Effect of meiotic stages and maturation protocols on bovine oocyte's resistance to cryopreservation

We investigated the effect of meiotic stages and two maturation protocols on bovine oocyte's resistance to cryopreservation. Oocytes at germinal vesicle breakdown (GVBD) and metaphase II (MII) stage as well as oocytes matured for 22 h in media supplemented with FSH or LH were vitrified by the open pulled straw method. After warming, oocytes underwent additional 16 h (GVBD group) or 2 h (MII group) maturation. Then they were subjected to in vitro fertilization and culture. Some oocytes that matured in the medium supplemented with LH were subjected to parthenogenetic activation after vitrification to determine their developmental potential in absence of fertilization. Survival of oocytes after vitrifying/warming was determined after 22 h in fertilization medium. Cleavage and blastocyst formation rates were used to assess their developmental competence. In both experiments, a portion of unvitrified MII oocytes were subjected to in vitro fertilization and culture as control groups. In Experiment 1, similar cleavage rates were obtained for both GVBD and MII oocytes (53.56 versus 58.01%, P > 0.05). However, significantly higher proportion of cleaved embryos from vitrified MII oocytes developed into blastocysts than those from vitrified GVBD oocytes (1.06 versus 8.37%, respectively, P < 0.01). In Experiment 2, vitrified MII oocytes matured in medium supplemented with LH were superior to vitrified MII oocytes matured in FSH supplementation not only in cleavage rates (61.13 versus 50.33%), but in blastocyst formation rates (11.79 versus 5.19%, P < 0.01) as well. Cleavage and blastocyst formation rates of parthenogenetically activated oocytes were similar to those that were fertilized. Nevertheless, the vitrifying/ warming process significantly compromised the oocytes' developmental capacity since the vitrified oocytes showed significant reduction in both cleavage and blastocyst rates compared to those of not vitrified controls in both experiments (P < 0.01). We showed that oocytes at different maturation stages respond to cryopreservation differently and MII stage oocytes have better resistance to cryopreservation than GVBD stage oocytes. The maturation protocols also influence oocyte's ability to survive cryopreservation. Poor developmental potential after vitrification seem to have resulted from the cryodamage to the oocyte itself. These results suggested the importance of maturation on the developmental competence of cryopreserved oocytes.     

Nuclear maturation and embryo development of porcine oocytes vitrified by cryotop: Effect of different stages of in vitro maturation

The present study was designed to evaluate the viability, meiotic competence and subsequent development of porcine oocytes vitrified using the cryotop method at different stages of in vitro maturation (IVM). Cumulus–oocyte complexes (COCs) were cultured in IVM medium supplemented with 1 mM dibutyryl cAMP (dbcAMP) for 22 h and then for an additional 22 h without dbcAMP in the medium. Germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), anaphase I/telophase I (AI/TI) and metaphase II (MII) were found to occur predominantly at 0–22, 26, 32, 38 and 44 h of IVM, respectively. Oocytes were exposed to cryoprotectant (CPA) or vitrified after different durations of IVM (0, 22, 26, 32, 38 and 44 h). After CPA exposure and vitrification, surviving oocytes that were treated before completion of the 44 h maturation period were placed back into IVM medium for the remaining maturation period, and matured oocytes were incubated for 2 h. CPA treatment did not affect the viability of oocytes matured for 26, 32, 38 or 44 h, but significantly decreased survival rate of oocytes matured for 0 or 22 h. CPA treatment had no effect on the ability of surviving oocytes to develop to the MII stage regardless of the stage during IVM; however, blastocyst formation following PA was severely lower (P < 0.05) than that in the control. At 2 h post-warming, the survival rates of oocytes vitrified at 26, 32, 38 and 44 h of IVM were similar but were higher (P < 0.05) than those of oocytes vitrified at 0 or 22 h of IVM. The MII rates of surviving oocytes vitrified at 0 and 38 h of IVM did not differ from the control and were higher (P < 0.05) than those of oocytes vitrified at 22, 26 or 32 h of IVM. After parthenogenetic activation (PA), both cleavage and blastocyst rates of vitrified oocytes matured for 22, 26, 32, 38 and 44 h did not differ, but all were lower (P < 0.05) than those matured 0 h. In conclusion, our data indicate that survival, nuclear maturation and subsequent development of porcine oocytes may be affected by their stage of maturation at the time of vitrification; a higher percentage of blastocyst formation can be obtained from GV oocytes vitrified before the onset of maturation.     

