ◽

Estradiol 17Β

Background: Ovarian follicular development and growth are controlled by many hormones and growth factors. Despite the fact that LH and estradiol-17β have been utilized for the in vitro culture of preantral follicles yet, the suitable time points of supplementation of LH and estradiol-17β is not known. Therefore this study aimed to investigate the influence of addition of LH and estradiol-17β at different time points on in vitro development of preantral follicles (PFs’) in sheep. Method: Preantral follicles isolated from the ovarian cortical slices using micro dissection method were cultured for six days in Bicarbonate buffered Tissue culture medium 199B (TCM 199B) or in a standard culture medium supplemented with LH (2 μg/ml) and estradiol-17β (5 ng/ml) at different points during the culture period. COCs isolated from the follicles at the end of six day culture in different treatments were subjected to in vitro maturation for additional 24h. Result: Supplementation of LH and estradiol-17β during last two days of the culture supported better proportion of PFs’ exhibiting growth whereas supplementation of LH and estradiol-17β during first two days of the culture supported better average increase in diameter and proportion of PFs’ exhibiting antrum formation at the end of six day culture. Further the oocytes in COCs isolated at the end of culture in these treatments and subsequently subjected to in vitro maturation (IVM) for 24hr developed at a higher frequency to MII (metaphase II) stage. Supplementation of LH and estradiol-17β to TCM 199B culture medium in early stages followed by standard medium alone in later stages supports better development of PFs’ in vitro. Following supplementation with LH and estradiol-17β for the first two days culture of PFs’ in standard medium appears to be advantageous for the development of preantral follicles in vitro.

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Effect of serum on the in vitro maturation of canine oocytes

Reproduction Fertility and Development ◽

10.1071/rd00012 ◽

1999 ◽

Vol 11 (8) ◽

pp. 387 ◽

Cited By ~ 35

Author(s):

Takeshige Otoi ◽

Maya Fujii ◽

Aya Ooka ◽

Masaki Tanaka ◽

Tatsuyuki Suzuki

Keyword(s):

Bovine Serum Albumin ◽

Bovine Serum ◽

In Vitro Maturation ◽

Culture Medium ◽

The Other ◽

Serum Supplement ◽

Meiotic Competence ◽

Positive Effect ◽

Serum Type

The meiotic competence of canine oocytes cultured for 72 h in medium supplemented with three different concentrations (5, 10 and 20%) of anoestrous, oestrous or metoestrous bitch serum, or with 0.3% bovine serum albumin (BSA), was examined. The oestrous serum supplement had a positive effect on the resumption of meiosis, compared with the other supplements (P<0.05). The number of oocytes that reached metaphase I (MI) and metaphase II (MII) was significantly higher (P<0.05) with the oestrous serum supplement than with the anoestrous serum supplement. There were no significant differences among the three different concentrations in each serum type with respect to the proportion of oocytes that completed meiosis (MI to MII). The number of oocytes that resumed meiosis in the 10% oestrous serum supplement was significantly higher (P<0.05) than those of each concentration of the anoestrous and metoestrous serum supplements, and of the 0.3% BSA supplement. Moreover, a higher number of oocytes reached MII in the presence of the 10% oestrous serum supplement than with the 10% anoestrous serum supplement. These results suggest that supplementation of the culture medium with 10% oestrous serum is the optimal treatment for in vitro maturation of canine oocytes.

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Inhibition of in vitro maturation of equine oocytes by interleukin 1 beta via specific IL-1 receptors

Reproduction ◽

10.1530/rep.0.1260509 ◽

2003 ◽

pp. 509-515 ◽

Cited By ~ 10

Author(s):

A Martoriati ◽

M Caillaud ◽

G Goudet ◽

N Gerard

Keyword(s):

