Groups Of Cumulus-oocyte Complexes Research Articles

124 CO-CULTURE WITH BOVINE INTACT CUMULUS - OOCYTE COMPLEXES DURING IN VITRO FERTILIZATION OF BUFFALO DENUDED OOCYTES COMPLETELY RESTORES THEIR FERTILIZING AND DEVELOPMENTAL COMPETENCE

Removal of cumulus cells is necessary for several technologies such as vitrification, intracytoplasmic sperm injection, and nuclear transfer. However, it is known that the presence of cumulus cells during IVF of buffalo oocytes is fundamental for fertilization and embryo development (Gasparrini et al. 2007 Anim. Reprod. Sci. 98, 335–342; Nandi et al. 1998 Theriogenology 50, 1251–1262). The aim of this work was to evaluate whether co-culture with intact bovine cumulus–oocyte complexes (COC) during IVF would restore the developmental competence of denuded buffalo oocytes. Due to the scarce availability of buffalo ovaries, the somatic support was provided by bovine cumulus cells. Abattoir-derived COC were matured in vitro according to our standard procedures (Gasparrini et al. 2006, Theriogenology, 65, 275–287) and randomly distributed in 3 fertilization groups: 1) a control group of COC (n = 122), 2) a negative control of denuded oocytes (DO; n = 119), and 3) DO co-cultured with in vitro matured bovine COC (DO+COC; n = 103) in a 1:1 ratio (3 bovine COC + 3 denuded buffalo oocytes/50 μL drop). Fertilization was carried out with frozen–thawed spermatozoa from a tested bull in TALP medium supplemented by 0.2 mM penicillamine, 0.1 mM hypotaurine, and 0.01 mM heparin at 38.5°C under a controlled gas atmosphere of 5% CO2 in humidified air. After fertilization the zygotes were cultured in SOF medium including essential and nonessential amino acids and 8 mg mL–1 BSA, at 38.5°C under humidified 5% CO2, 7% O2, and 88% N2, up to the blastocyst stage. On Day 5 and on Day 7 (Day 0 = IVF) cleavage and blastocyst rates were respectively recorded. Data were analysed by chi-square test. As expected, cleavage and blastocyst rates were lower (P < 0.01) in DO (36.1 and 9.2%, respectively) compared with the control (67.2 and 27.1%, respectively). However, co-culture during IVF (DO+COC) significantly increased (P < 0.01) both parameters compared with DO, giving cleavage (70.9%) and blastocyst (27.2%) rates similar to the control. The results of this study demonstrated that co-culture with bovine intact COC during IVF of buffalo denuded oocytes completely restores their fertilizing capability and blastocyst developmental competence. We conclude that this may be a suitable strategy for preserving the developmental competence of oocytes devolved to technologies, such as oocyte vitrification, that require cumulus removal.

Relationship between cumulus cell apoptosis, progesterone production and porcine oocyte developmental competence: temporal effects of follicular fluid during IVM

The objective of the present study was to determine the temporal effects of sow follicular fluid (FF) in vitro on cumulus cell viability and function, as well as oocyte nuclear and cytoplasmic maturation. Cumulus-oocyte complexes (COCs) recovered from the ovaries of prepubertal pigs were matured in medium with (+FF) or without (-FF) follicular fluid for the first 22 h of IVM. At 22 h of IVM, each group of COCs was then transferred to medium with or without FF and matured for another 22 h, forming four treatment groups (-FF/-FF, -FF/+FF, +FF/-FF and +FF/+FF). The concentration of progesterone in spent IVM medium and the incidence of cumulus cell apoptosis in individual COCs were determined at 22 and 44 h of IVM. Cumulus expansion was also recorded at 44 h of IVM. Finally, the ability of oocytes to complete meiosis to the MII stage and form blastocysts after IVF and embryo culture was assessed. Maturation with FF for part or the whole of IVM increased cumulus expansion and progesterone production and decreased the incidence of cumulus cell apoptosis compared with the -FF/-FF group (P < 0.05). The changes were greatest for the +FF/+FF group and intermediate for the -FF/+FF and +FF/-FF groups. Regression analysis revealed a negative association between cumulus cell progesterone production and the incidence of cumulus cell apoptosis (P < 0.001). Meiotic maturation was enhanced when FF was present during the first half of IVM. Oocytes matured in the presence of FF during the first and/or second half of IVM displayed an increased ability to form blastocysts compared with the -FF/-FF group (P < 0.05). The extent of the increase was similar for all FF-supplemented groups. The results show that FF exerts several beneficial effects at different times during IVM and suggest that a major role of FF is to provide protection from oxidative stress. We propose that the incidence of cumulus cell apoptosis in COCs must be kept below a certain threshold to ensure adequate functionality, including steroidogenic activity, is maintained for the acquisition of oocyte developmental competence.

