In vitro fertilization of bovine oocytes in protein-free culture system

We studied the possibility of fertilization of bovine oocyte-cumulus complexes, matured in vitro in a protein-free medium, in a protein-free culture system without preliminary capacitation of spermatozoa. The development of embryos to the morula-blastocyst and blastocyst stage was considered as a criterion of successful fertilization. It was shown that replacement of bovine serum albumin for polyvinyl alcohol or polyvinylpyrrolidone in Tyrode medium for fertilization did not affect significantly the development to the morula-blastocyst stage and the number of cells in blastocysts. It was also found that replacement bovine serum albumin for polyvinyl alcohol in all used media, Tyrode medium for washing of oocytes, medium for sperm preparation to fertilization, and Tyrode medium for fertilization, did not affect significantly the development to the morula-blastocyst and blastocyst stages, as well as on the number of cells in blastocysts. The results obtained suggest that in vitro fertilization of bovine oocyte-cumulus complexes is possible in a protein-free culture system without significant reduction in the capacity for in vitro development of the obtained embryos and number of cells in blastocysts.     

In vitro fertilization of bovine oocytes in in-vitro protein-free culture system

We studied the possibility of fertilization of bovine oocyte-cumulus complexes, matured in vitro in a protein-free medium, in a protein-free culture system without preliminary capacitation of spermatozoa. The development of embryos to the morula-blastocyst and blastocyst stage was considered as a criterion of successful fertilization. It was shown that replacement of bovine serum albumin for polyvinyl alcohol or polyvinylpyrrolidone in Tyrode medium for fertilization did not affect significantly the development to the morula-blastocyst stage and the number of cells in blastocysts. It was also found that replacement bovine serum albumin for polyvinyl alcohol in all used media, Tyrode medium for washing of oocytes, medium for sperm preparation to fertilization, and Tyrode medium for fertilization, did not affect significantly the development to the morula-blastocyst and blastocyst stages, as well as on the number of cells in blastocysts. The results obtained suggest that in vitro fertilization of bovine oocyte-cumulus complexes is possible in a protein-free culture system without significant reduction in the capacity for in vitro development of the obtained embryos and number of cells in blastocysts.     

In vitro-matured/in vitro-fertilized bovine oocytes can develop into morulae/blastocysts in chemically defined, protein-free culture media.

Development of bovine embryos derived from in vitro-matured/in vitro-fertilized (IVM/IVF) oocytes was examined in two culture media: hamster embryo culture medium (HECM), a relatively simple, chemically defined, protein-free medium containing 20 amino acids; and tissue culture medium (TCM)-199, a more complex medium designed for culture of somatic cells. The first experiment studied (1) effects of glucose and/or phosphate (Pi) using HECM and (2) the development of bovine IVM/IVF embryos in four different conditions: HECM, TCM-199, TCM-199 + 10% unheated bovine calf serum (BCS), and oviduct cell-conditioned TCM-199 + 10% BCS. After IVF, 45% of the inseminated oocytes developed to the morula/blastocyst stages (M&B) when cultured in HECM; blastocyst development was depressed in the presence of glucose and Pi when compared to Pi alone. When the four culture conditions were compared, there was no significant difference in M&B development (42-51% of inseminated oocytes). However, blastocyst development in TCM-199 supplemented with 10% BCS (29.7%) or with BCS + oviduct cell-conditioned medium (21.6%) was significantly greater than in nonsupplemented HECM (9.7%) or TCM-199 (13.8%). In the second experiment, the effects of serum supplementation and/or oviduct cell conditioning on HECM and TCM-199 were examined in a 2 x 2 x 2 factorial experiment. Oviduct cell conditioning of either HECM or TCM-199 without serum supplementation did not enhance bovine embryo development. Serum supplementation exhibited a biphasic effect, with inhibition at the first cleavage and stimulation of morula compaction and blastocyst formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro maturation of caprine oocytes in different culture media

Oocytes were aspirated from caprine ovaries, washed and cultured in TCM-199 containing penicillin, streptomycin and 10, 15, or 20% of fetal bovine serum (FBS), estrous sheep serum (ESS) or estrous goat serum (EGS). After 24–26 h culture, oocytes were separated from cumulus and corona cells by hyaluronidase and by passing through a fine pipette, fixed in acetic alcohol, stained with aceto orcein and observed under a phase-contrast microscope for evidence of maturation. High maturation rates (74–94%) were observed in all concentrations of the three different sera examined. No significant difference was observed between different concentrations and among different sera. Almost no maturation (4%) was observed in the medium lacking protein supplement. In conclusion, these sera with the concentrations examined can be substituted for one another for in vitro maturation of caprine oocytes.     