Oocyte Maturation ◽

In Vitro Maturation ◽

Culture Medium ◽

Interleukin 1 ◽

Inhibitory Effect ◽

Interleukin 1 Beta ◽

Nuclear Maturation ◽

Epidermal Growth ◽

Dose Dependent

Interleukin 1 beta (IL-1 beta) inhibits the LH-induced resumption of meiosis of equine oocytes in vitro. The present study was performed to clarify this inhibitory effect of IL-1 beta by testing increasing concentrations of IL-1 beta, and by measuring the effect of addition of IL-1 receptor antagonist (IL-1RA) to the culture medium. The effect of IL-1 beta on epidermal growth factor (EGF)-induced resumption of meiosis was also studied. Cumulus-oocyte complexes (COCs) were collected from subordinate follicles on ovaries obtained from an abattoir. In five distinct experiments, COCs were cultured for 30 h and nuclear maturation of oocytes was evaluated by DNA staining. In Expt 1, seven different media were tested: medium 1 (TCM199+BSA); medium 2 (medium 1+50 ng IL-1 beta ml(-1)); medium 3 (medium 1+eLH); and media 4, 5, 6 and 7 (medium 3 containing 0.1, 1.0, 10.0 and 50.0 ng IL-1 beta ml(-1), respectively). In Expt 2, four different media were tested: medium 1 (TCM199+BSA+eLH); medium 2 (medium 1+50 ng IL-1 beta ml(-1)); and media 3 and 4 (medium 2+IL-1RA at 50 and 100 ng ml(-1), respectively). In Expt 3, three different media were tested: medium 1 (TCM199+BSA+eLH); medium 2 (medium 1+50 ng IL-1RA ml(-1)); and medium 3 (medium 2+50 ng IL-1 beta ml(-1)). In Expt 4, four different media were tested: medium 1 (TCM199+BSA+eLH); and media 2, 3 and 4 (medium 1+IL-1RA at 50, 100 and 150 ng ml(-1), respectively). In Expt 5, three different media were tested: medium 1 (TCM199+BSA+EGF); medium 2 (medium 1+50 ng IL-1 beta ml(-1)); and medium 3 (medium 2+50 ng IL-1RA ml(-1)). In Expt 1, LH alone induced an increase in the rate of in vitro maturation (IVM) of equine oocytes (P<0.05), whereas IL-1 beta alone did not have any effect compared with medium 1. IL-1 beta (50 ng ml(-1)) significantly inhibited the eLH-induced IVM of oocytes (P<0.05) compared with medium 3. A decrease in rate of maturation was observed from a concentration of 10 ng IL-1 beta ml(-1) onwards. In Expt 2, the presence of IL-1RA in the culture medium inhibited the effect of IL-1 beta and restored the rate of oocyte maturation (P<0.05) observed in the presence of LH alone. In Expts 3 and 4 it was demonstrated that IL-1RA alone had no positive effect on the eLH-induced rate of maturation. In Expt 5, IL-1 beta inhibited the EGF-induced resumption of meiosis (P<0.05). The addition of IL-1RA inhibited this effect and restored the rate of oocyte maturation (P<0.05) observed with EGF alone. In conclusion, the present data confirm the inhibitory effect of IL-1 beta on IVM of equine oocytes induced by eLH and demonstrate its inhibitory effect on EGF-induced oocyte maturation. The rate of maturation decreased in a dose-dependent way and the lowest rate of maturation was observed at 50 ng IL-1 beta ml(-1) (P<0.05). The use of IL-1RA inhibited these effects, demonstrating that the action of IL-1 beta is receptor-mediated. Moreover, the results clearly show that, in equine species, IL-1 beta is involved in the physiology of COCs by regulating resumption of meiosis.

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Applying the nanobiotechnological methods for optimize the in vitro cultivation technology for cows oocytes

Faktori eksperimental'noi evolucii organizmiv ◽

10.7124/feeo.v22.958 ◽

2018 ◽

Vol 22 ◽

pp. 257-261 ◽

Cited By ~ 2

Author(s):

S. I. Kovtun ◽

A. B. Zyuzyun ◽

O. V. Shcherbak ◽

P. A. Trotskiy

Keyword(s):

In Vitro Culture ◽

In Vitro Maturation ◽

Culture Medium ◽

In Vitro Cultivation ◽

Vitro Fertilization ◽

Cultivation Technology ◽

And Control ◽

Positive Effect ◽

Generative Cells

Aim. Investigated effect nanomaterial of highly dispersed ultra fine silica (UFS) by carbohydrate – sucrose (UFS/sucrose) on the effectiveness of meiotic maturation cows oocytes in vitro. Methods. The fresh and frozen – thawed cow oocyte-cumulus complex (OCC) was divided into four groups: three experimental in which the cultivation was carried out in a medium containing of 0, 1; 0, 01 and 0,001 % UFS/sucrose and control without adding nanomaterial. Results. It was concluded that the addition of UFS/sucrose in 0.001 % concentration is effective for elevation level of oocytes maturation and provides reception of 76,8% oocytes that induced the metaphase II of meiosis. Adding carbohydrate – sucrose (UFS/sucrose) in 0.001 % concentration to the culture medium frozen – thawed cow generative cells, make positive effect on in vitro fertilization and provide embryos quantity enhancement to 33.3 %. Conclusions. Addition of UFS/sucrose in 0.001 % concentration to the culture medium have increase effect and promote level in vitro maturation of cows oocytes rising to 76.8 %. Usage of UFS/sucrose in 0.001 % concentration as part of in vitro culture medium for cows oocyte-cumulus complex conduce rising quantity of cattle embryos to 33.3 % after in vitro fecundation frozen – thawed and maturation oocytes. Keywords: oocytes, in vitro culture, embryos, nanomaterial, ultra fine silica (UFS).

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