The effects of roscovitine on cumulus cell apoptosis and the developmental competence of domestic cat oocytes

The developmental competence of cat oocytes matured in vitro is relatively poor when compared with that of in vivo oocytes. The study aimed to investigate the effect of roscovitine on the developmental competence of cat Felis catus oocytes matured in vitro. Cumulus-oocyte complexes (COCs) were classified as Grade I and II to III. Groups of COCs were cultured in 0, 12.5, 25, 50, 100, and 200 μM roscovitine for 24 h and were either fixed to assess the stages of nuclear maturation (Experiment 1) or additionally matured in vitro for 24 h before fixation (Experiment 2). In Experiment 3, cumulus cells from the COCs treated with roscovitine were examined for apoptosis. Experiment 4 examined the developmental competence of cat oocytes after roscovitine treatment and in vitro fertilization in terms of cleavage and morula and blastocyst formation rates. Roscovitine reversibly arrested cat oocytes at an immature stage in a dose-dependent manner. Roscovitine at 12.5 and 25 μM demonstrated less efficiency compared with that of other doses. However, higher doses of roscovitine induced cumulus cell apoptosis and resulted in a high number of degenerated oocytes after in vitro maturation. Roscovitine at 12.5 and 25 μM were therefore used to evaluate their effect on embryo development. Pretreatment with 12.5 and 25 μM roscovitine prior to in vitro maturation decreased the developmental competence of cat oocytes compared with that of non–roscovitine-treated controls. In conclusion, roscovitine reversibly maintained cat oocytes at the germinal vesicle stage without detrimental effect on nuclear maturation. However, it negatively affected cumulus cell viability and developmental competence.

343 DISTRIBUTION OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE AND IN VITRO MATURATION OF PORCINE OOCYTE - CUMULUS COMPLEXES FROM SMALL AND MEDIUM FOLLICLES