Vitrification of immature and mature equine and bovine oocytes in an ethylene glycol, ficoll and sucrose solution using open-pulled straws

Studies were conducted to compare viability of immature and mature equine and bovine oocytes vitrified in ethylene glycol. Ficoll using open-pulled straws. Oocytes from slaughterhouse ovaries (N=50/group) with >2 layers of compact cumulus cells were vitrified immediately after collection (immature groups) or vitrified after 36 to 40 (equine) or 22 to 24 (bovine) h of maturation (mature groups). Immature oocytes were matured after thawing. Before vitrification, oocytes were exposed to TCM-199 + 10 FCS + 2.5 M ethylene glycol + 18% Ficoll + 0.5 M sucrose (EFS) for 30 sec and then to 5 M ethylene glycol in EFS for 25 to 30 sec at 37 degrees C. Oocytes were loaded into straws in approximately 2 microL of cryoprotectant and plunged directly into LN2. Warming straws and dilution of cryoprotectant was at 37 degrees C in TCM-199 + 10% FCS + 0.25 M sucrose for 1 min and then TCM-199 + 10% FCS + 0.15 M sucrose for 5 min. Non-vitrified oocytes undergoing the same maturation protocol for both species were used as controls. Oocytes were stained with orcein for nuclear maturation and live/dead status was determined using Hoechst 33342. Maturation of oocytes to MII after thawing was similar (P>0.05) among groups within species. All equine treatment groups had lower (P<0.01) maturation rates than bovine groups. Live/dead status did not differ among vitrification treatments within species. The percentage of oocytes that survived and reached MII did not differ (P>0.05) within treatment groups of each species. Rates of mature cortical granule distribution did not differ (P>0.05) within species; however, more bovine oocytes (P<0.05) had mature cortical granule distribution and nuclear maturation than equine oocytes. When concurrent cortical granule distribution and nuclear maturation were examined, there was no difference within species; however, only 30% of equine oocytes had nuclear and cytoplasmic maturation compared with 70% of bovine oocytes (P<0.05). In summary, both immature and mature equine and bovine oocytes survived cryopreservation using vitrification in open-pulled straws. However, survival rates were lower for equine than for bovine oocytes.     

Effect of nuclear stages during IVM on the survival of vitrified-warmed bovine oocytes

The effect of nuclear stages during IVM on the survival of vitrified-warmed bovine oocytes was investigated. Oocytes with compact cumulus cells were cultured for 0, 6, 12 and 24 h in TCM199 supplemented with 5% fetal bovine serum (FBS) in 3% CO2 in air. The oocytes were first exposed to 20% ethylene glycol solution and were subjected to vitrification in a solution containing 40% ethylene glycol, 18% Ficoll-70 and 0.3 M sucrose. After warming in 20 degrees C water, oocytes which had been vitrified at less than 24-h of IVM were again cultured to complete the 24-h of IVM period. Oocytes were then incubated with frozen-thawed spermatozoa in Brackett and Oliphant (BO) medium containing 60 micrograms/ml heparin and 0.25% BSA for 20 h. In vitro fertilization rates of oocytes vitrified-warmed at 0, 6, 12 and 24-h IVM were 75.2, 68.0, 82.0 and 72.4%, respectively, comparable to the rates for unvitrified control oocytes (80.6%). A higher incidence of polyspermic fertilization was observed in oocytes vitrified at 24-h IVM (44.9 vs 22.6% in the control group, P < 0.05). Vitrification of oocytes at 12-h IVM seemed to be better than that of other IVM groups, since the normal fertilization rate of all treated oocytes was the highest (36.0%) among the vitrification groups. Developmental competence of the oocytes following vitrification and in vitro fertilization (12-h IVM group) was examined by cell-free culture of presumptive zygotes up to 9 d in modified synthetic oviduct fluid (mSOF) in 5% CO2, 5% O2 and 90% N2. The cleavage rate of zygotes from vitrified oocytes 48 h after insemination was 29.8%, which was lower than that of the control group (57.0%, P < 0.05). Development to blastocysts from the vitrified oocytes (4.8%) was much lower than that of the control group (27.0%, P < 0.05). These results indicate that cryopreservation of bovine oocytes by vitrification may be affected by their maturation stage in vitro, and that developmental competence to blastocysts of cleaved oocytes following vitrification may be impaired compared with unvitrified control oocytes.     

