The presence of synthetic polymers in the maturation medium affects the cryotolerance and developmental capacity after parthenogenic activation of vitrified goat oocytes

The purpose of this present study is to assess if addition of the synthetic polymers in maturation medium can influence cryotolerance and subsequently embryonic development of mammalian oocytes. We examined the roles of two polymers, including polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP), on in vitro maturation (IVM), embryonic developmental capacity, and cryotolerance of goat oocytes. The present study includes two parts. At first, goat cumulus−oocyte complexes (COCs) were matured in a medium supplemented with 10% fetal bovine serum (FBS), 3 mg/ml PVP, or 1 mg/ml PVA, respectively. Data of oocyte with first polar body, cleavage, and blastocyst following parthenogenetic activation (PA) were recorded. Secondly, after maturation in the above medium, oocytes were vitrified using the Cryotop technique and then the morphology, cleavage and blastocyst formation of vitrified oocytes have been checked. The results demonstrated that the adding of PVP or PVA in maturation medium can't affect IVM of goat oocytes in comparison with FBS, as concern cumulus cell expansion, first polar body formation, and embryonic development. Additionally, without plunging into liquid nitrogen, only exposure to the vitrification and warming solutions cannot also influence the quality of oocytes, in terms of morphology, cleavage, and blastocyst formation. However, after IVM with synthetic polymers and vitrification, the ratio of oocytes with standard morphology in PVP or PVA group was only 59.47% ± 3.56% or 54.86% ± 5.19%, respectively, and was significantly less than that in the FBS group (89.37% ± 4.52%, P < 0.05). Furthermore, the cleavage ratio of oocytes in PVP or PVA group was 37.41% ± 4.17% or 27.71% ± 3.91% and was considerably less than that in the FBS group (64.97% ± 4.69%, P < 0.05). In addition, the cleavage ratio in PVP group was statistically higher than that in PVA group (P < 0.05). In terms of blastocyst development, a significant difference was observed between the synthetic polymer group and the FBS group (24.96% ± 3.62%, P < 0.05). However, the blastocyst ratio in the PVA group (7.51% ± 1.68%) was statistically less than the PVP groups (13.20% ± 4.59%, P < 0.05) and the FBS group (P < 0.05). In conclusion, two potential serum replacements, either PVP or PVA, can support IVM and embryonic development of goat oocytes at the concentration used in this study. But IVM with synthetic polymers supplemented to maturation medium may reduce the cryotolerance of oocytes. Additionally, the supportive function of PVP on embryonic development of vitrified oocytes might be better than that of PVA.     

Replacement of serum and hormone additives with follicular fluid in the IVM medium: effects on maturation, fertilization and subsequent development of buffalo oocytes in vitro

Buffalo follicular fluid was used in the IVM medium in place of serum and hormone additives for stimulating nuclear and cytoplasmic maturation of buffalo oocytes in vitro. Follicular fluid (buFF) was aspirated from visible surface follicles from buffalo ovaries. Cumulus oocyte complexes (COCs) were matured for 24 to 26 h at 38.5 degrees C, 5% CO(2) in air in the maturation medium (TCM-199). When used, the concentration of fetal bovine serum (FBS) was 10% and that of FSH-P was 5 mug/ml. In Experiment 1 TCM-199 was supplemented with 1) FBS, 2) FBS + FSH-P, 3) 20% buFF and 4) 40% buFF. The matured oocytes were denuded and stained with Giemsa stain to study nuclear maturation. The proportion of oocytes which completed nuclear maturation was similar in medium containing FSH (74%) and 20 or 40% buFF (67%), which was higher (P < 0.05) than in medium with FBS but without FSH or buFF (47%). In Experiment 2, which was aimed at examining the effects of buFF on cumulus expansion and rates of fertilization and subsequent development to the blastocyst stage after IVF, the maturation medium was supplemented with 1) FBS + FSH-P, 2) 20% buFF and 3) 40% buFF. The COCs matured in medium containing 20 or 40% buFF had significantly higher (P < 0.01) cumulus expansion than those matured in medium with FBS + FSH-P. Of the COCs matured in medium with FBS + FSH-P and 20 or 40% buFF, the fertilization rates indicated by the incidence of cleavage (56, 51 and 52%, respectively) and the proportion of cleaved COCs developing to morula (58, 54 and 57%, respectively) and blastocyst stage (30, 31 and 35%, respectively) were not significantly different. In Experiment 3, supplementation of the maturation medium with 1) FBS + FSH-P and 2) FBS + FSH-P + 20% buFF resulted in similar rates of morulae (41 and 38%, respectively) and blastocysts (31 and 25%, respectively), indicating that simultaneous presence of FBS, FSH-P and buFF did not have an additive effect on embryo yield. The results show that the gonadotropin and serum source in the IVM medium can be replaced by buFF at the 20% level to achieve comparable morula and blastocyst yields.     