Glucose metabolism through the pentose phosphate pathway (PPP) plays a critical role in meiotic maturation and fertilization. However, the relationship between the distribution of a PPP key enzyme, glucose-6-phosphate dehydrogenase (G6PD), in cumulus–oocyte complexes (COCs) and the in vitro maturation (IVM) of the oocytes is not clear. In the present study, we examined the distribution of G6PD, the morphological characteristics in OCCs derived from small (d2 mm in diameter) and medium (3 to 6 mm in diameter) follicles, and the rate of oocyte maturation. Porcine COCs were collected from small or medium follicles of slaughterhouse ovaries. The COCs were cultured in a maturation medium, BSA-free NCSU37 supplemented with 10% porcine follicular fluid, eCG, and hCG, for 20 h and then in the absence of hormones for 24 h. To determine the distribution of G6PD, the COCs were treated with 13 �M brilliant cresyl blue (BCB) in TL-HEPES-PVA for 90 min. Results from 3–6 replicates were analyzed by ANOVA and Duncan&amp;apos;s multiple range test. The mean diameters for COCs collected from small follicles (136.7 &amp;micro;m for the outer zona and 103.1 &amp;micro;m for ooplasm) were significantly less than for those derived from medium follicles (164.1 &amp;micro;m and 122.0 &amp;micro;m, respectively). G6PD activity was detected in the cumulus cells for most of the COCs derived from medium follicles, but it was not significantly different from that of COCs derived from small follicles. In the second group of COCs, very little G6PD activity was found in both the cumulus cells and the oocytes (34.7 &amp;plusmn; 11.5&amp;percnt; and 18.0 &amp;plusmn; 6.7&amp;percnt; in COCS derived from small and medium follicles, respectively). After stimulation by eCG and hCG, the percentages of COCS in which G6PD activity was detected in the cumulus cells, but not in the oocytes, were 56.2 &amp;plusmn; 23.8&amp;percnt; and 72.9 &amp;plusmn; 6.1&amp;percnt; for small and medium follicles, respectively. The percentage of oocytes at the metaphase II stage (53&amp;percnt; and 63.9&amp;percnt; in oocytes from small and medium follicles, respectively) was higher for the COCs that showed higher G6PD activity in their cumulus cells. In conclusion, although no statistical differences were detected in the distribution of G6PD between COCs from small and medium follicles, due to a large variation, a higher percentage of mature oocytes seems to be the result of COCs where the G6PD activity is detected in the cumulus cells, but not in the oocyte, during IVM.

205 DEVELOPMENT OF OOCYTES FROM COWS TREATED WITH RETINOL IS COMPROMISED PRIOR TO IMPLANTATION

In the cow, sheep, and gilt, a retinoid support enhances the oocyte development (reviewed by Hidalgo C et al. 2003 Reproduction 125, 409–416). The shortest effective time interval for these animals injected with retinol (ROH) is four days, but the reasons for this have not been described. Furthermore, the development after transfer of embryos derived from oocytes of donors treated with ROH is not known. The objective of the present work was to elucidate the two above questions. We analyzed the ROH and retinyl palmitate (REP) contents in plasma and follicular fluid (FF). The estrus cycles of heifers (n = 12) were synchronized with progestagen and PGF2α. Blood samples were taken at progestagen removal (Day 0). Animals were injected with 1 × 106 ROH units (n = 6) or corn oil (vehicle; n = 6) on Days 0, 1, and 4. Contents of follicles between 3 and 10 mm were aspirated on Day 4 by an ultrasound-guided procedure. All samples were analyzed for ROH and REP by HPLC. To produce embryos for transfer, follicles were aspirated from donors treated four times with 1 × 106 ROH units/week or vehicle, starting four days before the first aspiration. Both groups of cumulus-oocyte complexes (COCs) were matured in vitro with or without 5 nM 9-cis-retinoic acid (RA). The presence of RA prolonged the exposure to retinoids (cows treated with ROH) or acted as a positive control (heifers with vehicle). Oocytes were fertilized and cultured in mSOF + 5% FCS. Embryo transfer (ET) to recipients was performed with fresh (one) or vitrified (two) good-morphology Day 7 embryos, and pregnancy monitored on Days 21, 35, and 60. Data analysis was by GLM (ROH and REP concentrations) or CATMOD (pregnancy monitoring), and Duncan's test (a,bP &lt; 0.05; v,x,y,zP &lt; 0.02). Average FF volume recovered were 351 ± 127 μL (controls; range 100–700) and 393 ± 127 μL (ROH; range 80–950). Concentrations of REP were unaffected by timing, follicle or blood, and ROH treatment (data not shown). Concentrations of ROH (μg/dL) for vehicle and ROH-treated cows were (LSM ± SE) 42.0 ± 1.8vx and 42.0 ± 2.4vx (plasma-Day 0, before ROH injection), 37.3 ± 1.8v and 47.64 ± 2.4x (plasma-Day 1), 42.6 ± 1.9vx and 45.5 ± 2.3vx (plasma-Day 4), and 6.1 ± 3.0y and 16.8 ± 2.6z (FF-day 4), respectively. Cumulatively, embryos from donors receiving ROH did not exhibit pregnancy on Day 21 (0/15x; confirmed on Day 35), which differed from vehicle donors on Day 21 (44%, 8/18y) and Day 35 (33%, 6/18y), and tended to differ (P = 0.07) on Day 60 (22%, 4/18). Pregnancy rates were independent of fresh or vitrified embryos within ETs. Injected ROH elevated blood concentrations of ROH, although values became normalized on Day 4. However, a coasting period of 4 days for ROH administration seems to be justified by increased intrafollicular levels of ROH. Development into blastocysts is disrupted before implantation, showing that ROH directly affects the oocyte during its intrafollicular growth. This work was supported by MCYT-AGL-2002-0117.