Holding bovine oocytes in the absence of maturation inhibitors: kinetics of in vitro maturation and effect on blastocyst development after in vitro fertilization

Holding immature oocytes before maturation simplifies the transport of oocytes and aids in scheduling later manipulations. We examined the effect of holding bovine oocytes in the absence of meiotic inhibitors on their subsequent meiotic and developmental competence. Oocytes were matured immediately after recovery (control) or were held in a mixture of 40% TCM 199 with Earle's salts, 40% TCM 199 with Hanks' salts, and 20% FBS, at room temperature for 16 to 18h (EH-held) and then matured. Chromatin status was determined at 0, 10, 14, 18, and 22h of maturation culture. Oocytes were fertilized in vitro after either 18 or 22-24h maturation. The EH treatment maintained oocytes at the germinal vesicle stage (79.3%, vs. 87.7% for control oocytes at 0h; P>0.05). Upon culture, held oocytes matured more quickly than did control oocytes. The proportions of mature oocytes were not significantly different between groups at 18h (EH-held, 80.6% and control, 79.3%); however, after 22h significantly more EH-held than control oocytes had degenerated (24.1% vs. 4.5%, P<0.0001). Blastocyst development was similar between groups for oocytes fertilized after 18h maturation (EH-held, 29.6% and control, 27.8%). When oocytes were fertilized after 22-24h maturation, EH-held oocytes yielded lower blastocyst development than did control oocytes (16.5% vs. 29.3%, P<0.05). In conclusion, bovine oocytes may be effectively held in the EH treatment before maturation without adversely affecting meiotic or developmental competence. However, holding affects the kinetics of maturation and this must be taken into account when subsequent manipulations are performed.     

Bovine oocyte vitrification: effect of ethylene glycol concentrations and meiotic stages

Success in oocyte cryopreservation is limited and several factors as cryoprotectant type or concentration and stage of oocyte meiotic maturation are involved. The aim of the present study was to evaluate the effect of maturation stage and ethylene glycol (EG) concentration on survival of bovine oocytes after vitrification. In experiment 1, kinetics of oocyte in vitro maturation (IVM) was evaluated. Germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), and metaphase II (MII) oocytes were found predominantly at 0, 0–10, 10–14, and 18–24 h of IVM, respectively. In experiment 2, in vitro embryo development after in vitro fertilization (IVF) of oocytes exposed to equilibrium (ES) and vitrification solution VS-1 (EG 30%), or VS-2 (EG 40%) at 0, 12 or 18 h of IVM was evaluated. Only blastocyst rate from oocytes vitrified in SV-2 after 18 h of IVM was different from control oocytes. Hatched blastocyst rates from oocytes vitrified in VS-1 after 12 and 18 h, and SV-2 after 18 h of IVM were different from unvitrified oocytes. In experiment 3, embryo development was examined after IVF of oocytes vitrified using VS-1 or VS-2 at 0, 12 or 18 h of IVM. Rates of blastocyst development after vitrification of oocytes in VS-1 at each time interval were similar. However, after vitrification in VS-2, blastocyst rates were less at 18 h than 0 h. Both cleavage rates and blastocyst rates were significantly less in all vitrification groups when compared to control group and only control oocytes hatched. In conclusion, both EG concentration and stage of meiotic maturation affect the developmental potential of oocytes after vitrification.     

Factors affecting nuclear maturation, cleavage and embryo development of vitrified bovine cumulus-oocyte complexes

The objective was to investigate the effects of cryodevice, vitrification solutions, and equilibration time on in vitro maturation, cleavage, and embryo development of vitrified bovine oocytes. In Experiment 1, the nuclear maturation (MII) rate of immature bovine COCs vitrified was compared between two equilibration times (0 vs 10 min) in vitrification solution 1 (VS1) and two cryodevices (cryotop vs 0.25 mL straw). The MII rate was higher in the non-vitrified control group than in vitrified groups (61 vs 16%, P < 0.0001). Equilibration time did not affect MII rate (P = 0.964); however, the MII rate was higher for COCs vitrified on cryotops than in straws (23 vs 9%, P = 0.007). In Experiment 2, bovine COCs were vitrified on cryotops using two equilibration times (0 vs 5 min) in VS1 and two kinds of vitrification solutions (freshly prepared vs frozen). Cleavage and blastocyst rates were higher (P < 0.0001) in the non-vitrified control group than vitrified groups (cleavage rate 93 vs 42% and blastocysts rate 31 vs 0.4%). Cleavage rate of COCs vitrified using frozen solutions with 5 min equilibration was higher (P = 0.05) than other treatment groups. However, blastocyst rate did not differ (P = 0.993) among treatment groups. In conclusion, cryotop was a better cryodevice than 0.25 mL straw for vitrification of bovine COCs. Furthermore, 5 min equilibration in VS1 improved cleavage. Compared with control, the vitrification procedure per se damaged bovine COCs, resulting in poor nuclear maturation and embryo development. However, vitrification did not immediately kill oocytes, as the cleavage rate was acceptable.     