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

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

Bovine oocyte vitrification before or after meiotic arrest: effects on ultrastructure and developmental ability

The nuclear stage at which oocytes are cryopreserved influences further development ability and cryopreservation affects ultrastructure of both cumulus cells and the oocyte. In this work, we analyze the effects of vitrification at different nuclear and cytoplasmic maturation stages on the oocyte ultrastructure and developmental ability. Culture in TCM199 + PVA with roscovitine 25 M during 24h led to meiotic arrest (MA) in cumulus-oocyte complexes (COCs), while permissive in vitro maturation (IVM) was performed in TCM199, 10% FCS, FSH-LH and 17beta-estradiol for 24 h. Oocytes were vitrified using the open pulled straw method (OPS) with minor modifications. Fresh and vitrified/warmed COCs were fixed as immature, after IVM, after meiotic arrest (MA) and after MA + IVM. Vitrification combined with MA followed by IVM produced the highest rates of degeneration, regardless of the vitrification time. As a consequence, lower proportions of embryos cleaved in these groups, although differences were eliminated at the five-eight cell stage. Development rates up to day 8 were similar in all experimental groups, being significantly lower than those in fresh controls. Only oocytes vitrified after IVM were able to give blastociysts. The morphological alterations observed can be responsible for compromised development. More research is needed to explain the low survival rates of the bovine oocyte after vitrification and warming.     

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.     

Bone Morphogenetic Protein 15 in the Pro-Mature Complex Form Enhances Bovine Oocyte Developmental Competence

Developmental competence of in vitro matured (IVM) oocytes needs to be improved and this can potentially be achieved by adding recombinant bone morphogenetic protein 15 (BMP15) or growth differentiation factor (GDF9) to IVM. The aim of this study was to determine the effect of a purified pro-mature complex form of recombinant human BMP15 versus the commercially available bioactive forms of BMP15 and GDF9 (both isolated mature regions) during IVM on bovine embryo development and metabolic activity. Bovine cumulus oocyte complexes (COCs) were matured in vitro in control medium or treated with 100 ng/ml pro-mature BMP15, mature BMP15 or mature GDF9 +/− FSH. Metabolic measures of glucose uptake and lactate production from COCs and autofluorescence of NAD(P)H, FAD and GSH were measured in oocytes after IVM. Following in vitro fertilisation and embryo culture, day 8 blastocysts were stained for cell numbers. COCs matured in medium +/− FSH containing pro-mature BMP15 displayed significantly improved blastocyst development (57.7±3.9%, 43.5±4.2%) compared to controls (43.3±2.4%, 28.9±3.7%) and to mature GDF9+FSH (36.1±3.0%). The mature form of BMP15 produced intermediate levels of blastocyst development; not significantly different to control or pro-mature BMP15 levels. Pro-mature BMP15 increased intra-oocyte NAD(P)H, and reduced glutathione (GSH) levels were increased by both forms of BMP15 in the absence of FSH. Exogenous BMP15 in its pro-mature form during IVM provides a functional source of oocyte-secreted factors to improve bovine blastocyst development. This form of BMP15 may prove useful for improving cattle and human artificial reproductive technologies.     

Generation of Live Piglets from Cryopreserved Oocytes for the First Time Using a Defined System for In Vitro Embryo Production

We report the successful piglet production from cryopreserved oocytes for the first time by using a simple, high capacity vitrification protocol for preservation and a defined system for in vitro embryo production. Immature cumulus-oocyte complexes (COCs) from prepubertal gilts were vitrified in microdrops and stored in liquid nitrogen. After warming, COCs were subjected to in vitro maturation (IVM), fertilization (IVF), and subsequent culture (IVC). Adjusting warmplate temperature to 42°C during warming prevented temperature drops in a medium below 34.0°C and significantly increased the percentage of oocyte survival and thus blastocyst yields obtained from total vitrified oocytes compared with that of warming at 38°C (87.1% vs 66.9% and 4.4% vs 2.7%, respectively). Nuclear maturation and fertilization of oocytes were not affected by vitrification and warming temperature. Blastocyst development on day 7 (day 0 = IVF) of the surviving oocytes after warming at 38°C and 42°C was not different but lower (P<0.05) than those of non-vitrified control oocytes (4.6%, 5.2% and 17.9%, respectively). However, blastocyst cell numbers in the control and vitrified groups were similar irrespective of warming temperature. Omitting porcine follicular fluid (pFF) from IVM medium (POM) did not affect maturation, fertilization and embryo development of vitrified-warmed oocytes. Transfer of blastocysts obtained on day 5 from vitrified oocytes matured either with or without pFF into 4 recipients (2 for each group) resulted in 4 pregnancies and the delivery of a total of 18 piglets. In conclusion, optimization of warming temperature was a key factor for achieving high survival rates, and surviving oocytes could be utilized in vitro using defined media. Using these modifications, live piglets could be obtained from cryopreserved oocytes for the first time.     

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

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

In vitro maturation 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.     

