Kinetics of nuclear maturation and effect of holding ovaries at room temperature on in vitro maturation of camel (Camelus dromedarius) oocytes.

Experiments were conducted to investigate kinetics of in vitro nuclear maturation and the effect of storing ovaries at room temperature on initial chromatin configuration and in vitro maturation of dromedary camel oocytes. Cumulus oocyte complexes (COCs) were collected from slaughterhouse ovaries and matured in vitro for 4-48h. At every 4h interval (starting from 0 to 48 h), groups of oocytes were fixed, stained and evaluated for the status of nuclear chromatin. Oocytes were categorized as germinal vesicle (GV), diakinesis (DK), metaphase-I (M-I), anaphase-I (A-I), metaphase-II (M-II) stage and those with degenerated, fragmented, activated or without a visible chromatin as others. At the start of culture, 74% (66/89) oocytes were at GV stage, 13% (12/89) at DK and 12% (11/89) were classified as others. Germinal vesicle breakdown started spontaneously in culture and at 20 h of culture 97% oocytes had already completed this process. After 8 and 16 h of maturation the highest proportion of oocytes (42%, 48/114 and 41%, 51/123) were at DK and M-I stage, respectively. The proportions of oocytes reaching M-II stage at 32 (42%, 50/118), 36 (45%, 47/104), 40 (49%, 57/117), 44 (52%, 103/198) and 48 h (46%, 55/120) of culture were not different from each other (P>0.05). The proportion of oocytes categorized as others, however, increased after 40 h of culture and was higher (P<0.05) at 48 h compared with other maturation periods. There was no difference (P>0.05) in the proportion of oocytes reaching M-II stage from the ovaries collected and stored in normal saline solution (NSS) at room temperature for 12h (43%, 64/148) and those collected in warm NSS (37 degrees C) and processed immediately after arrival in laboratory (49%, 57/117). However, low number of oocytes reached M-II stage from ovaries collected in warm NSS but stored at room temperature (29%, 37/128) compared with other two groups (P<0.05). It may be concluded that dromedary oocytes require 32-44h of in vitro culture to have an optimum number of oocytes in M-II stage. However, further studies are required to find out the most appropriate maturation period, which will result in the further development of these oocytes after IVF, ICSI, parthenogenetic activation or nuclear transfer. Ovaries can be collected and stored in normal saline solution at room temperature for 12h without any appreciable effect on the nuclear maturation of the oocytes.

[Successful direct transfer of a deep frozen-thawed equine embryo]. / Erfolgreicher Direkttransfer eines tiefgefrorenen Embryos beim Pferd.

Embryos were flushed on day 7 after ovulation from two mares, and frozen using a conventional slow freezing procedure in phosphate buffered (PBS) saline supplemented with 10% FCS, 1.5 mol/L ethylene glycol and 0.25 mol/L sucrose. One of the two embryos was thawed after 10 months of storage in liquid nitrogen and transferred directly (without dilution of the cryoprotectant and quality examination) to a synchronized recipient. This transfer resulted in the birth of a live female foal. To our knowledge, this is the first live foal born after direct transfer of a frozen-thawed equine embryo.

Effect of freezing rate and exposure time to cryoprotectant on the development of mouse pronuclear stage embryos.

BACKGROUND: The effects of exposure time (20 versus 45 s) to a high concentration of cryoprotectant (7.0 mol/l ethylene glycol with 0.5 mol/l sucrose) and freezing rates (1200-10 300 degrees C/min) during rapid freezing of mouse pronuclear stage embryos on survival and development to blastocysts were investigated. Different freezing rates were achieved by directly plunging the straws (rapid freezing) and open pulled straws (OPS) in liquid nitrogen (OPS freezing) and by plunging the straws (super rapid) and OPS (super OPS) in a super cooled liquid nitrogen chamber (at -212 degrees C) before storage in liquid nitrogen. METHODS: Morphologically intact mouse zygotes (n = 891) pre-equilibrated in 1.5 mol/l ethylene glycol for 5 min were either loaded in 0.25 ml straws containing cryoprotectant or loaded in OPS with 2 microl cryoprotectant. After 20 or 45 s of loading the straws or mixing in cryoprotectant and loading in OPS, they were plunged either directly in to liquid nitrogen or were plunged first in to liquid nitrogen in a super cooled chamber and then stored in liquid nitrogen. Zygotes were thawed and intact embryos cultured in vitro. RESULTS: The rate of survival was higher (91%, P < 0.01) when zygotes were frozen with rapid freezing compared with super rapid, OPS and super OPS freezing rates with an exposure time of 20 s (70, 65, and 76% respectively). When zygotes were exposed to cryoprotectant for 45 s and frozen with rapid freezing rates, the survival was higher (86%, P < 0.01) compared with those frozen with OPS (62%) but was not different from those frozen with super rapid and super OPS freezing rates (81 and 75%). A higher rate of survival was observed when zygotes were exposed to cryoprotectant for 45 s and frozen with super OPS than with OPS freezing (75 versus 62%; P < 0.05). The rate of cleavage and development of intact zygotes to blastocysts was not different among the different groups. CONCLUSION: Exposure of zygotes to a high concentration of cryoprotectant (7.0 mol/l ethylene glycol with 0.5 mol/l sucrose) for 20 or 45 s did not influence their survival and development and increasing the freezing rate from 1200-10 300 degrees C/min was of no advantage when using a rapid freezing procedure for freezing mouse pronuclear stage embryos.