Effect of holding at room temperature on initial chromatin configuration and in vitro maturation rate of equine oocytes

The relationship of holding time in media at room temperature (approximately 22 degrees C) to initial chromatin configuration and rate of in vitro maturation (IVM) of equine oocytes was determined. Only oocytes having a complete, compact cumulus were used in this study. Oocytes were removed from ovaries 3.5-8 h after slaughter and were put into one of four treatment groups: (1) immediate/fix (IF) = immediate fixation following removal from the ovary; (2) delay/fix (DF) = fixation after oocytes were held 1-4 h in medium at room temperature; (3) immediate/mature (IM) = immediate placement into maturation medium at 5% CO2 at 38.2 degrees C; and (4) delay/mature (DM) = placement into maturation medium at 5% CO2 at 38.2 degrees C after oocytes were held 1-4 h in medium at room temperature. Chromatin configurations in fixed oocytes were classified as fluorescent nucleus (FN), condensed chromatin (CC), fibrillar, intermediate, or fibrous germinal vesicle (GV). Other classifications were Metaphase I, II, or degenerating/abnormal. Oocytes held at room temperature before fixation (DF) had a lower proportion of oocytes in the fibrous GV, fibrillar and intermediate configurations than did oocytes fixed immediately (IF; 1/54, 2% versus 15/51, 30%, respectively, P < 0.001). Oocytes held before fixation tended to have a higher percentage of both the CC and FN configurations than did oocytes fixed immediately (CC: 22/40, 55% versus 11/36, 31%, respectively, P = 0.056; FN: 17/40, 43% versus 10/36, 28%, respectively, P = 0.066). Holding of oocytes did not affect the rate of resumption of meiosis or the rate of degeneration in culture; however, of oocytes resuming meiosis, more oocytes in the delayed than in the immediate maturation group had reached MII by 24h culture (14/15, 93% versus 8/15, 53%, respectively, P = 0.018).     

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.     

Kinetics of nuclear maturation and protein profiles of oocytes from prepubertal and adult cattle during in vitro maturation

The aim of this present study was to compare the kinetics of nuclear maturation between calf and cow oocytes in order to determine if there are differences between the 2 groups which could explain their disparate developmental capacity. The constitutive and neosynthetic protein patterns of cow and calf oocytes and of their corresponding cumulus cells were also compared during in vitro maturation. A total of 397 calf oocytes and 406 cow oocytes was matured in M199 + 10 ng/mL EGF. The first group of oocytes (n = 30) was immediately fixed and stained after removal from the follicle, and represent 0 h. The remaining oocytes were removed from the maturation medium at 4, 8, 12, 16, 20 and 24 h respectively. Half were denuded, fixed and stained for nuclear status; while the remainder were radiolabeled with methionine-(35S). Immediately after isolation, all the oocytes were at the GV stage. By 8 h, GVBD had occurred in most oocytes (calf: 97%; cow: 100%) and some had reached pro-metaphase I (calf: 49%; cow: 51%). By 12 h, most of the oocytes were at metaphase I (calf: 84%; cow: 94%). By 16 h, 54% of calf oocytes had reached telophase I or beyond compared with 71% of cow oocytes. This difference between the 2 groups became significant by 20 h, with 89% of cow oocytes (P < 0.05) at metaphase II and 71% of calf oocytes. By 24 h of culture, GVBD had occurred in all cases. Most oocytes completed meiosis I and were arrested at metaphase II with the first polar body extruded (calf: 72%; cow: 86%). No differences were noted in the constitutive and the neosynthetic protein profiles of cumulus cells in relation to the age of animal. Changes in neosynthetic protein patterns were observed both in cow and calf cumulus during IVM, and several proteins showed stage-specific synthesis. For the constitutive protein patterns of cow and calf oocytes, there were quantitative (38 and 40 kD) and qualitative (4, 10, 16, 17, 24, 25 and 26 kD) differences between the 2 groups. Only a few differences were observed in neosynthetic proteins between cow and calf oocytes, but there were changes in relation to nuclear status both in cow and calf oocytes. In conclusion, the difference in developmental capacity between cow and calf oocytes may be explained by a difference in the kinetics of nuclear maturation, which was significant at 20 h of culture (with 89% of cow oocytes at metaphase II and 71% of calf oocytes). At the biochemical level, our results indicate that nuclear progression during in vitro maturation of bovine oocytes is linked to changes in protein synthesis by the oocyte itself, while cumulus protein synthesis may either stimulate or modulate the process of oocyte maturation.     

