Influence of maturation culture period on the development of canine oocytes after in vitro maturation and fertilization

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-h culture group. In the 48-h culture group, 3.9% of fertilized oocytes developed to the 16-cell stage or beyond, but none of the cleaved embryos in the 72- and 96-h culture groups developed to the same stage. These results indicate that full nuclear maturation of oocytes collected from ovaries at the follicular phase occurs after 72 h of in vitro culture. However, an optimum maturation period (48 h) for the in vitro development of canine oocytes after IVF may be different from the period necessary to reach the maximal oocyte maturation rate, when based on the developmental stage of the cleaved embryos.     

Effects of canine serum collected from dogs at different estrous cycle stages on in vitro nuclear maturation of canine oocytes

Canine oocytes are ovulated at prophase of the first meiotic division and undergo maturation in the distal part of the oviduct for at least 48-72 h. Because of these differences from other domestic mammals, the efficiency of in vitro maturation (IVM) of canine oocyte is very low. The present study was conducted to evaluate the effects of canine serum on IVM of canine oocytes recovered from ovaries in various reproductive states (follicular, luteal or anestrous stages). Oocytes were recovered by mincing ovaries from bitches presented for ovariohysterectomy at various stages of the estrous cycle. Heat-inactivated canine serum was prepared with blood taken from dogs at the anestrous, estrous or diestrous stage of the estrous cycle as determined by progesterone concentration and vaginal cytology. Oocytes were cultured for 72 h in tissue culture medium (TCM)-199 supplemented with 10% canine anestrous, estrous or diestrous serum or fetal bovine serum (FBS) (experiment 1), or supplemented with 0 (control), 5%, 10% or 20% canine estrous serum (experiment 2). In experiment 1, IVM of oocytes collected at the follicular stage of the estrous cycle to metaphase II (MII) stage was higher (p < 0.05) with canine estrous serum (14.2%) than with canine anestrous (5.2%) or diestrous serum (6.3%), FBS (2.2%) or in the control (2.2%). In experiment 2, oocytes collected at the follicular stage of the estrous cycle cultured in TCM-199 with 10% canine estrous serum showed a higher maturation rate to MII stage (13.5%, p < 0.05) compared with those cultured with 5% (1.3% MII) or 20% canine estrous serum (5.1% MII) or the control (2.7% MII). In conclusion, our results demonstrate that supplementing culture medium with 10% canine estrous serum improves IVM of canine follicular stage oocytes.     

Embryonic development of in vitro matured and in vitro fertilized dog oocytes

In several studies, early cleavage stage canine embryos have been derived from in vitro fertilized oocytes cultured under various conditions. Despite these results, IVF protocols for canine oocytes have yielded low fertilization rates. In this study, Experiment I compared the effects of tissue culture medium (TCM)-199 supplemented with either (A) 1 microg/ml estradiol or (B) 20 microg/ml estradiol + 1 microg/ml human somatotropin (hST) on the in vitro nuclear maturation rate of canine oocytes. Meiotic progression to the metaphase I and II (MI/MII) stages at 72 hr of in vitro culture (IVC) was 10.2% (11/108) in medium A versus 14.1% (30/142) in medium B (P = 0.802). In Experiment II, cleavage rate was determined among oocytes recovered from ovaries of bitches at different reproductive stages. Oocytes (n = 888) were retrieved from bitches at the follicular, anestrous, and luteal stages and selected for high morphological quality. Oocytes were matured for 48 hr in TCM-199 supplemented with 1 microg/ml hST + 20 microg/ml estradiol. Oocytes were in vitro fertilized with fresh canine spermatozoa that had been isolated on a Percoll gradient, and were cultured in synthetic oviduct fluid (SOF) medium with bovine serum albumin (BSA; 4 mg/ml) up to 5 days in 5% CO(2) in air at 37 degrees C. A proportion of oocytes (30.6%) with identifiable nuclear material had cytoplasm penetrated or fertilized by sperm. The percentage of oocytes developing into early stage embryos was 10.1% (27/267). Although pronuclear development was observed to be higher for oocytes recovered at the follicular phase, the cleavage rate was similar among oocytes recovered from bitches at the follicular, anestrus, and luteal stages. There was no correlation between the proportion of capacitated or acrosome reacted spermatozoa and pronuclei formation and/or percent cleavage. It was concluded that TCM-199 supplemented with 1 microg/ml hST and estradiol (20 microg/ml) supports nuclear and cytoplasmic maturation of canine oocytes. In this study, meiotic competence was verified by the in vitro production (IVP) and development of embryos up to the 8 cell-stage. Furthermore, the results indicate that, under the described conditions and despite the influence of reproductive status of the bitch on the developmental competence of in vitro fertilized oocytes to the pronuclei stage, cleavage was independent of donor's reproductive estrous cycle stage.     