In vitro maturation and fertilization of bovine oocytes and in vitro culture of presumptive zygotes in the presence of bovine pestivirus

A pathogen which has been shown to commonly contaminate in vitro bovine embryo production system is bovine pestivirus (bovine viral diarrhea virus). Three experiments were designed to evaluate the in vitro maturation (experiment I), fertilization (experiment II) and embryo development (experiment III) of immature oocytes, inseminated oocytes and presumptive zygotes in the presence of a bovine pestivirus (non-cytopathic, nCP type 1). The virus inoculum used was derived from a persistently infected cow. In experiment I, follicular oocytes (n=1257) recovered from slaughterhouse derived ovaries were randomly assigned to either a control group (n=578) which did not become exposed to bovine pestivirus and a treatment group (n=679) which was inoculated with bovine pestivirus (2.20-3.69 log(10) TCID(50)/50 microl) at the time of commencement of in vitro maturation. Overall, there was no significant difference between the control and pestivirus inoculated oocytes in either the cumulus cell expansion rate (79+/-7.5% versus 74+/-10.7%) or the nuclear maturation rate (89+/-4.8% versus 85+/-7.4%), respectively. In experiment II, in vitro matured oocytes (n=607) were inseminated either in the absence (control; n=301) or the presence of bovine pestivirus (4-4.6 log(10) TCID(50)/50 microl; n=306). A significant (P<0.01) reduction in the overall number of fertilized oocytes with two well formed male and female pronuclei was observed in the treatment group compared to the control group (58.5+/-5.8% versus 73.3+/-3.6%, respectively). In experiment III, after in vitro maturation and fertilization, presumptive zygotes were randomly assigned to either a control group (n=139) which was not exposed to bovine pestivirus or a treatment group which was inoculated with bovine pestivirus (2.97-4.47 log(10) TCID(50)/30 microl; n=139). The zygotes were then cultured under mineral oil in an atmosphere of 88% N(2), 7% O(2) and 5% CO(2) at 39 degrees C. The morphologic appearance of the embryos was assessed 48 h after the commencement of culture, and then every 48 h up to days 7-8 after insemination. The 22% (31/139) and 3.6% (5/139) of the presumptive zygotes developed to the morula or blastocyst stage in the control and the bovine pestivirus inoculated groups, respectively (P<0.001). This study demonstrates that bovine pestivirus has a significant detrimental effect on in vitro fertilization and early in vitro embryo development.     

In vitro maturation, fertilization and early cleavage rates of bovine fetal oocytes

The in vitro developmental competence of oocytes harvested from 3 to 6 mm follicles from ovaries of 7.5 months to term fetuses and adult cows was compared. Cumulus oocyte complexes (COCs) were washed and placed in 200 microl droplets of maturation medium 199, supplemented with 10 microg/ml FSH, 10 microg/ml LH, 1.5 microg/ml estradiol, 75 microg/ml streptomycin, 100 IU/ml penicillin, 10 mM Hepes, and 10% fetal bovine serum (FBS) under oil and incubated for 24 h at 39 degrees C and 5% CO2. Matured oocytes were exposed to frozen-thawed TALP swim-up, heparin-capacitated sperm (20 h, 39 degrees C, 5% CO2). Presumptive zygotes were cultured in medium 199 containing 8 mg/ml BSA-V, 100 IU/ml penicillin G, 75 microg/ml streptomycin, and 10 mM Hepes (48 h, 39 degrees C, 5% CO2). Oocytes/embryos were fixed, stained with DAPI, and evaluated under fluorescent microscopy to assess maturation, fertilization, and subsequent embryonic development. There was a difference (P<0.05) between fetal and adult cow oocytes for in vitro maturation (IVM; 80.1% versus 92.0%), fertilization (69.3% versus 79.9%), and cleavage rates (36.7% versus 49.9%), respectively. Poor IVM, fertilization and embryonic development of fetal oocytes may be due to a higher incidence of blockage at germinal vesicle (GV) and metaphase-I (M-I) stage after IVM (12.0% versus 2.3% for fetal versus adult oocytes, respectively, P<0.05). Although the IVF results with fetal oocytes are poorer than with adult cow oocytes, they were still high enough to be considered for use in research and when death of the dam and/or fetus is pre-mature or sudden.     