Refreezing of murine intact and biopsied embryos by rapid-freezing procedure.

The in-vivo development of murine morula stage embryos frozen-thawed once or twice and embryos biopsied after one freezing cycle and refrozen was studied. Embryos (n = 860) were cryopreserved using a rapid-freezing procedure. At least 24 h after freezing, embryos were thawed and cultured in vitro for 3 h. In experiment I, morphologically intact embryos were either transferred (n = 180) into recipients or refrozen (n = 160). Unfrozen embryos (control group, n = 180) and refrozen embryos stored for at least 24 h and then thawed, were transferred into recipients. In experiment II, embryos frozen once were thawed and biopsied or sham-biopsied (n = 230 and 180 respectively) and refrozen (n = 226 and 179 respectively). They were thawed and transferred (n = 192 and 160 respectively) into recipients. Recipient mice were either killed on day 15 after embryo transfer and number of implantation sites and live fetuses recorded or pregnant recipients (n = 6, experiment II) were allowed to carry the fetuses to term. There was no difference in the survival rate of embryos at thawing between those frozen once or twice (91 versus 93%). The implantation rate and number of live fetuses in the pregnant recipients at necropsy among those transferred with unfrozen embryos (57% and 51%; 8/9), embryos frozen once (55% and 45%; 8/9) or twice (51% and 48%; 6/8) was not different. There was no difference in the survival rate of refrozen embryos biopsied or sham-biopsied after one freezing cycle (89 versus 87%). The implantation rate and number of live fetuses in pregnant animals transferred with biopsied or sham-biopsied embryos was not different (64 and 41% versus 57 and 37% respectively). All six pregnant animals allowed to carry the fetuses to term delivered normal live fetuses (n = 39). On mating 12 females with six males of the progeny born out of biopsied embryos, all became pregnant and delivered live fetuses. It may be concluded that murine biopsied and intact embryos can be successfully refrozen by rapid-freezing procedure.

The protective action of polyvinyl alcohol during rapid-freezing of mouse embryos.

Biological products like serum and BSA are routinely used in embryo freezing solutions. These products are undefined and can potentially expose the embryos to infectious agents. Therefore, this experiment was designed to evaluate in vitro and in vivo survival of mouse embryos frozen in solutions supplemented with a chemically defined macromolecule, polyvinyl alcohol (PVA). Morula-stage embryos from superovulated mice were collected, frozen by a rapid freezing procedure, and cryoprotectant diluted out (after thawing) in media supplemented with either 10% fetal calf serum (FCS), 0.1 mg/mL PVA, or a combination of 10% FCS and 0.1 mg/mL PVA. Frozen-thawed (good to excellent quality) and nonfrozen (control, collected in FCS supplemented medium) embryos were cultured in medium M16 (32) supplemented with either 4 mg/mL BSA or 0.1 mg/mL PVA for 72 h. Embryos frozen in solutions supplemented with FCS or PVA and nonfrozen embryos were transferred to pseudopregnant recipients. Recipients were humanly killed on Day 15 after transfer, and the rate of implantation and percentage of live fetuses were recorded. The supplementation of collection, freezing and cryoprotectant dilution solutions with FCS, PVA or FCS plus PVA did not influence (P > 0.05) the rate of survival and in vitro development of embryos to hatched/hatching blastocyst-stage. However, a higher (P < 0.01) in vitro development rate to hatching/hatched-stage was recorded when frozen-thawed embryos were cultured in medium supplemented with BSA than with PVA. There was no difference (P > 0.05) in the rate of implantation (68 vs 72%) or percentage of live fetuses (62 vs 60%) between pregnant recipients with embryos frozen in medium with FCS or PVA. The rate of implantation and development of embryos frozen in medium supplemented with PVA or FCS was comparable (P > 0.05) to that of nonfrozen embryos. It may be concluded that PVA can be substituted for FCS in medium for freezing mouse embryos; however, it can not be completely substituted for BSA in the in vitro culture of embryos to the hatched blastocyst stage.