Effect of conditioned medium of mouse embryonic fibroblasts produced from EC-SOD transgenic mice in nuclear maturation of canine oocytes in vitro

The rate of in vitro maturation (IVM) of canine oocytes has not improved in comparison to that of other mammalian species. This study aims to improve the efficiency of canine oocytes IVM using the antioxidant, extracellular superoxide dismutase (EC-SOD). Thus, the effect of conditioned medium of EC-SOD transgenic mouse embryonic fibroblasts cultured with MEF culture medium (DMEM + 5% FBS) for in vitro nuclear maturation in canine oocytes was investigated. In experiment I, oocytes were collected from the ovaries of domestic bitches, which were allotted to one of two groups: (1) TCM199 + 1% FBS (n = 108) or (2) DMEM + 5% FBS (n = 112), cultured for 48 h and investigated for in vitro nuclear maturation of canine oocytes using Hoechst staining. Meiotic progression to metaphase II in group 1 was 1.8% compared to 1.8% in group 2. In experiment II, EC-SOD levels were examined in NTg-CMEF and Tg-CMEF at 0, 2 and 4 days obtained from EC-SOD transgenic mice generated in our laboratory. The concentration of EC-SOD in Tg-CMEF at day 2 (371.7 +/- 3.1 ng/ml) was the highest for all groups (P < 0.05). EC-SOD levels in Tg-CMEF were higher than in NTg-CMEF; therefore, the efficiency of Tg-CMEF for IVM was investigated. In experiment III, oocytes were allotted to one of three groups: (1) Tg-CMEF at day 0 (n = 84), (2) Tg-CMEF at day 2 (n = 92) or (3) Tg-CMEF at day 4 (n = 98), cultured for 48 h and the IVM of canine oocytes investigated. The mean percentage of MII oocytes in IVM was 2.4, 4.4 and 2.0% for groups 1, 2 and 3, respectively. In experiment IV, the effects of conditioned medium of EC-SOD transgenic mouse embryonic fibroblasts (Tg-CMEF) cultured in MEF culture medium were compared with conditioned medium acquired from non-transgenic mouse embryonic fibroblasts (NTg-CMEF) on IVM of canine oocytes. In this experiment, meiotic progression to metaphase II was 7.1% in Tg-CMEF versus 0% in NTg-CMEF (P < 0.05). Tg-CMEF was more effective than NTg-CMEF. In conclusion, it was verified that canine oocytes were able to effectively progress to metaphase II in IVM when cultured in Tg-CMEF.     

Effect of co-culturing with embryonic fibroblasts on IVM, IVF and IVC of canine oocytes