Successful vitrification of bovine blastocysts,derived by in vitro maturation and fertilization

Bovine blastocysts were produced through maturation, fertilization, and development in vitro. For vitrification, solutions designated EFS, GFS, and PFS were prepared; these were 40% ethylene glycol, 40% glycerol, and 40% propylene glycol, respectively, diluted in modified phosphate-buffered saline (PBS) containing 30% Ficoll + 0.5 M sucrose. The embryos were exposed to the solutions in one step at room temperature, kept in the solutions for various times, vitrified in liquid nitrogen, and warmed rapidly. When the embryos were vitrified in EFS solution after 1 or 2 min exposure, the postwarming survival rate, assessed by the reexpansion of the blastocoel, was 74–77%. However, when the exposure time was extended to 3 min or longer, this rate dropped to 7–0%. This reduction was attributed to the toxicity of ethylene glycol. Of the embryos vitrified in GFS solution, 53% survived when they were cooled after 1 min exposure; as the duration of the exposure increased, the survival rate increased, reaching a peak (72%) at 4 min. The rate then decreased gradually with exposure time. In PFS solution, embryos surviving after vitrification were recovered only with 1 min exposure (33%), reflecting the high toxicity of propylene glycol. After vitrification in EFS or GFS solution, two embryos were nonsurgically transferred into each of 14 recipient animals. Of the 14 recipients, ten (71%) became pregnant; two resulted in early stillbirths, four recipients delivered twins (four alive and four stillborn), and two delivered single live calves, demonstrating the effectiveness of this simple vitrification method for the cryopreservation of in-vitro-produced bovine blastocysts. © 1993 Wiley-Liss, Inc.     

In vitro maturation and fertilization of goat oocytes

Goat oocytes were isolated from 3-5 mm diam. follicles. The oocytes with compact cumulus mass were matured and fertilized in vitro. Three different media, viz. modified Krebs-Ringer bicarbonate, Dulbecco's and Ham's F-12 with three different additives (bovine serum albumin, BSA; follicle stimulating hormone, FSH and fetal calf serum, FCS) were tested. The three basal media gave almost similar results with Ham's F-12 being slightly better. Addition of BSA (10 mg/ml) increased the rates of maturation and penetration. FSH + BSA (2.5 micrograms/ml + 10 mg/ml) further enhanced the rates while FCS (10%) proved to be even more effective. In modified Krebs-Ringer bicarbonate and Dulbecco media with additives FCS + BSA, around 60% oocytes matured to metaphase II of which 53% were penetrated by capacitated goat spermatozoa while in F-12 medium 70% reached metaphase II and 63% were penetrated. Ham's F-12 medium with additives FCS + BSA was slightly better for maturation and penetration of goat oocytes in comparison to two other media tested.     

In vitro maturation and fertilization of goat oocytes

Follicular cumulus-enclosed goat oocytes were matured in vitro in the presence of granulosa cells, follicle stimulating hormone (FSH), luteinizing hormone (LH) and estradiol-17beta. While 86% of the oocytes from follicles 2 to 6 mm in diameter achieved meiotic maturation, only 24% of the oocytes from follicles 1 to 2 mm in diameter progressed to Metaphase II. Exposure of follicle-enclosed cumulus-oocyte complexes to 20 degrees C prior to culture resulted in 11.5% of the oocytes exhibiting abnormal meiotic spindle. This indicated that immature goat oocytes are particularly sensitive to temperature. Ejaculated spermatozoa were capacitated according to the technique previously proposed for ram sperm (1). The fertilization rates of ovulated and mechanically denuded in vitro-matured oocytes were 85 and 82.8%, respectively; 59.7% of ovulated and 57.1% of in vitro-matured oocytes were normally fertilized, as shown by the presence of both the female and the male pronucleus as well as by the remnants of the sperm tail in the ooplasm, 17 hours after insemination. Polyspermy was the main abnormality detected, and it affected almost 20% of the inseminated oocytes. The cleavage rate (two to fourcell stage) 41 hours after insemination of in vitro-matured and fertilized oocytes was 58%.     

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.     

