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.     

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.     

In vitro canine oocyte nuclear maturation in homologous oviductal cell co-culture with hormone-supplemented media

The aim of the present research was to verify the influence of oviductal cell co-culture previously supplemented with steroids (estrogen, progesterone, or both) on IVM rates for oocytes from anestrous bitches that were cultured in vitro for 48, 72 and 96 h. Oocytes harvested from anestrous bitches were selected and allocated into four groups: Group 1 (co-culture in oviductal epithelial cells without hormonal supplementation-control); Group 2 (estrogen supplementation); Group 3 (progesterone supplementation); Group 4 (estrogen+progesterone supplementation). The oviductal epithelial cell culture was established 72 h prior to oocyte co-culture. After periods of 48, 72 and 96 h, the degree of oocyte nuclear maturation was assessed. Co-culture in oviductal epithelial cells with estrogen was not as beneficial for canine IVM as supplementation with progesterone and estrogen, or progesterone supplementation alone. Therefore, it was feasible to use co-culture with oviductal epithelial cells obtained from anestrous bitches for IVM (monolayer culture with oviduct cells previously supplemented with progesterone). Final stages of oocyte maturation were achieved at 72 and 96 h of culture; therefore, the duration of maturation for oocytes obtained from bitches in different stages of the estrous cycle should be taken into account.     

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.     

Mitochondrial distribution and meiotic progression in canine oocytes during in vivo and in vitro maturation

The objective was to evaluate mitochondrial distribution, and its relationship to meiotic development, in canine oocytes during in vitro maturation (IVM) at 48, 72, and 96 h, compared to those that were non-matured or in vivo matured (ovulated). The distribution of active mitochondria during canine oocyte maturation (both in vitro and in vivo) was assessed with fluorescence and confocal microscopy using MitoTracker Red (MT-Red), whereas chromatin configuration was concurrently evaluated with fluorescence microscopy and DAPI staining. During IVM, oocytes exhibited changes in mitochondrial organization, ranging from a fine uniform distribution (pattern A), to increasing clustering spread throughout the cytoplasm (pattern B), and to a more perinuclear and cortical distribution (pattern C). Pattern A was mainly observed in germinal vesicle (GV) oocytes (96.4%), primarily in the non-matured group (P < 0.05). Pattern B was seen in all ovulated oocytes which were fully in second metaphase (MII), whereas in IVM oocytes, ∼64% were pattern B, irrespective of duration of culture or stage of nuclear development (P > 0.05). Pattern C was detected in a minor percentage (P < 0.05) of oocytes (mainly those in first metaphase, MI) cultured for 72 or 96 h. In vitro matured oocytes had a minor percentage of pattern B (P < 0.05) and smaller mitochondrial clusters in IVM oocytes than ovulated oocytes, reaching only 4, 11, and 17% of MII at 48, 72, and 96 h, respectively. Thus, although IVM canine oocytes rearranged mitochondria, which could be related to nuclear maturation, they did not consistently proceed to MII, perhaps due to incomplete IVM, confirming that oocytes matured in vitro were less likely to be competent than those matured in vivo.     

In vitro maturation, in vitro fertilization and embryonic development of canine oocytes

In this study we have investigated the efficiency of in vitro maturation (IVM) as a basic way to study the development of canine oocytes after in vitro fertilization (IVF). We decided, therefore, to perform two-part experiments. Firstly, experiment I compared the effects of TCM199 without fetal bovine serum (FBS) with TCM199 supplemented with 5% FBS on the in vitro nuclear maturation rate of canine oocytes. For the efficiency of meiotic development to the metaphase II (MII) stage, we found that 4.7% (4/64) of all oocytes grown in TCM199 without FBS developed to the MII stage compared with only 1.7% (1/59) of those grown in TCM199 with 5% FBS for 48 h. Therefore, FBS did not increase in vitro nuclear maturation. In experiment II, the cleavage rate of canine oocytes used for IVF was investigated following heparin treatment. Canine oocytes were fertilized in four groups: Fert-TALP medium without heparin (Fert I) or Fert-TALP medium supplemented with 10, 20 or 30 microg/ml heparin (Fert II, Fert III, Fert IV, respectively). Oocytes that were grown for 24 h in Fert I following fertilization showed the highest rate of all of the groups, 6.5% (5/77) and developed to the early morula stage. Markedly, the oocytes cultured in Fert I for 24 h following insemination had a higher rate of embryonic development than other groups. We can assert that, unlike findings in other mammals, heparin treatment in canine IVF does not increase the efficiency of the fertilization rate and is therefore not an important factor.     