We studied the effects of mouse embryonic fibroblasts (MEF) and canine embryonic fibroblasts (CEF) on IVM, IVF and IVC of canine oocytes. Cumulus-oocyte complexes were harvested from ovaries by slicing, and in vitro maturation was evaluated in three different conditions: culture media only (control), co-culture with MEF, or co-culture with CEF. The oocytes were cultured for 48 or 72 h. Only oocytes larger than 100 microm in diameter with a homogeneous dark cytoplasm and two or more layers of cumulus cells were used. The culture medium was TCM 199+10% fetal bovine serum (FBS) with 100 IU/mL penicillin and 100 microg/mL streptomycin. After 48 h of IVM, the oocytes were fertilized in vitro with fresh canine spermatozoa that had been selected by a swim-up method, and the oocytes and spermatozoa were co-cultured in modified Krebs-Ringer bicarbonate solution (TYH) for up to 20 h in 5% CO2 in air at 38.5 degrees C. After insemination, oocytes were transferred to three different conditions (the same as for IVM) and were cultured. After 48 or 72 h of maturation in vitro, the maturation rate of MII oocytes cultured in co-culture of MEF and CEF was higher than for oocytes cultured in control (P<0.05). Although the rate that reached the MII stage was not different in the 48 and 72 h cultures, the percentage of degenerated oocytes was greater at 72 h in all three treatment groups. The proportion of monospermic and polyspermic oocytes was not different among the three treatment groups. Cleavage rates were higher in the MEF and CEF treatment groups than in the control group (P<0.05). Co-culture with CEF developed the embryo up to the 16-cell stage, and with MEF up to morula stage. In conclusion, co-culture of embryonic fibroblast cells enhanced nuclear and cytoplasmic maturation of canine oocytes.     

Effects of estradiol-17beta and progesterone supplementation on in vitro nuclear maturation of canine oocytes

Unlike in other domestic animals, in vitro maturation (IVM) of canine oocytes has had limited success. The present study investigated the effect of the estrous cycle and estradiol-17beta (E2) or progesterone (P4) supplementation on in vitro nuclear maturation of canine oocytes recovered from domestic dog ovaries in various reproductive states (follicular, luteal or anestrous stages). Oocytes were cultured in serum-free tissue culture medium (TCM)-199 supplemented with various concentrations of E2 (Exp. 1: 0, 0.1, 1.0 or 2.0 microg/ml) or P4 (Exp. 2; 0, 0.5, 1.0 or 2.0 microg/ml) for 72 h to determine the effective concentration of hormones. In Exp. 3, in order to investigate the synergistic effect of E2 and P4 supplementation, three groups of oocytes were cultured with 2 microg/ml E2 plus various concentrations of P4 (0, 0.5, 1.0 or 2.0 microg/ml). As results, the rate of maturation to metaphase II (MII) stage was significantly higher (P < 0.05) in oocytes from the follicular stage supplemented with 2 microg/ml E2 (14.7%) compared to the other groups (1.5-8.2%). Significantly higher (P < 0.05) maturation rate to MII stage was observed in oocytes from the follicular stage supplemented with 1.0 (10.0%) or 2.0 microg/ml (10.8%) P4 compared to the other groups (0-4.8%). Furthermore, more (P < 0.05) oocytes from the follicular stage supplemented with 2.0 microg/ml of E2 and P4 (16.6%) were matured to MII stage compared to oocytes from the follicular stage supplemented with 2.0 microg/ml E2 alone (10.4%) or the other groups of oocytes (0-7.8%). Interestingly, compared to 2.0 microg/ml E2 alone (10.4%), supplementation of 2 microg/ml E2 + 0.5 microg/ml P4 (3.4%) decreased the maturation of oocytes from the follicular stage to MII stage. In conclusion, the present study demonstrated that supplementation of the culture medium with E2 or P4 alone significantly increased maturation of canine oocyte to MII and that P4 supplementation with E2 further promote or decrease oocyte maturation compared to E2 alone depending on P4 concentration.     