In vitro maturation and fertilization of prepubertal goat oocytes

The aim of this work was to study the IVM-IVF of prepubertal goat oocytes collected from a slaughterhouse as an alternative source of oocytes to those of FSH-primed adult goats. In Experiment 1, IVM of prepubertal goat oocytes in co-culture with granulosa cells were compared with IVM in 50 microl microdrops of medium. There was no significant difference in the percentage of maturation (72.0 vs 76.9%) between the 2 groups. In Experiment 2, a low percentage of normal fertilization (24.4%) was observed for prepubertal goat oocytes matured with granulosa cells from prepubertal goats. This result was significantly lower than that obtained for ovulated (62.2%) or in vitro-matured (48.7%) oocytes from adult goats. There were no significant differences with respect to the oocytes from adult goats matured in vitro when prepubertal goat oocytes were cultured with adult goat granulosa cells (33.3%) or in microdrops (29.7%). No differences were observed among the treatments in the percentage of oocytes showing evidence of fertilization (normal fertilization + abnormal fertilization + polyspermy). In Experiment 3, it was shown that there were no differences in the percentage of normally fertilized oocytes after in vitro maturation in microdrops containing oocytes with 1 to 2 and 3 or more complete layers of cumulus cells (32.1 and 33.3% respectively). In conclusion, the ovaries of prepubertal slaughterhouse goats were found to be an economical alternative for an abundant source of oocytes for IVM-IVF research. In vitro maturation of oocytes in microdrops yielded maturation and fertilization rates comparable to those obtained with oocytes from FSH-primed adult goats. Moreover, similar maturation and fertilization rates were obtained using oocytes with 1 to 2 layers or 3 or more layers of cumulus cells.     

In vitro maturation and fertilization of follicular oocytes in cattle

This work aims towards developing research concerning the improvement of animal reproduction, embryo development and genetic engineering. In our laboratory, an attempt has been made to standardize in vitro conditions able to optimally support bovine oocyte maturation and fertilization in order to yield viable embryos. Ovaries from cows and heifers, obtained from local slaughter-house, were used for recovery of oocytes from antral follicles. Cumulus-oocyte complexes were statically cultured for 24h at 39 degrees C in medium TCM 199 supplemented with fetal calf serum inactivated, hormones, glucose and granulosa cells under a 5% CO2 and 95% humidity atmosphere. A first group of oocytes was used for fixing and staining procedure for evidence of in vitro maturation. After culture 69.4% (77/111) of oocytes reached full maturation showing cumulus expansion, first polar body extrusion and the 2nd metaphase plate. A 2nd group was used for in vitro fertilization. In vitro semen capacitation was obtained with swim-up system (8.9) with separation of high motility fraction in Talp Hepes medium. Oocytes and spermatozoa were coincubated for 18-20h in Talp medium at 39 degrees C with 5% CO2 and 95% humidity. At the end of culture stereoscope and microscope observations were made for evidence of fertilization. After IVF 67.4% (58/86) resulted fertilized. Most of them showed two pronuclei and residual sperm tail. In few cases oocytes with 1 pronucleus and the swollen sperm head or with syngamy or polyspermic were found. In these experiments high percentages of in vitro matured and in vitro fertilized oocytes have been obtained. These bovine zygotes can be considered an essential step to develop new technologies in cattle breeding.     

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.     

Optimum gas atmosphere for in vitro maturation and in vitro fertilization of bovine oocytes

The objective of this study was to determine optimal gas atmosphere conditions for in vitro maturation (IVM) and in vitro fertilization (IVF) of bovine oocytes. In Experiment 1, groups of 10 to 12 cumulus-oocyte complexes (COCs) were matured (24 h) and fertilized (18 h) under 1) 5% CO(2), 5% O(2;) 2) 5% CO(2), 10% O(2) or 3) 5% CO(2), 20% 0(2.) The COCs were cultured in 50 microl drops of maturation medium (TCM-199 + 10% bovine calf serum + oLH, oFSH and estrogen) or fertilization medium (TALP + swim-up separated spermatozoa +1 microg/ml heparin sulfate) under a layer of 10 ml paraffin oil at 39 degrees C with saturated humidity. Half of the oocytes in each drop were assigned randomly for maturation scoring and the remainder were inseminated. Reduced atmospheric O(2) drastically decreased proportions of oocytes reaching MII (71.4, 26.9 and 9.3% with 20, 10 and 5% O(2), respectively; P < 0.05). The percentages of total fertilization in 10 and 20% O(2) were similar and considerably higher than in 5% O(2) (80.3, 87.0 and 53.1%, respectively; P < 0.05). In addition, the percentage of polyspermy markedly increased when IVF was conducted in reduced O(2) (26.6 and 28.8% in 5 and 10% O(2) vs 15.4% in 20% O(2;) P < 0.05). Experiment 2 was similar to Experiment 1 except that CO(2) was the variable: 1) 2.5% CO(2) in air, 2) 5% CO(2) in air and 3) 10% CO(2) in air. The proportion of MII oocytes did not differ across treatments (64.9, 68.9 and 61.9%, respectively; P > 0.05). Although the percentages of total fertilization among treatments were not different (75.4, 80.9 and 76.1%, respectively), the proportion of normal fertilization was significantly reduced in 10% C0(2) (55.1%) when compared with that of either 2.5% CO(2) (62.7%) or 5% CO(2) (68.7%; P < .05). This study indicates that low O(2) is detrimental for IVM/IVF of bovine oocytes and that optimal atmospheric conditions are either 2.5 or 5% CO(2) and 20% O(2).     