Influence of co-culture with oviductal epithelial cells on in vitro maturation of canine oocytes

The process of oocyte maturation in the canine species is unique among mammals: oocytes are immature at ovulation and the resumption and progression of meiotic maturation occur in the oviduct. This study was performed to investigate (i) the effect of co-culture with infundibulum and ampullar oviductal epithelial cells on the in vitro maturation of canine oocytes and (ii) the culture time necessary to reach full meiotic maturation. For this purpose the oocytes, collected from the ovaries of bitches undergoing ovariectomies, were divided into three groups and cultured for 48 and 72 h with the following systems: (A) TCM 199 + 10% oestrus bitch serum + FSH (0.1 IU.mL(-1)), LH (0.1 IU.mL(-1)) + progesterone (1 microg.mL(-1)) + oestradiol (1 microg.mL(-1)) + cysteamine (100 microM); (B) medium A plus infundibulum cells; (C) medium A plus ampullar cells. Infundibulum and ampullar cells were recovered from the oviducts of bitches at the oestrus stage of their cycle. The results showed that after 48 h of incubation, a significantly higher meiotic resumption (P < 0.01) was observed in the oocytes cultured with infundibulum (59%) and ampullar cells (60.0%), than in the control group (40.0%). There was also a significantly (P < 0.01) higher meiotic progression to the MII in systems B and C (15.6% and 16.7%) than in system A (4.0%). After 72 h of culture, the percentages of meiotic resumption and progression were unchanged. These results showed that both the infundibulum and the ampullar oviductal epithelial cells positively influence the meiotic resumption and progression of canine oocytes and that 48 h are sufficient for the completion of nuclear maturation.     

Epidermal growth factor enhances meiotic resumption of canine oocytes in the presence of BSA

Despite many attempts to improve the in vitro maturation (IVM) of canine oocytes using various culture conditions, the efficiency of canine IVM remains very low compared with that of other domestic animals. In the present study we examined the effect of ovarian estrus stage on oocyte quality, and the effect of epidermal growth factor (EGF) in the presence and absence of macromolecules on the IVM of canine oocytes. More oocytes >or=100 microm in diameter were obtained from follicular ovaries than from ovaries at other estrus stages. After 72 h of culture, significantly more oocytes recovered from follicular ovaries than from anestrous and luteal ovaries were in germinal vesicle break down (GVBD). Bovine serum albumin (BSA) or fetal bovine serum (FBS) supplementation improved meiotic resumption as compared to polyvinyl alcohol (PVA) supplementation; however, there was no difference between the BSA and FBS supplements. The oocytes matured in North Carolina State University (NCSU) 37 medium containing 0.4% BSA and 100 ng/ml EGF showed the highest rates of development to the metaphase II (MII) stage when compared with the control treatment (P < 0.05). These results suggest that the estrous cycle of bitches influences the meiotic resumption of oocytes cultured in vitro, and EGF increases the meiotic resumption of canine oocytes in the presence of BSA in vitro.     