Effect of 17beta-estradiol on the in vitro maturation of bovine oocytes

Although 1 microg/ml of 17beta-estradiol (E2) is often used in routine in vitro maturation (IVM) and in vitro fertilization (IVF), its effect remains controversial. The objective of our study was to investigate the effects of E2 on bovine oocyte IVM and subsequent embryo development, using a defined medium. Bovine cumulus oocyte complexes (COCs), aspirated from 2 to 8 mm follicles of slaughterhouse ovaries, were matured in TCM199 in the presence of 1 microg/ml E2 with or without 0.05 IU/ml recombinant hFSH. Cultures without E2, FSH or both served as controls. COCs were matured for 22 h at 39 degrees C in a humidified atmosphere of 5% CO2 in air. To investigate the effect of E2 with and without FSH on nuclear maturation, COCs were fixed after maturation and the nuclear stage was assessed following DAPI staining. Similarly, denuded oocytes (DO) were matured in the presence of E2 and the nuclear stage assessed after 22 h. To investigate the effect of E2 with and without FSH during IVM on subsequent embryo development, in vitro matured COCs were fertilized in vitro and after removal of the cumulus cells, the presumed zygotes were cocultured on BRL monolayer for 11 days. At Day 4, the number of cleaved embryos, and at Days 9 and 11, the number of blastocysts, were assessed. Addition of 1 microg/ml E2 to TCM199 significantly decreased the percentage of Metaphase II (MII) compared to control (56.3 and 74.0%, respectively), and increased the percentage of nuclear aberrations compared to control (13.3 and 2.1%, respectively). The negative effect of E2 on nuclear maturation was stronger when DO were matured; 25.1 and 60.0% of the oocytes reached MII stage for the E2 and control groups, respectively. When COCs were matured in TCM199 supplemented with FSH, the addition of 1 microg/ml E2 did not influence the proportion of MII oocytes, although a higher percentage of nuclear aberrations as compared to control was observed. Presence of E2 during IVM also decreased the blastocyst rate (14.4 and 10.0% for control and E2 groups, respectively). However, when FSH was present, the addition of E2 had no effect on the cleavage rate and blastocyst formation (20.3 and 21.7% for control and E2 groups, respectively). In conclusion, supplementation of 1 microg/ml E2 to a serum free maturation medium negatively affects bovine oocyte nuclear maturation and subsequent embryo development. Although these effects are attenuated in the presence of FSH, we strongly suggest omission of E2 in routine maturation protocols of bovine oocytes.     

In vitro maturation,fertilization and development of domestic cat oocytes recovered from ovaries collected at three stages of the reproductive cycle

This study was conducted to examine the effect of the donor cat's reproductive cycle stage on in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro development of oocytes recovered from ovaries that were collected and stored at 35 degrees C for a short period (1-6 h). Based on the presence or absence of follicles and corpora lutea, the ovarian pairs collected were classified into inactive, follicular, or luteal stages. Nuclear status of 161 cumulus-oocyte complexes (COCs) were examined immediately after recovery; 91.3% of the oocytes were found to be at the immature germinal vesicle (GV) stage, and 3.7% of the oocytes were at metaphase II (MII) stage. The percentage of the oocytes at the GV stage was significantly lower in the follicular stage than in the inactive stage (P < 0.01). Of the oocytes from the follicular stage, 9.1% were at MII stage. After culture for 24 h, however, the proportions of oocytes that reached metaphase I and MII were not different among the reproductive cycle stages of the ovaries collected (P > 0.05). After co-incubation with sperm, 63.1% of oocytes were fertilized, but there were no significant differences among the reproductive cycle stages of the ovaries with respect to the proportions of normal and polyspermic fertilization. However, the number of oocytes reaching cleavage stage and development to the morula and blastocyst stages from follicular stage ovaries were significantly lower (P < 0.05) than those obtained from inactive and luteal stage ovaries. These results indicate that the reproductive cycle stage of donor cat ovaries, stored at 35 degrees C, has no apparent effects on the frequencies of maturation and fertilization of oocytes, but influences developmental competence of the oocytes following IVM or IVF.     

In vitro maturation and fertilization of oocytes from Norwegian semi-domestic reindeer (Rangifer tarandus )

Reindeer oocytes were submitted to in vitro maturation, fertilization and culture (IVM,IVF,IVC) using the established procedures for bovine in vitro embryo production. The study was conducted outside the main breeding season. Semen was collected from epididymides immediately after slaughter, and was diluted in Tris-fructose-citric acid extender containing 6% glycerol and 20% egg-yolk and then frozen in liquid nitrogen. Following 24 h of maturation, cumulus expansion was complete, and 71% of the oocytes reached Metaphase II (MII), with extrusion of the first polar body. Of the remaining oocytes, 22% were at the germinal vesicle stage (GV), 2% at diakinesis and 5% at Metaphase I (MI). The percentages of fertilization and cleavage were 36.0 and 31.8%, respectively. Two of the fertilized oocytes developed to the morula stage after 7 d of culture.     