A simple and high-throughput method to assess maturation status of bovine oocytes: Comparison of anti-lamin A/C-DAPI with an aceto-orcein staining technique

A precise, accurate, nonambiguous and high-throughput method is required to assess nuclear maturation of mammalian oocytes. The objectives of this study were to compare the efficiency and ease of use of a simplified fluorescence imaging (anti-lamin A/C and 4′,6-diamidino-2-phenylindole [DAPI]) technique to the existing technique (aceto-orcein staining) for the evaluation of nuclear maturation of bovine oocytes, and to determine the kinetics of bovine oocyte maturation using an anti-lamin A/C-DAPI technique. In Experiment 1, oocytes were matured in vitro and stained with aceto-orcein and anti-lamin A/C-DAPI staining techniques. The proportions of oocytes lost during procedures and those that could not be classified (because of ambiguous morphology) during evaluation were lower (P < 0.0001) in oocytes stained with anti-lamin A/C-DAPI (9% and 2%) than those stained with aceto-orcein (31% and 13%), respectively. Anti-lamin A/C-DAPI was a quick procedure which could be completed within 7 h after completion of the maturation (compared with > 24 h for the aceto-orcein method). Furthermore, > 200 oocytes could be stained in one batch with anti-lamin A/C-DAPI technique. In Experiment 2, nuclear maturation kinetics of bovine oocytes at various time intervals (0, 6, 12, and 22 h) during in vitro maturation (IVM) was evaluated using the anti-lamin A/C-DAPI technique. Germinal vesicle, germinal vesicle breakdown, metaphase I, and metaphase II oocytes were predominant at 0, 6, 12, and 22 h of IVM, respectively. We concluded that the anti-lamin A/C-DAPI was an efficient and simple technique for nonambiguous evaluation of nuclear maturation status of large numbers of oocytes in a short interval.     

Effects of bovine follicular fluid on maturation of bovine oocytes

Three experiments were conducted to determine the effects of follicular fluid and media on bovine oocyte maturation. Experiments 1 and 3 test the effects of follicular fluid obtained at different times after the LH surge on bovine oocyte maturation in vitro, while Experiment 2 was designed to compare TALP and Medium 199 as serum-free maturation media. Bovine follicular fluid (BFF) was obtained from preovulatory follicles either before (0 h BFF) or at 4, 8, 12 or 20 h after a GnRH-induced LH surge. Oocytes were obtained from follicles 1 to 6 mm in diameter from ovaries retrieved from a slaughterhouse. In Experiment 1, both 0 h and 4 h BFF inhibited resumption of meiosis, whereas BFF collected at 8, 12 and 20 h did not. When oocytes were cultured in media that contained equal portions of 0 and 8 h BFF, meiosis was not inhibited. In Experiment 2, Medium 199 supplemented with bovine serum albumin (BSA) was superior to Tyrode's medium with albumin, lactate and pyruvate for oocyte maturation. In Experiment 3, a higher percentage (P<0.05) of oocytes cultured for 18 h in 40% 20 h BFF in Medium 199 reached Metaphase-II (64%) than those cultured in 0 h BFF (41%) or control medium (39%). There was a transient meiotic arrest due to 0 h BFF as evidenced by the higher percentage of oocytes with germinal vesicles at 8 h of incubation (35% with 0 h vs 20% with 20 h; P<0.05). Furthermore, expansion of cumulus cells was induced in 8 and 20 h BFF, but not 0 h BFF.     