Development to live young from bovine small oocytes after growth, maturation and fertilization in vitro

Bovine oocytes (90 to 99 microns in diameter) were isolated from early antral follicles (0.5 to 0.7 mm in diameter). Cumulus-oocyte complexes (COC) with pieces of parietal granulosa were embedded in collagen gels and cultured for 14 d. After in vitro growth culture, oocytes recovered from the collagen gels were further matured, fertilized and cultured in vitro, and then were transferred to recipient cows. After 14 d of growth culture, 37% of the oocytes (203/556) showed normal morphology in the collagen gels. The mean diameter of the oocytes was 110.1 +/- 6.0 microns, significantly larger (P < 0.01) than before growth culture (94.8 +/- 2.7 microns), and 77% were at the germinal vesicle stage while 23% had undergone germinal vesicle breakdown. After 24 h of maturation culture followed by insemination, 27% of in vitro-grown oocytes reached the second metaphase, and 42% of the oocytes were normally fertilized. After insemination, 18.2% of in vitro-grown oocytes cleaved and 3.7% developed to the blastocyst stage. Three blastocysts obtained from in vitro-produced 90- to 99-micron oocytes were transferred to 3 recipients. One recipient subsequently became pregnant and delivered a live calf on Day 277. These results demonstrated for the first time that 90 to 99-micron oocytes from early antral follicles can complete growth and acquire full developmental competence in vitro so that live young can be produced after maturation, fertilization, subsequent culture in vitro, and transfer to recipient cows.     

Antioxidant requirements for bovine oocytes varies during in vitro maturation,fertilization and development

Antioxidants may be beneficial additives to synthetic culture media because these well defined media lack serum or other macromolecules that serve as reactive oxygen species scavengers. In this study, three separate experiments were performed to determine the effects of antioxidants on the development of oocytes to the morula and blastocyst stage when added during in vitro maturation (IVM) of bovine oocytes, during in vitro fertilization (IVF), and during embryo culture for the first 72 h of the development period. Bovine oocytes were matured, fertilized (under 20% O(2)), and embryos were cultured (under 7% O(2)) in defined conditioned medium in vitro with or without supplementation with the antioxidant cysteine, N-acetyl-L-cysteine (NAC), catalase and superoxide dismutase (SOD). Significant improvements in the proportion of oocytes undergoing morula and blastocyst development (33.3% versus 20.3%, P<0.05) were achieved when cysteine (0.6 mM) was added to the maturation medium as compared to control medium without antioxidant supplementation. However, the addition of NAC (0.6mM), catalase (5 or 127 U/ml) or SOD (10 or 1000 U/ml) to the maturation medium did not improve the proportion of oocytes undergoing morula and blastocyst development. During the IVF period, addition of antioxidants (cysteine or NAC 0.6mM, catalase 127U/ml, SOD 100U/ml) significantly reduced the subsequent rate of bovine embryo development to the morula and blastocyst stage (P<0.05). In a defined medium for embryo culture (7% O(2)), the addition of cysteine improved the development of bovine embryos while NAC, catalase and SOD had no positive effect on embryonic development. Our study showed that medium supplementation with cysteine during IVM and in vitro culture (IVC) improved the rate of bovine embryo development, in contrast to extracellular antioxidants like catalase and SOD that caused no improvement.     