Meiotic arrest maintained by cAMP during the initiation of maturation enhances meiotic potential and developmental competence and reduces polyspermy of IVM/IVF porcine oocytes

We investigated effects of invasive adenylate cyclase (iAC), 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cyclic AMP (dbcAMP) on porcine oocyte in vitro maturation (IVM), in vitro fertilisation (IVF) and subsequent embryonic development. Porcine oocytes were collected in Hepes-buffered NCSU-37 supplemented with or without 0.1 microg/ml iAC and 0.5 mM IBMX. IVM was performed in a modified NCSU-37 supplemented with or without 1 mM dbcAMP for 22 h and then without dbcAMP for an additional 24 h. After IVF, oocytes were cultured in vitro for 6 days. After 12 h of IVM, no difference in nuclear status was observed irrespective of supplementation with these chemicals during collection and IVM. At 22 h, most (95%) of the oocytes cultured with dbcAMP remained at the germinal vesicle (GV) stage, whereas 44.3% of the oocytes cultured without dbcAMP underwent GV breakdown. At 36 h, oocytes cultured with dbcAMP had progressed to prometaphase I or metaphase I (MI) (32.6% and 49.3%, respectively), whereas non-treated oocytes had progressed further to anaphase I, telophase I or metaphase II (MII) (13.6%, 14.3% and 38.0%, respectively). At 46 h, the rate of matured oocytes at MII was higher in oocytes cultured with dbcAMP (81%) than without dbcAMP (57%), while the proportion of oocytes arrested at MI was lower when cultured with dbcAMP (15%) than without dbcAMP (31%). The rate of monospermic fertilisation was higher when oocytes were cultured with dbcAMP (21%) than without dbcAMP (9%), with no difference in total penetration rates (58% and 52%, respectively). The blastocyst rate was higher in oocytes cultured with dbcAMP (32%) than without dbcAMP (19%). These results suggest that a change in intracellular level of cAMP during oocyte collection does not affect maturational and developmental competence of porcine oocytes and that synchronisation of meiotic maturation using dbcAMP enhances the meiotic potential of oocytes by promoting the MI to MII transition and results in high developmental competence by monospermic fertilisation.     

The effect of okadaic acid on meiotic maturation of canine oocytes of different size

The present study was conducted to determine the effect of okadic acid (OA), a potent inhibitor of seronine/treonine 1 and 2A phosphatase, on meiotic resumption and progression in canine oocytes with different diameters. Cumulus-oocyte complexes were collected from ovaries of bitches at different oestrous phases. In Experiment 1, to determine the optimal concentration of OA (0.5 or 2 μM), the oocytes were pre-incubated for 1, 3, and 20 h in TCM 199 supplemented with 20% SCE and thereafter cultured in the same medium without OA. In Experiment 2, the selected oocytes were divided into three groups according to their diameter: <110 μm, 110-120 μm, >120 μm, and pre-incubated in OA 0.5 μM for 1 h. Oocytes were cultured in vitro as previously described. After 72 h of IVM, in Experiment 1, significantly more oocytes reached MII stage with 0.5 μM for 1 h (30.8% P <0.001%) for oocytes cultured in other OA condition and in control group. In Experiment 2, OA induced a significantly higher incidence of MII oocytes in the 110-120 μm and >120 μm groups (P <0.001) compared to control group, but a significantly higher proportion of the oocytes >120 μm pre-incubated with OA progressed to MII (51.3% P <0.001). In contrast, smaller oocytes (<110) did not develop to MII stage with or without OA. In conclusion, treatment of canine oocytes with 0.5 μM for 1 h, improves meiotic maturation. The culture of fully grown (>120 μm) oocytes with OA at the onset of in vitro maturation can result in a higher frequency of meiotic maturation.     

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.     

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.     

Intracytoplasmic sperm injection of in vitro matured oocytes of domestic cats with frozen-thawed epididymal spermatozoa