Effect of cryoprotectants and their concentration on post-thaw survival and development of expanded mouse blastocysts frozen by a simple rapid-freezing procedure.

Experiments were conducted to develop a simple rapid-freezing protocol for expanded mouse blastocyst-stage embryos. The effect of type of cryoprotectant (ethylene glycol and propylene glycol) and its concentrations (4.5, 6.0 and 7.0 mol/L each with 0.5 mol/L sucrose) on morphological survival and development in vitro were studied. The survival and development of embryos frozen with best concentration of each cryoprotectant pre-exposed to either a low concentration (1.5 mol/L with 0.25 mol/L sucrose) of the respective cryoprotectant or ascending concentrations of sucrose were also compared. The in vivo development of embryos frozen with best protocol (pre-exposure to 1.5 mol followed by 7.0 mol ethylene glycol) was compared with nonfrozen embryos. The rate of re-expansion and hatching was influenced by the type and concentration of the cryoprotectant. A significantly higher re-expansion and hatching rate was achieved at 7.0 mol of both cryoprotectants compared with 4.5 and 6.0 mol of the respective cryoprotectants. When comparing 2 cryoprotectants, a higher (P < 0.05) rate of hatching was obtained with ethylene glycol at 7.0 mol compared with a similar concentration of propylene glycol. The highest re-expansion (91%) and hatching (86%) of expanded blastocysts was achieved with pre-exposure of embryos to a low concentration of ethylene glycol followed by freezing in the same cryoprotectant at 7.0 mol. The transfer of embryos frozen using this protocol resulted in the development of live fetuses. The proportion of live fetuses in the pregnant recipients with frozen-thawed embryos were not different from those transferred nonfrozen embryos (49 vs 57%). It may be concluded that simple rapid-freezing with dehydration in ascending sucrose concentrations or pre-equilibration in a low concentration of ethylene glycol or propylene glycol followed by exposure to the respective cryoprotectant at 7.0 mol resulted in high survival and development of expanded blastocysts. Ethylene glycol at 7.0 mol with pre-equilibration is, however, most effective for cryopreservation of this stage in the mouse.

Effect of cryoprotectants and their concentration on post-thaw survival and development of rapid frozen-thawed pronuclear stage mouse embryos.

Experiments have been conducted to develop a simple rapid-freezing protocol for pronuclear stage mouse embryos. The effect of type of cryoprotectant (dimethyl-sulphoxide (DMSO) or 1,2-propanediol (PROH)) and concentration (ranging from 3.5 to 8.0 mol/l with 0.5 mol/l sucrose) on the post-thaw morphological survival rate, on cleavage rate on development to the blastocyst stage were studied. Further, in-vivo viability of embryos frozen using the most effective cryoprotectant concentration (PROH at 7.0 mol/l) was compared with viability of non-frozen embryos. The type of cryoprotectant and its concentration influenced the survival and development of embryos to the blastocyst stage in vitro. The best development with PROH was achieved at 7.0 mol/l (66%, 128/193), whereas with DMSO the best development was achieved at 6.0 mol/l (42%, 71/171). The rates of survival and cleavage did not differ between the two best cryoprotectant concentrations (P > 0.01) but the proportion of embryos which developed to blastocyst was higher (P < 0.001) with PROH at 7.0 mol/l compared with DMSO at 6.0 mol/l. The rates of survival and development were higher (P < 0.001) with DMSO at 3.5 and 6.0 mol/l compared with similar concentrations of PROH. The cleavage and development, however, was higher (P < 0.001) at 7.0 mol/l PROH compared with the same concentration of DMSO. At 8.0 mol/l the survival and development was not different (P > 0.01) between DMSO and PROH. The rate of implantation and the percentage of live fetuses at autopsy of the recipients receiving non-frozen embryos was higher (63 and 41% respectively) than in those receiving frozen-thawed embryos (53 and 37% respectively), but not significantly different. It may be concluded that the concentration range of cryoprotectants which allows acceptable embryo viability after freezing and thawing is very narrow. The rapid protocol using dehydration in 0.25 mol/l and 0.5 mol/l sucrose followed by exposure to 7.0 mol/l PROH and 0.5 mol/l sucrose for 45 s was the most effective for cryopreservation of pronuclear stage mouse embryos.