Effects of water quality on in vitro fertilization and development of bovine oocytes in protein-free medium

Examination was made of the effects of water quality in medium preparation on fertilization and early development of bovine in vitro matured (IVM) oocytes in a protein-free medium. The IVM oocytes were inseminated and cultured for 7 d in protein-free media prepared with 4 different types of water preparations: tap, deionized, twice-distilled, and purified water using the Milli-Q system (Milli-Q water). High frequencies (70 to 83%) of normal fertilization were obtained in media prepared with all types of water. However, the frequency of development to the blastocyst stage in media prepared with Milli-Q water (31 +/- 3%) was significantly higher than with the 3 other types of water (11 to 13%). Moreover, the effects of storage period of Milli-Q water on early development of bovine embryos was also examined. The frequency of development to the blastocyst stage in media prepared with Milli-Q water immediately after preparation (fresh Milli-Q water; 35 +/- 4%) was significantly higher than for Milli-Q water stored for 1 wk (18 +/- 4%) or 2 wk (18 +/- 3%). Effects of commercially available purified water on early development of bovine embryos were also examined. The frequency of development to the blastocyst stage in media prepared with Milli-Q water (33 +/- 5%) was significantly higher than for purified water purchased from 3 different suppliers (Brand A; 21 +/- 6%, Brand B; 21 +/- 2%, Brand C; 21 +/- 4%). Each water sample was analyzed by the measurement of electrical conductivity, organic compounds and/or inorganic ion and endotoxin concentrations to evaluate purity. Fresh Milli-Q water showed the lowest level of electrical conductivity and contained the lowest concentration of organic compounds. These results indicate that in vitro fertilization of bovine oocytes is not affected by the water quality in the preparation of medium; however, early development of bovine embryos is seriously affected by the purification method and the storage period of water used for medium preparation.     

In vitro fertilization and development of frozen-thawed bovine oocytes.

Bovine oocytes were vitrified (V-oocytes) or frozen slowly (S-oocytes) at the germinal vesicle (GV) stage or after maturation in vitro (IVM) and their survival assessed morphologically and also by in vitro fertilization (IVF) and culture. The morphological survival of S-oocytes was 30.7% after freezing at the GV stage and 53.3% after IVM. The corresponding survival rates of V-oocytes were significantly lower, viz. 14.6 and 14.0%, respectively. The fertilization rate of S-oocytes frozen after IVM (51.0%) was lower than that of unfrozen controls (75.8%), but higher than after other treatments. Development continued in 16.0% of the fertilized S-oocytes, compared to 39.4% of control IVF zygotes and 1.6% developed into morulae or blastocysts (4.5% in controls). Only 0.8% of frozen-thawed GV stage oocytes and 4.6% of post-IVM V-oocytes cleaved after IVF and none formed morulae or blastocysts. Transfer of four embryos (two morulae and two blastocysts) derived from post-IVM S-oocytes into a recipient heifer resulted in pregnancy and the birth of twin calves.     

In vitro fertilization and cleavage of in vivo matured bovine oocytes

The effect of the interval between onset of estrus and oocyte collection on in vitro fertilization (IVF) rates has been investigated. The oocytes were surgically collected 6-18 h (Group I), 19-24 h (Group II), 25-29 h (Group III) and 30-36 h (Group IV) after the beginning of estrus. Recognizable stages of nuclear maturation were identified in 54.9% of the oocytes used for IVF (5.9% at germinal vesicle, 31.4% at metaphase I, 17.6% at metaphase II); the other 45.1% were degenerate. Considerable between- and within-cow variation in oocyte morphology, oocyte maturation and IVF results was observed. The cverall fertilization and cleavage rates (to four-cell stages) were 26.5 and 6.0%, respectively. The fertilization rate increased as the interval between onset of estrus and collection increased and was optimal 30-36 h after onset. Thus, onset of estrus proved an effective means of timing oocyte collection for IVF.     

Effects of non-steroidal anti-inflammatory drugs in the maturation and fertilization media on bovine oocytes

In the present investigation the effects of 2 non-steroidal anti-inflammatory drugs commonly used in veterinary practice, flunixin-meglumin (FM) and phenylbutazone (PB), were studied. These drugs were added to the in vitro maturation medium of bovine oocytes. The effect of FM on in vitro fertilization was also tested. The results indicated that in vitro concentrations corresponding to maximal concentrations of FM in the plasma after therapeutic doses did not influence the in vitro maturation or fertilization rates of oocytes. However, doses higher than physiologic concentrations resulted in significant decreases in oocyte maturation and complete failure of fertilization. Concentrations of PB corresponding to concentrations commonly found in the plasma after therapeutic doses resulted in a significant decrease in the number of matured oocytes.