In vitro maturation of bovine oocytes in the presence of growth hormone accelerates nuclear maturation and promotes subsequent embryonic development

Regulatory effect of GH on follicular growth and development in the cow is well documented. The aim of this study was to investigate the role of GH on in vitro bovine oocyte maturation. Therefore bovine cumulus oocyte complexes (COCs) were cultured in M199 without FCS and gonadotropins and in the presence of 10, 100, or 1,000 ng/ml bovine GH (NIH-GH-B18). The COCs were incubated at 39°C in a humidified atmosphere with 5% CO₂ in air and nuclear stage was assessed after 2, 4, 8, 16, 22, and 24 hr of incubation using DAPI staining. To assess the effect of GH on developmental capacity of the oocytes, COCs were incubated in the presence of GH for 22 hr, followed by IVF and in vitro embryo culture. Cultures without GH served as controls. For subsequent development, the embryos were cultured in M199 supplemented with 10% FCS on a monolayer of BRL cells. Embryos were scored morphologically and the efficiency of the culture system was evaluated as (1) the percentage of cleaved embryos 4 days after IVF, (2) the percentage of blastocysts on day 9 expressed on the basis of the number of oocytes at the onset of culture, and (3) the percentage of hatched blastocysts on day 11 expressed on the basis of the total number of blastocysts present at day 9. GH (100 and 1,000 ng/ml) significantly accelerated nuclear maturation (P < 0.001). A 4 and 8 h the percentage of oocytes in GV stage after GH treatment (54% and 19%) was significantly lower than the control (64% and 41%). Similarly at 16 and 22 h the percentage of oocytes in MII stage was significantly higher in the GH-treated group; (58% and 77%) and (46% and 62%) for GH and control respectively. The number of oocytes in MII beyond 22 hr of culture did not differ; 100 and 1,000 ng/ml GH induced significant cumulus expansion (P < 0.05), which was not observed in the absence of GH. Addition of 100 and 1,000 ng/ml GH during maturation significantly (P < 0.01) enhanced subsequent cleavage rate from (64% and 67%) in control to (75% and 81%) in GH-treated group; embryonic development in terms of day 9 blastocyst formation was also significantly increased in the presence of GH (29% and 34%) compared to the control (18% and 24%). The hatchability of the blastocysts was not influenced by GH. From the present data, it can be concluded that GH present during IVM has a beneficial effect on subsequent development. © 1996 Wiley-Liss, Inc.     

Penetration in vitro of bovine oocytes during maturation by frozen-thawed spermatozoa

Bovine immature oocytes cultured for various times in TC-199 medium were inseminated with frozen-thawed spermatozoa in Medium BO with caffeine (5 mM) and heparin (10 micrograms/ml). Very high penetration rates (95-100%) were obtained in all oocytes which had been cultured for 0-20 h. When oocytes cultured for 0 and 4 h were inseminated, 100% of them were penetrated and had a decondensing sperm head and most of the oocytes remained at the stage of condensed germinal vesicle (GV) to telophase-I 20-22 h after insemination. The formation of male and female pronuclei was first observed in oocytes inseminated 8 h after culture. The proportions of polyspermy and average number of spermatozoa in penetrated oocytes gradually decreased as oocyte maturation proceeded. Penetration of at least one spermatozoon with a decondensing head into oocytes at the GV stage (without culture) was almost completed up to 8 h after insemination and at that time most of the penetrated oocytes were still at the stage of GV or condensed GV. These results indicate that maturation of bovine oocytes is not required for sperm penetration into the vitellus or for sperm nuclear decondensation under the in-vitro conditions used.     

Effects of temperature gradients on in vitro maturation of bovine oocytes

The effect of temperature gradients on in vitro maturation of bovine oocytes was examined in this study. Six treatment groups were made by combining 3 different maturation periods (0 to 10 h, 10 to 18 h and 18 to 24 h) with 2 different culture temperatures (37.0 degrees C and 38.5 degrees C). The frequency of oocytes matured to the metaphase II stage was apparently gradually increased as the culture temperature was increased from 37.0 degrees C to 38.5 degrees C at 0, 10 and 18 h after the onset of culture (75.2 vs 80.5, 82.3 and 84.3%, respectively), but this difference was not significant. Neither was the minor decrease in the proportion of oocytes reaching metaphase II when the temperature was decreased from 38.5 degrees C to 37.0 degrees C at 10 and at 18 h after the onset of maturation (84.3 vs 82.4 and 78.0%, respectively). However, more oocytes cleaved (79.2%; P = 0.0653) and developed to morulae (43.6%; P = 0.0019) and blastocysts (27.4%; P = 0.1568) when they were in vitro matured at 38.5 degrees C between 0 and 10 h, and then at 37.0 degrees C from 10 to 24 h. Although only the morula group was statistically different, cleavage- (79.2 vs 69.8, 72.5, 74.2, 76.3, 74.3%, respectively) and blastocyst formation (27.4 vs 23.2, 24.6, 25.2, 19.6, 21.9%, respectively) from this group was the highest among the 6 treatments.     