In vitro culture of goat morulae to blastocysts before freezing.

The results of transfer of frozen-thawed caprine embryos that were collected either as blastocysts or morulae and cultured to the blastocyst stage prior to freezing were compared. After thawing, the embryos collected as blastocysts appeared to be of marginally better quality than those that had been cultured from morulae (89 vs 72% rated as good; P > 0.05). The transfer of 24 frozen-thawed embryos collected as blastocysts to 12 recipients resulted in a pregnancy rate of 83% (10/12) and an embryo survival rate of 67%. Corresponding results for frozen-thawed blastocysts that had been cultured from morulae and were transferred to 11 recipients were 54% (6/11) and 41%, respectively. Since an earlier investigation had shown that the transfer of frozen caprine morulae yields very poor results, in our laboratory all morulae are now cultured to the blastocyst stage before being cryopreserved.

Meiotic competence of marmoset monkey oocytes is related to follicle size and oocyte-somatic cell associations.

This study was conducted to investigate the relationships between oocyte meiotic competence, follicle size, and occyte-somatic cell associations in the marmoset monkey (Callithrix jacchus). Follicles were excised from ovaries of nonstimulated adult cyclic females (n = 6) collected on Day 7 of the follicular phase. Follicles were separated into size groups: large preantral (260-400 microns), periantral (420-640 microns), small antral (660-1000 microns), large antral (1020-2000 microns), and preovulatory (> 2000 microns). Partially naked and cumulus/granulosa-enclosed oocytes (n = 473) were released from follicles and cultured in Waymouth's medium with 10% fetal calf serum, 1 microgram/ml human (h) FSH, and 10 micrograms/ml hLH. Somatic cells remaining after 46 h were removed, and oocytes were fixed after 48 h and mounted for viewing. Chromatin staining and microtubulin fluorescence labeling were used to assess progression of meiotic maturation and spindle normality. The follicle size distribution and oocytesomatic cell associations are reported. Competencies of oocytes to achieve germinal vesicle breakdown (GVBD) and metaphase II (MII) increased significantly (p < 0.001) with follicular size but not with the association of somatic cells. Marmoset oocytes from antral follicles resumed (GVBD) and completed (MII) meiotic maturation with high frequencies (98% and 72%, respectively), with no significant differences among size groups of antral follicles. GVBD competence was virtually absent in oocytes from preantral follicles (2%) and was acquired coincidentally with antrum formation (60%), although MII competence was attained after the completion of antrum formation. Partially naked oocytes from small antral follicles matured with a high incidence of spindle and meiotic abnormalities (44%). Marmoset oocyte meiotic competencies are notably higher than in any other nonhuman primate species studied, and a possible explanation for this phenomenon in relation to the stage of antrum formation is offered.

Superovulation of goats with purified pFSH supplemented with defined amounts of pLH.

The superovulatory response of goats treated with purified pFSH supplemented with 30, 40 or 50% pLH was compared. Sixty-four Boer goat does were synchronized by progestagen-containing ear implant, randomly allotted to 3 groups and, beginning 2 d before implant removal, treated with purified pFSH supplemented with 30, 40 or 50% pLH. Each animal received 16 Armour Units of pFSH administered in 6 descending doses at 12-h intervals. Along with the last 2 injections, the does received 5 mg PGF(2alpha). Embryos were flushed either surgically or after slaughter on Day 5 or 6 after the last day of standing estrus. The percentage of animals responding to treatment was not different among groups treated with pFSH supplemented with 30, 40 or 50% pLH (76, 71 and 63%, respectively). The corresponding data for number of ovulations was 11.3 +/- 1.6, 16.3 +/- 1.8 and 16.4 +/- 2.6, for number of ova and embryos recovered 8.1 +/- 1.9, 12.0 +/- 1.5 and 13.5 +/- 2.9 and for number of transferable embryos 6.6 +/- 1.9, 9.1 +/- 1.5 and 7.1 +/- 2.1 (x +/- SEM). Results confirm the earlier finding of a good response of goats to pFSH preparations with a high FSH:LH ratio, and, although group differences were statistically nonsignificant (P > 0.05), they suggest that supplementation with approximately 40% pLH may be close to the optimum.