Serum free embryo culture medium improves in vitro survival of bovine blastocysts to vitrification

The aim of this study was to examine the effects of co-culture with Vero cells during the in vitro maturation (IVM) and three culture media, B2+5% fetal calf serum (FCS) on Vero cells, synthetic oviduct fluid (SOF)+5% FCS, and SOF+20 gL(-1) bovine serum albumin (BSA), on the developmental competence of the embryos and their ability to survive vitrification/warming. We also tested the effect of morphological quality and the age of the embryo on its sensitivity to vitrification. The IVM system neither affects the embryo development up to Day 7 nor survival rates after vitrification. The culture of embryos in SOF+FCS and in Vero cells+B2 allowed obtaining more Day 6 and Day 7 blastocysts, and a higher % of Day 7 blastocysts vitrified than culture in SOF+BSA. Contrarily, on Day 8, more blastocysts were vitrified in SOF+BSA than in SOF+FCS. Blastocysts quality affected development after vitrification/warming, and Day 7 embryos showed higher survival rates than their Day 8 counterparts. Day 7 blastocysts produced in Vero cells or in SOF+BSA survived at higher rates than those produced in SOF+FCS at 24 and 48 h after warming. Embryo culture with BSA allows obtaining hatching rates after vitrification/warming higher than those obtained after co-culture with Vero cells in B2 and FCS. Moreover, this system provides hatching rates from Day 8 blastocysts comparable to those obtained on Day 7 in Vero cells. Further studies, including embryo transfer to recipients, are needed to clarify factors affecting the freezability of in vitro produced bovine embryos.     

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

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

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.     

Low concentrations of MEM vitamins during in vitro maturation of porcine oocytes improves subsequent parthenogenetic development

To investigate the effects of water-soluble vitamin supplementation for IVM/IVC of porcine oocytes and evaluate maturation and developmental capacity in vitro, porcine cumulus oocyte complexes (COCs) was matured in NCSU-23-based medium with water-soluble vitamins for 44 h and then cultured in PZM-3 for 7 days following activation. The COCs were allocated into five treatment groups and matured in various concentrations of MEM vitamins (control, 0.05, 0.1, 0.2, 0.4, and 1x). Metaphase II plates of the cumulus-free oocytes were observed following Hoechest 33258 staining. The COCs were allocated into four treatment groups, matured in various concentrations of MEM vitamins (control, 0.05, 0.1, 0.2, and 0.4x) and cultured in PZM-3 following activation. Also, COCS were matured without MEM vitamins and cultured in PZM-3 with various concentrations (control, 0.1, 0.4, 1.0, and 2.0 x) of MEM vitamins. Furthermore, 2 x 2 factorial (IVM/IVC) experiments were performed in IVM medium with or without 0.05 x MEM vitamins and IVC medium with or without 0.4x MEM vitamins to examine the in vitro development of parthenogenetic embryos. Maturation rates of COCs treated with MEM vitamins did not differ significantly among groups. However, compared to the control group, oocytes matured with the addition of 0.05 x MEM vitamins developed to blastocysts at a higher percentage (P<0.05) following activation and culture in PZM-3 without MEM vitamins. Total cell number of blastocysts was significantly higher in the 0.05 x group. Addition of 0.4x MEM vitamins decreased (P<0.05) cleavage and blastocyst developmental rates compared with 0.05 x MEM vitamins-treated group. In contrast, addition of vitamins to PZM-3 medium for in vitro culture of activated porcine oocytes did not affect development. In conclusion, addition of a low concentration of MEM vitamins to IVM medium for porcine oocytes enhanced subsequent development and improved embryo quality.     

The effects of bovine serum albumin and fetal bovine serum on the development of pre- and postcleavage-stage bovine embryos cultured in modified CR2 and M199 media

The effects of protein supplementation on bovine embryo development in vitro was evaluated using a 4 × 2 factorial arrangement with ten replications. A total of 6438 oocytes collected from abattoir ovaries were used. Bovine serum albumin (BSA) and fetal bovine serum (FBS) were added in various combinations to simple (modified CR2) and complex (M199) media during culture of precleavage-stage IVM/IVF-derived ova from 18 h after insemination to 72 h and postcleavage-stage embryos after 72 h of culture. Cleavage rates did not differ (p > 0.05) between media supplemented with FBS or with BSA. However, the postcleavage development to the blastocyst stage of in vitro-derived bovine embryos is better in media supplemented with FBS than BSA. A greater (p < 0.05) proportion of cleaved occytes developed to blastocysts and hatched blastocysts in media supplemented with FBS during postcleavage culture. The percentage of embryos that stopped development at the morula stage was significantly (p < 0.05) greater in media supplemented with BSA during postcleavage culture. Viability of blastocysts produced in CR2 and M199 supplemented with FBS were further assessed by transfer to recipients. In CR2, 25 transferred blastocysts resulted in seven pregnancies and the birth of three normal calves. In M199, 24 transferred blastocysts resulted in five pregnancies and the birth of two normal calves. There was no difference (p > 0.05) in rate of embryo development between CR2 and M199.