Chromosome configuration during in vitro maturation of goat, sheep and buffalo oocytes

The competence of meiotic chromosome configuration at the time of co-culture of oocytes with spermatozoa is an essential prerequisite for successful in vitro fertilization (IVF). Although this technology has been used in several livestock species, various intrinsic and extrinsic factors affecting the high repeatablity of IVF have yet to be understood. The present study was conducted to determine the appropriate time for coculture of oocytes and spermatozoa in order to optimize the fertilization rate in sheep, goats and buffalo. Oocytes were collected from the ovaries of slaughtered animals. The oocytes were divided into 10 groups and cultured for maturation in TCM-199 supplemented with estrous cow serum for different durations at 38.5 x 0.5/C in a CO(2) incubator. Sheep and goat oocytes were removed from culture medium after 0,6,12,22,24,26,28,30,32 and 36 and buffalo oocytes after 0,6,12,16,20,22,24,26,28, and 36 h. The oocytes were treated with hypotonic solution (0.75 M KCl) and fixed in Carony's fixative on glass slides. The fixed oocytes were stained with Giemsa solution, and the meiotic chromosomes were evaluated under a compound microscope at x 1000 magnification. Observations were recorded on a total of 1328 oocytes (sheep, 409; goat, 727 and buffalo, 192). The sequential configurations of diffused chromatin, pachytene, diplotene (along with nucleoli), diakinesis and metaphase II (MII) were analyzed at different durations of culture. Control oocytes (fixed at 0 h without incubation) were mostly at the pachytene stage, and as the duration of culture increased the instances of diplotene, diakinesis and finally MII increased. Oocytes at the MII stage of meiosis are known to be at the optimal stage of development for co-culture with spermatozoa and successful in vitro fertilization. On the basis of sequential configuration of chromosomes, it was found that the optimal duration of in vitro maturation of oocytes is 32, 30 and 24 h for sheep, goats and buffalo, respectively.     

Heparin effect on in vitro nuclear maturation of bovine oocytes

Oocytes undergo numerous biochemical and morphological changes during their development from preantral to preovulatory phases. In vitro studies have suggested several compounds that might induce oocyte maturation. Heparin is a natural component of ooplasm, follicular fluid and uterine fluid and previous studies indicated that it might act as a chromatin maturation factor in bovine oocytes. We tested this hypothesis in vitro by timing germinal vesicle breakdown (GVBD) and first polar body (PB) formation without any other natural or introduced factors that might influence the rate of oocyte maturation. We also determined if these oocytes could be fertilized. Bovine oocytes were incubated in a salt medium and TCM 199 supplemented with different concentrations of heparin for 24 h at 37.5 degrees C in a humidified atmosphere of 5% CO2. With 1.0 and 6.5 mg/ml heparin, the time of GVBD was reduced from 4.7+/-1.1 h to about 1.5 h and the time of first PB formation was reduced from 22.0+/-1.1 h to 9.0-11.0 h in salt medium. In TCM 199, only 6.5 mg/ml heparin significantly reduced the time of PB formation. In both incubation media, 1.0 and 6.5 mg/ml heparin induced GVBD, extrusion of the first PB and formation of the metaphase II nucleus. Moreover, heparin did not interfere with the fertilization of oocytes matured in TCM 199. Based on the results, we propose that heparin plays an important role in the rearrangement of the oocyte chromatin and acts as an oocyte maturation factor.     

Comparison of cytoskeletal integrity,fertilization and developmental competence of oocytes vitrified before or after in vitro maturation in a porcine model