Effect of angiotensin II with follicle cells and insulin-like growth factor-I or insulin on bovine oocyte maturation and embryo development

The objective was to evaluate the effects of angiotensin II (Ang II), insulin-like growth factor-I (IGF-I) and insulin on the nuclear and cytoplasmic maturation of bovine oocytes in the presence of follicular cells. Cumulus-oocyte complexes (COCs) were cultured for 22h in the presence of follicular cells (control with cells) and Ang II, IGF-I or insulin (treatments), or in the absence of follicular cells (control without cells). Using these five groups, Experiment 1 was conducted with and without the addition of gonadotrophins. Only oocytes in the Ang II group resumed meiosis at rates (88.2+/-1.8% and 90.7+/-4.3% for oocytes cultured in the absence or presence of LH/FSH, respectively) similar to those observed for oocytes cultured in the absence of follicular cells (89.7+/-0.3% and 92.6+/-2.6%; P<0.01). In Experiment 2, the effect of Ang II alone and in combination with IGF-I or insulin on oocyte maturation for 7h (germinal vesicle breakdown), 12h (metaphase I) and 22h (metaphase II) was evaluated in a design similar to that of the first experiment. Ang II plus IGF-I or insulin induced the resumption of meiosis, irrespective of the presence of gonadotrophins (P<0.01). Experiment 3 used groups similar Experiment 2 to determine the rate of subsequent embryo development, using fetal calf serum (FCS) in the culture medium. The COCs were cultured in maturation medium for 1h (1+23h), 12h (12+12h) or 24h in the presence of follicular cells and the respective treatments and for the remaining period in the absence of follicular cells to complete 24h. In Experiment 4, BSA was used in lieu of serum in the maturation medium in a 12+12h maturation system. Oocytes matured using the 12+12h system with BSA or FCS in the presence of Ang II+IGF-I had higher rates of blastocyst formation than the other treatments (P<0.05). In conclusion, Ang II reversed the inhibitory effect of follicular cells on nuclear maturation of bovine oocytes, irrespective of the presence of gonadotrophins, IGF-I and insulin. However, oocyte cytoplasmatic maturation (i.e., subsequent embryo development), was higher when Ang II and IGF-I were present in the maturation medium containing follicular cells cultured for 12+12h.     

Cryopreservation of immature bovine oocytes by vitrification in straws

The aim of this study was to cryopreserve by vitrification by ethylene glycol (EG) and dimethyl sulfoxide (DMSO) immature bovine oocytes in straws and to investigate the effects of vitrification on post-thaw oocyte maturation. A total of 575 cumulus oocyte complexes were obtained by follicle aspiration from 238 ovaries of cows slaughtered at a local abattoir. Following selection, oocytes with compacted cumulus cells and evenly granulated ooplasm were vitrified using one of the three different solutions with a non-vitrified group served as control. The first step vitrification solution contained 20% EG while the second step solution contained 40% EG+1M sucrose in a basic media used in group EG. Oocytes were matured in N-2-hidroxyethyl piperazine-N-2-ethanosulfonic acid (HEPES) buffered tissue culture medium (TCM) 199 for 24h at 39 degrees C in a humidified atmosphere of 5% CO2 in air. Oocytes were fixed following evaluation for polar body formation, stained with Giemsa solution and nuclear maturation was examined. The numbers of oocytes which were observed at Metaphase II (MII) stage were 41 (34.1%), 17 (14.9%), 29 (20.7%) and 78 (79.6%) in groups EG, DMSO, Mix and Control, respectively. Maturation rate distribution in group Mix was not statistically different when compared to maturation rate distributions in groups EG and DMSO (p>0.05). Differences between other groups were significant (p<0.001). However, better results were obtained in EG group compared to DMSO and mix groups. Maturation rates were lower in all treatment groups than the control group. The lowest maturation result was obtained in DMSO group. Maturation rate in group Mix was between maturation rates of EG and DMSO groups. Immature bovine oocytes can be vitrified in straws, but maturation success differs with the cryoprotectant and it seems that to obtain better maturation rates, new cryopreservation techniques specific for immature bovine oocytes are needed.     