The ability to mature and fertilize oocytes of endangered species may allow us to sustain genetic and global biodiversity. The first objective of this study was to compare the effect of two different culture media and two different incubation times on in vitro maturation (IVM) of domestic cat oocytes. The second objective was to determine the developmental competence of in vitro matured cat oocytes after intracytoplasmic sperm injection (ICSI) with cat spermatozoa. Oocytes recovered from ovaries of ovariectomized cats were cultured either in TCM 199 medium or in synthetic oviductal fluid (SOF), both of which were supplemented with cysteamine, BSA, FSH, LH. Nuclear maturation was assessed after 24 h and 40 h of incubation. Results of IVM showed that the percentage of oocytes reaching MII after 24 h and 40 h of incubation were significantly higher (P<0.001) after culture with SOF (88/110, 80% and 159/192, 82.8%) than TCM 199 (86/129, 66.7% and 58/90, 64.4%). Oocytes (n = 231) matured in vitro in SOF for 24 h were fertilized by ICSI with frozen-thawed epididymal cat spermatozoa. After ICSI, one group of oocytes (n = 129) was activated with ethanol, and a second group (n = 102) was not activated. The developmental competence of all ICSI oocytes was examined after 7 days of in vitro culture. After 28 h of culture, the cleavage frequency of ICSI-activated oocytes was significantly higher (P<0.001) than that of IC     

Cytogenetic analysis of bovine parthenotes after spontaneous activation in vitro

We conducted a cytogenetic study of bovine parthenotes derived from oocytes matured and cultured in vitro. In vitro maturation was carried out by culturing follicular oocytes for 24 h in TCM199 supplemented with estrous cow serum (ECS) and hormones at 39 degrees C in 5% CO2. Matured oocytes were incubated for 20 h in sperm TALP without the addition of spermatozoa, after which they were cultured in maturation droplets for 48 to 72 h. Spontaneous activation occurred in 9.5% of the matured oocytes. Cytogenetic analysis of 24 parthenotes revealed that 62.5% exhibited a normal, diploid chromosome complement. The remaining 37.5% had various ploidy anomalies: haploidy (25%), triploidy (4.2%) and tetraploidy (8.3%). Parthenotes exhibited different developmental stages. The number of blastomeres ranged from 2 to 8 within a parthenote. Only 1 parthenote was comprised 9 to 16 cells. The results showed that spontaneous parthenogenetic activation which occurs in an IVM/IVF system may interfere with embryo production efficiency.     

Influence of caffeine pretreatment on biphasic in vitro maturation of dog oocytes

Phosphodiesterase (PDE) inhibitors have been utilized for in vitro maturation (IVM) of oocytes to manipulate the meiotic resumption and progression. Premature chromatin condensation and DNA replication of the oocytes, immediately after the decrease in the cAMP level, are the difficulties in canine IVM. Caffeine, a nonselective competitive PDE inhibitor, due to its structural similarity to adenosine molecule maintains the cAMP level by occupying PDE enzymes such as PDE-3A inside the oocyte and PDE-4 and PDE-5 in the cumulus cells. In this study, the effects of 12-hour caffeine pretreatment in a biphasic IVM protocol were assessed on maturation rates of canine oocytes. Sixty hours of culture after a 12-hour of 10 mM caffeine pretreatment resulted in 16.9% ± 2.4 of the oocytes reaching metaphase II stage (MII) and 25.9% ± 5.2 degeneration rate compared with the control group with 2.2% ± 2.2 MII and 37.6% ± 4.3 degeneration rates (P < 0.05). Caffeine pretreatment induced higher mitogen-activated protein kinases (MAPK1 and MAPK3) phosphorylation and maturation-promoting factor activity at 12 hours and activated MAPK1 and maturation-promoting factor at 48 hours after culture in cumulus-oocyte complexes (COCs) compared with the control group (P < 0.05). Fresh canine COCs were also analyzed before IVM using brilliant cresyl blue (BCB) staining. Oocytes showed difference in meiotic resumption (MI-MII) (BCB+ = 16.11% ± 5.5, BCB− = 9.86% ± 5.0; P < 0.05) after 60 hours of culture following 12-hour caffeine pretreatment. The BCB+ canine oocytes had higher MII rate than the BCB− group under caffeine pretreatment (10.2% ± 2.9 vs. 1.1% ± 1.1, respectively; P < 0.05). Results indicated that 12-hour caffeine pretreatment of canine COCs improves the MII maturation rates at 72 hours and BCB+ oocytes have higher competency in vitro for nuclear maturation.     

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.     