Aim of the study was to investigate the effect of vitrification on viability, cytoskeletal integrity and in vitro developmental competence after in vitro fertilization (IVF) of oocytes vitrified before or after in vitro maturation (IVM) using a pig model. Oocytes from abattoir-derived porcine ovaries were vitrified at either the germinal vesicle (GV) or metaphase II (MII) stage by modified solid surface vitrification (SSV). Oocyte viability was evaluated by stereomicroscopic observation whereas their nuclear stage and morphology of microtubules and F-actin were observed by confocal microscopy after immunostaining. Fertilization was assessed by orcein staining. The survival rate after vitrification was higher for MII-stage than for GV-stage oocytes. However, the ability of surviving oocytes to reach the MII stage after vitrification at the GV stage (GV-vitrified oocytes) was similar to that of control oocytes. Furthermore, after IVM, GV-vitrified oocytes had better spindle and F-actin integrity than oocytes vitrified at the MII stage (MII-vitrified oocytes). In accordance with this result, GV-vitrified oocytes had better ability to extrude the second polar body and support male pronucleus formation after in vitro fertilization (IVF), in comparison to MII-vitrified oocytes. Fertilization rates did not differ among groups. Finally, the ability of GV-vitrified oocytes to develop into embryos was superior to that of MII-vitrified oocytes. However, both vitrified groups showed reduced blastocyst development compared with the control group. In conclusion vitrification of porcine oocytes at the GV stage is advantageous in conferring better cytoskeletal organization and competence to develop to the blastocyst stage in comparison with vitrification at the MII stage.     

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.     

Synthetic oviductal fluid and oviductal cell coculture for canine oocyte maturation in vitro

The perfection of in vitro maturation in the bitch has yet to be achieved, and is an essential prerequisite for gamete salvage programmes in endangered canine species. In contrast to most mammals, the bitch ovulates an immature oocyte which undergoes meiotic maturation within the oviduct. A model of the oviductal environment may therefore be useful for performing in vitro maturation. This study was performed to investigate the effect of introducing an oviductal element to the culture environment, first with the use of a synthetic oviductal fluid (SOF), and secondly, using coculture with isolated canine oviductal epithelial cells, upon the rate of oocyte maturation in vitro. It was found that there was no difference in the proportion of oocytes undergoing germinal vesicle breakdown (GVBD) after 48 h in culture between SOF containing 0.3% bovine serum albumin (BSA, 45%), containing 4% BSA (36%) and control medium 199 (27%). There was also no difference in oocyte nuclear maturation to metaphase I/anaphase I/metaphase II (MI/AI/MII) after 48 h in culture between SOF containing 0.3% BSA (5%), containing 4% BSA (7%) and control medium 199 (6%). In addition, there was no difference in oocyte nuclear maturation to MI/AI/MII after 96 h between SOF containing 0.3% BSA (0), containing 4% BSA (7%) and control medium 199 (11%). In contrast, the proportion of oocytes undergoing GVBD after 96 h in culture was affected by the treatment used, with 27% in SOF + 0.3% BSA, 62% in SOF + 4% BSA and 63% in medium 199. It was found that there was no difference in the proportion of oocytes undergoing GVBD between the coculture treatments 199 (33%), 199 + cells (37%), coculture medium (30%) and coculture medium + cells (49%), and for oocyte nuclear maturation to MI/AI/MII, between medium 199 (2%), 199 + cells (0), coculture medium (6%) and coculture medium + cells (2%) after 48 h in culture. In addition, there was no difference in oocyte nuclear maturation to GVBD after 96 h between 199 (61%), 199 + cells (59%), coculture medium (65%) and coculture medium + cells (53%). In contrast, the proportion of oocytes maturing to MI/AI/MII after 96 h in culture was affected by the treatment used, with a significant difference between 199 (0), 199 + cells (9%), coculture medium (0) and coculture medium + cells (0). It was shown, therefore, that the culture of oocytes in the SOF improved oocyte nuclear maturation when supplemented with a high concentration of protein and that culture in the presence of oviductal epithelial cells improved oocyte maturation, but only after a prolonged period of time.     

Nuclear maturation and development of IVM/IVF canine embryos in synthetic oviductal fluid or in co-culture with buffalo rat liver cells

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.     