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.     

Effect of incubation temperature on in vitro maturation of porcine oocytes: nuclear maturation, fertilisation and developmental competence.

The present study examined the effect of low culture temperature during in vitro maturation (IVM) of pig oocytes on their nuclear maturation, fertilisation and subsequent embryo development. In experiment 1, oocytes were cultured at 35 or 39 degrees C for 44 h in modified tissue culture medium 199 supplemented with 10 ng/ml epidermal growth factor, 0.57 mM cysteine, 75 microg/ml potassium penicillin G, 50 microg/ml streptomycin sulphate, 0.5 microg/ml LH and 0.5 microg/ml FSH to examine the nuclear maturation status. In experiment 2, oocytes were cultured at 35 degrees C for 44 or 68 h and nuclear maturation was examined. In experiment 3, oocytes matured for 44 or 68 h at 39 degrees C and for 68 h at 35 degrees C were co-incubated with frozen-thawed spermatozoa for 5-6 h. Putative embryos were transferred into North Carolina State University (NCSU) 23 medium containing 0.4% bovine serum albumin. At 12 h after insemination, some oocytes were fixed to examine the fertilisation rate and the remaining embryos were examined at 48 and 144 h for cleavage and blastocyst formation rate, respectively. Compared with 39 degrees C, culture of oocytes at 35 degrees C for 44 h significantly (p < 0.05) reduced the metaphase II (M II) rate (79% vs 12%). However, extension of culture time to 68 h at 35 degrees C significantly increased (p < 0.05) the M II rate (7% vs 58%). In experiment 3, compared with other groups, fewer (p < 0.05) oocytes reached M II when cultured at 35 degrees C for 68 h (69-81% vs 49%). Extension of culture duration to 68 h at 39 degrees C stimulated spontaneous activation (28%) of oocytes. No difference in cleavage rates was observed among different groups. Compared with oocytes matured for 44 h at 39 degrees C (31%), the proportion of blastocysts obtained was low (p < 0.05) for oocytes matured at 35 degrees C (13%) or 39 degrees C (3%) for 68 h. The results indicate that lower culture temperature can delay nuclear maturation of pig oocytes. However, extension of culture time can stimulate nuclear maturation and these oocytes are capable of fertilisation and development to the blastocyst stage at moderate rates.     

In vitro fertilization of ovine oocytes vitrified by solid surface vitrification at germinal vesicle stage

Cryopreservation of immature oocytes at germinal vesicle (GV) stage would provide a readily available source of oocytes for use in research and allow experiments to be performed irrespective of seasonality or other constraints. This study was designed to evaluate the recovery, viability, maturation status, fertilization events and subsequent development of ovine oocytes vitrified at GV stage using solid surface vitrification (SSV). Cumulus oocyte complexes (COCs) obtained from mature ewes were randomly divided into three groups (1) SSV (oocytes were vitrified using SSV), (2) EXP (oocytes were exposed to vitrification and warming solutions without vitrification) or (3) Untreated (control). Following vitrification and warming, viable oocytes were matured in vitro for 24h. After that, nuclear maturation was evaluated using orcein staining. Matured oocytes were fertilized and cultured in vitro for 7days. Following SSV, 75.7% 143/189 oocytes were recovered. Of those oocytes recovered 74.8%, 107/143 were morphologically normal (viable). Frequencies of in vitro maturation were significantly (P<0.01) decreased in SSV and EXP groups as compared to control. In vitro fertilization rates were significantly (P<0.01) decreased in SSV (39.3%) group as compared to EXP (56.4%) and control (64.7%) groups. Cleavage at 48h post insemination (pi) and development to the blastocyst stage on day 7 pi were significantly (P<0.001) decreased in SSV oocytes as compared to EXP and control groups. In conclusion, immature ovine oocytes vitrified using SSV as a simple and rapid procedure can survive and subsequently be matured, fertilized and cultured in vitro up to the blastocyst stage, although the frequency of development is low.