The promotory effect of growth hormone on the developmental competence of in vitro matured bovine oocytes is due to improved cytoplasmic maturation

In a previous study we have shown that the addition of growth hormone (GH) during in vitro maturation accelerates nuclear maturation, induces cumulus expansion, and promotes subsequent cleavage and embryonic development. The aim of this study was to investigate whether the promotory effect of GH on subsequent cleavage and blastocyst formation is due to an improved fertilization and whether this effect is caused by an improved cytoplasmic maturation of the oocyte. Therefore, bovine cumulus oocyte complexes (COCs) were cultured for 22 hours in M199 supplemented with 100 ng/ml bovine GH (NIH-GH-B18). Subsequently the COCs were fertilized in vitro. Cultures without GH served as controls. To verify whether the promoted fertilization is caused by the effect of GH on cumulus expansion or oocyte maturation, cumulus cells were removed from the oocytes after in vitro maturation (IVM) and denuded MII oocytes were selected and fertilized in vitro. Both IVM and in vitro fertilization (IVF) were performed at 39°C in a humidified atmosphere with 5% CO₂ in air. At 18 hours after the onset of fertilization, the nuclear stage of the oocytes was assessed using 4,6-diamino-2-phenylindole (DAPI) staining. Oocytes with either an metaphase I (MI) or MII nuclear stage and without penetrated sperm head were considered unfertilized; oocytes with two pronuclei, zygotes, and cleaved embryos were considered normally fertilized; and oocytes with more than two pronuclei were considered polyspermic. To evaluate cytoplasmic maturation, the distribution of cortical granules 22 hours after the onset of IVM, and sperm aster formation 8 hours after the onset of fertilization were assessed. In addition, to assess the sperm-binding capacity, COCs were fertilized in vitro, and 1 hour after the onset of fertilization the number of spermatozoa bound to the oocytes was counted. The addition of GH during IVM significantly (P < 0.001) enhanced the proportion of normal fertilized oocytes. Removal of the cumulus cells prior to fertilization and selection of the MII oocytes did not eliminate the positive effect of GH on fertilization. No effect of GH on the sperm-binding capacity of the oocyte was observed. In addition, GH supplementation during IVM significantly (P < 0.001) enhanced the migration of cortical granules and sperm aster formation. It can be concluded that the promotory effect of GH on the developmental competence of the oocyte is due to a higher fertilization rate as a consequence of an improved cytoplasmic maturation. Mol. Reprod. Dev. 49:444–453, 1998. © 1998 Wiley-Liss, Inc.     

Effects of ovarian cortex cell co-culture during in vitro maturation on porcine oocytes maturation, fertilization and embryo development

The objective of the experiments was to evaluate the effects of porcine ovarian cortex cells (pOCCs) during in vitro maturation (IVM) of porcine oocytes on IVM of porcine oocytes, in vitro fertilization (IVF) parameters and subsequent embryo development. The pOCCs was cultured in the 500 microl TCM199 without hormone until the confluence, and then cultured in 500 microl TCM199 supplemented with hormone for 12 h before the oocytes added. Porcine oocytes were co-cultured with the pOCCs monolayers in the co-culture system for 44 h, following fertilized in the mTBM for 6 h. Finally, the presumptive zygotes were cultured for 144 h in the NCSU-23 supplemented with 0.4% BSA. The results showed that matured M II oocytes in the co-culture group were higher than that in the control group (P<0.05). Although penetration did not differ between the co-culture and control groups (P=0.481), polyspermy declined in the co-culture group (P<0.05), whereas male pronucleus (MPN) formation was improved in the co-culture group compared with the control group (P<0.05). More blastocysts developed in the co-culture group than that in the control group (P<0.05); however, the cleavage rates and the mean number cells per blastocyst showed no significant difference between the treated group and the control group (P=0.560 and 0.873, respectively). In conclusion, the presence of the pOCCs monolayers during IVM enhanced the maturation quality of the porcine oocytes, reduced the polyspermy, increased the percentages of MPN formation and blastocyst, but the blastocyst quality was not improved.     

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.