Effect of duration of in vitro maturation on nuclear maturation and fertilizability of feline oocytes

This study was conducted to improve in vitro production of embryos from domestic cats using TCM-199 as an IVM medium. The time sequence of nuclear maturation and the optimal timing of in vitro insemination were examined. Most oocytes were at the germinal vesicle stage immediately after collection; however, 8.3% had already resumed meiosis before IVM culture. After 30 h of IVM culture, the percentage of oocytes at metaphase II (MII) reached a peak (75.5%) and did not change (P>0.05) from 30 to 48 h after IVM culture. The percentage of oocytes with two pronuclei was higher (P<0.05) for oocytes matured for 30 and 36 h (38.2 and 33.0%, respectively) than for those after IVM culture for only 24 h (18.5%). Total sperm penetration rate was highest (P<0.05) for oocytes that had been matured for 30 h (46.1%). After 30 h of IVM and 18 h of IVF culture, 66.3 and 24.8% of inseminated oocytes had cleaved and developed to the blastocyst stage, respectively. We concluded that IVM of feline oocytes for 30 h in TCM-199 resulted in optimal nuclear maturation and sperm penetration.     

The effect of harvesting technique on efficiency of oocyte collection and different maturation media on the nuclear maturation of oocytes in camels (Camelus dromedarius)

The purpose of this investigation was to develop an efficient method for harvesting oocytes from dromedary camel ovaries and to examine the effect of different maturation media on their subsequent maturation in vitro. Oocytes were collected by aspirating the follicular contents using a needle attached to a syringe (Method I, n=163 ovaries) or to a constant aspirating pressure, applied by a vacuum pump (Method II, n=117 ovaries). Individual follicles were excised from ovaries and follicles were punctured with two needles (Method III, n=117). Oocytes were matured in vitro for 40-42 h. At the end of maturation period, oocytes were denuded of cumulus cells and the proportion of oocytes in metaphase-II (MII) stage was determined. In the second experiment, oocytes collected by the dissection method were matured in Tissue Culture Medium199 (TCM), CR1 or modified Connaught Medical Research Laboratories medium-1066 (CMRL) and their nuclear maturation was evaluated after 40-42 h. The recovery rate of oocytes was higher (P<0.01) with Method III compared with Method I or II (94, 31 and 33%, respectively). A higher proportions of oocytes collected with Method I or II were either completely or partially denuded compared with Method III (31, 14% versus 1%). The proportions of viable oocytes (78, 60 and 70%, respectively) and those showing metaphase II was not different (39, 50 and 46%, respectively, P>0.05) among the three treatment groups. Oocyte maturation rate was higher (P<0.05) when TCM was used compared with CMRL or CR1 medium. There was, however, no difference in the maturation rate for oocytes cultured in CMRL or CR1 medium. It may be concluded that a higher proportion of cumulus enclosed oocytes may be recovered by follicle dissection method compared to aspiration using syringe or pump. The higher recovery rate with a comparable proportion of viable and matured oocytes resulted in the overall increase in the number of matured (MII) oocytes/ovary with follicle dissection procedure compared with aspiration procedures. For in vitro maturation of oocytes, TCM is superior to CR1 and CMRL as basic maturation medium for this species.     

In vitro maturation of porcine oocytes in follicular fluid with subsequent effects on fertilization and embyo yield in vitro

The objective of the present study was to test the ability of porcine follicular fluid (pFF) to improve maturation of porcine cumulus-oocyte-complexes (COC) in vitro and to observe subsequent effects on fertilization and development to late morula/blastocyst stages under in vitro conditions. The COC were incubated in Tissue Culture Medium (TCM) 199, supplemented with 1% fetal calf serum (FCS), 10% pFF collected from immature follicles (2 to 5 mm), with or without addition of 1microg/ml FSH. Control groups were matured in TCM 199 with or without FSH. Follicular aspirates were centrifuged (1700 x g, 5min.) and the supematants were stored at -20 degrees in 1.5-ml Eppendorff cups until used. On 7 experimental days a total of 3849 immature COC was aspirated from follicles ranging from 2 to 5 mm in diameter. A total of 1117 COC was selected for the experiments, and 239 COC were fixed and stained with 1.5% aceto-orcein after 48 h of in vitro maturation at 39 degrees C with 5% CO(2) in humidified air. Germinal vesicle breakdown (GVBD; 91.7%) and development to metaphase II (60.4%) were superior (P 相似文献   