Abnormal in vitro development of ovarian follicles explanted from mice exposed to tetrachlorvinphos.

A system of mouse ovarian follicle culture in which follicles can be grown from a preantral stage of development through antral formation has been developed and modified recently by Nayudu and colleagues. Follicles have been shown to grow in this culture system at a relatively constant rate and show responsiveness to LH at the end of the culture by ovulation of mature oocytes. Reported here are the distinctly different in vitro growth patterns of follicles explanted from 22- to 24-day-old mice during a period when the colony was being treated for skin parasites with tetrachlorvinphos (TCVP) (Rabond). There is to date no information on the effects of this compound on the mammalian female reproductive system. For follicles from the TCVP treated group, the duration of growth as intact follicles was markedly reduced in comparison to mice of the same strain and source not treated with TCVP. In the treated group, premature termination of follicular growth was also associated with the spontaneous expulsion of oocytes with immature nuclei and without cumulus cells. For those follicles from treated mice that did remain in culture until the day luteinizing hormone was given, the ovulatory response was poor and the maturation response of the oocytes was low in comparison with the follicles from untreated mice. The effect of the treatment on the follicles was further characterized by obvious differences in the patterns of growth. Follicles in the untreated group grew in a linear pattern at around 25 microns/day; a single phase, fast pattern for the whole culture period.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of cryoprotectant concentration, equilibration time and thawing procedure on survival and development of rapid frozen-thawed mature mouse oocytes.

Experiments were conducted to develop a simple rapid-freezing protocol for mature mouse oocytes that would yield a high proportion of oocytes with developmental potential. The effects of concentration (3.5, 4.5 and 6.0 M dimethyl sulfoxide (DMSO) all with 0.5 M sucrose) and the duration of exposure (2.5 min vs 45 sec) of oocytes to the cryoprotectant and its extraction after thawing in 2, 3 or 4 steps of descending sucrose concentration were studied. The most effective of the rapid-freezing and thawing protocols (4.5 M DMSO; 45 sec exposure and 3-step thawing) was compared to slow freezing protocols using 1.5 M DMSO and 1.0 M 1,2 propanediol as cryoprotectants. The DMSO concentrations had an effect on survival, fertilization and embryo development using short (45 sec) but not long (2.5 min) exposure. The rate of morphological oocyte survival was significantly higher using 4.5 M DMSO than 3.5 or 6.0 M (92% vs 82 and 73%, respectively). The development of fertilized embryos to blastocysts was also significantly higher at 4.5 M than at 3.5 or 6.0 M (68% vs 42 and 53%, respectively). The extraction of cryoprotectant in 3 or 4 steps of descending sucrose concentration resulted in higher survival (P < 0.01) and fertilization than in 2 steps. The best survival, fertilization and development was achieved with the 3-step procedure. Optimal combinations of conditions were 4.5 M DMSO at 45 sec prefreeze exposure and 3-step extraction of the cryoprotectant. Oocytes frozen by conventional methods had a survival, fertilization and development to blastocyst rate significantly lower than those frozen under the optimal rapid conditions. Thus rapid freezing of mature mouse oocytes with 4.5 M DMSO + 0.5 M sucrose and short prefreeze exposure is effective and has the additional advantage of being less time-consuming than slow freezing methods.

Transfer of split goat embryos without zonae pellucidae either fresh or after freezing.

Embryos in the morula to blastocyst stage were collected from superovulated goats either surgically or after slaughter. Embryos with good or fair morphology were bisected with a microblade without the aid of a holding pipette or other microinstruments. Of 103 morulae and 77 blastocysts that were split, 200 (97%) and 151 (98%) demi-embryos, respectively, with no major morphological aberrations were obtained. Zona-free demi-embryos derived from blastocysts were incubated in vitro for 2 h, and those derived from morulae for 24 h so that they would reach the blastocyst stage. Demi-embryos were transferred either fresh or after freezing and thawing. After 2 h of incubation, a significantly higher proportion of zona-free demi-embryos derived from blastocysts were of good quality than were zona-free demi- embryos derived from morulae (75 vs 45%). Seventy-six percent of zona-free demi-embryos derived from morulae developed to the blastocyst stage after 24 h in vitro. The quality of the embryos before splitting had a significant effect on the development of the demi-embryos in vitro. Transfer of 11 pairs of zona-free demi-embryos resulted in the birth of five twin and three singleton kids. Transfer of 11 pairs of demi-embryos frozen-thawed without zonae pellucidae resulted in the delivery of two singleton kids.