Supplements to in vitro maturation media affect the production of bovine blastocysts and their apoptotic index but not the proportions of matured and apoptotic oocytes

The objective of this study was to compare the effect of different supplements to the basic IVM medium (TCM199) on the efficiency of cattle oocyte maturation and blastocyst production, and the incidence of apoptosis in both oocytes and blastocysts. Two protein supplements (FBS and fafBSA) and a macromolecule (PVP40) were compared in a 3 treatmentsx9 replicates design. Cumulus-oocyte complexes (COCs) aspirated from slaughterhouse ovaries were matured for 24h in TCM199 medium supplemented with 10% FBS, 6% fafBSA or 4% PVP40 (50-70 COCs in each treatment/replicate), then inseminated and cultured in vitro for 8 days. Immature and mature oocytes as well as Day 8 blastocysts were subjected to TUNEL analysis. Cleavage rate was monitored on Day 2 post-insemination (pi), whereas blastocyst yield on Day 8 pi. The composition of maturation media did not affect zygotic cleavage rate on Day 2 (on average 71.0%), however the blastocyst rate on Day 8 pi was significantly lower (P<0.001) for embryos derived from oocytes matured with PVP40 (16.0%) than for those matured with FBS (22.4%) or fafBSA (22.1%). The rate of TUNEL positive oocytes differed significantly between immature (1.4%) and mature (11.2%) oocytes (P<0.01). Supplements to maturation medium were not related to the incidence of apoptosis in mature oocytes (11.2%) and the rate of oocytes at the second metaphase stage (71.5%). Cumulus cell expansion was reduced by maturation in medium supplemented with PVP40. This macromolecule was also correlated with higher apoptotic index in blastocysts (5.8%) when compared to FBS (3.2%) and fafBSA (3.1%; P<0.001). In conclusion, lower blastocyst rate and elevated apoptotic index in embryos derived from oocytes matured with PVP40 may suggest that synthetic macromolecule provides less balanced environment for oocyte maturation and therefore should be treated with caution.     

Effects of sperm treatments on the in vitro development of bovine oocytes in semidefined and defined media

Bovine in vitro matured oocytes were inseminated with frozen-thawed spermatozoa prepared by A) swim-up through Fert-TALP supplemented with hyaluronic acid (HYA, 1 mg/ml), heparin (5.0 microg/ml) and bovine serum albumin (BSA, 6 mg/ml) or B) washing by centrifugation in modified Brackett-Oliphant medium (mBO) supplemented with 10 mM caffeine-sodium benzoate. For Method A, in vitro fertilization (IVF) was performed in Fert-TALP supplemented with 6 mg/ml BSA, 5.0 microg/ml heparin, 20 microM D-penicillamine, 10 microM hypotaurine and 1 microM epinephrine. For Method B it was performed in mBO medium supplemented with 10 mg globulin-free BSA/ml and 10 microg heparin/ml. Presumptive zygotes were cultured in 1 of 3 culture media: 1) BSAITS - TCM 199 supplemented with 10 mg/ml BSA and ITS (5 microg/ml insulin, 5 microg/ml transferrin, and 5 ng/ml sodium selenite); 2) BECM - bovine embryo culture medium; and 3) BECM supplemented with ITS. Altogether, a significantly higher proportion of oocytes developed to the blastocyst stage after insemination with spermatozoa prepared by Method A than by Method B (17.9 vs 7.1%, respectively; P < 0.001). For Method A, the cleavage rate and the proportion of zygotes with >2 cells 48 h after insemination did not differ significantly between any of the 3 culture media assayed, but blastocyst formation was significantly stimulated in BSAITS and BECMITS compared with that in BECM (20.7 and 22.1% vs 10.7%, respectively; P < 0.05). For Method B, the cleavage rate and the proportion of zygotes with >2 cells were significantly lower in BSAITS than in BECM and BECMITS (56.4 and 28.7% vs 71.6 and 42.1%; and 70.2 and 51.1%, respectively; P < 0.05). However, no significant differences were recorded in blastocyst development rates between any of the culture media assayed (6.4 to 7.4%; P > 0.05).