294 EFFECT OF CYSTEAMINE ADMINISTRATION DURING EQUINE OOCYTE MATURATION ON GLUTATHIONE CONTENT, NUCLEAR MATURATION, AND DEVELOPMENTAL CAPABILITY AFTER INTRACYTOPLASMIC SPERM INJECTION

In the recent years, assisted reproduction methods have produced only limited success in equine species in comparison with other domestic mammals. A major factor affecting oocyte viability during in vitro culture is oxidative stress. Oxidative modifications could be responsible for oocyte-defective in vitro maturation and consequently compromise subsequent fertilization and embryonic development. Low-molecular-weight thiol compounds such as cysteamine, added during in vitro culture of bovine, porcine, and ovine oocytes, increase intracellular glutathione (GSH) synthesis, which prevents oxidative damages and consequently improves in vitro maturation and embryo development. The present study was aimed at investigating whether equine oocyte maturation efficiency and embryonic developmental capability following ICSI benefit from the addition of cysteamine during in vitro maturation (IVM). Cumulus oocyte complexes (COCs) were collected from slaughtered ovaries and cultured for 30 h at 38.5°C in 500 μL of control medium (TCM199 + 0.4% BSA + 0.1 IU/mL rhFSH + 50 ng/mL EGF) either supplemented with 100 μM cysteamine or not. After culture, nuclear stage was assessed by Hoechst 33342 staining after cumulus cell removal, and MII oocytes were analyzed for GSH content (Baker MA et al. 1990 Anal. Biochem. 190, 360–365). Groups of COCs matured under the same conditions were denuded with hyaluronidase and only oocytes with a visible polar body were fertilized by ICSI. The number of embryos that reached the 2–4 cell stage was assessed by nuclear staining with propidium iodide after 72 h of culture in SOF supplemented with 5% calf serum at 38.5°C in a modified atmosphere (5% CO2, 5% O2, and 90% N2). Our data indicated that oocytes cultured in the presence of cysteamine had a nuclear maturation rate similar to those cultured in control medium (Table 1). Intraoocyte GSH content increased during IVM, and the addition of cysteamine induced a significant GSH accumulation in matured oocytes. After ICSI, a similar proportion of zygotes in each group developed beyond the two-cell stage after 72 h of culture. The results of this study demonstrate that the addition of cysteamine to the IVM medium increases GSH content in equine oocytes. However, this affects neither the maturation rate nor the capability to reach the early embryonic development after ICSI. We hypothesize that factor(s) other than GSH content are responsible for the limited in vitro developmental capability of equine oocyte. Table 1. Effect of cysteamine administration on maturation rate, oocyte GSH content (pmol/oocyte), and early embryonic development after ICSI This work was supported by a 2003 UniMi Grant.

Low oxygen tension during in vitro maturation of porcine follicular oocytes improves parthenogenetic activation and subsequent development to the blastocyst stage

To establish a reliable in vitro maturation system for activation and subsequent development as nuclear recipients for the effective production of pig clones, we assessed maturation, activation and parthenogenetic development in response to the following: (1) type of immature oocytes (cumulus–oocyte complexes (COCs) or parietal granulosa plus cumulus–oocyte complexes (GCOCs)); (2) oxygen (O 2) tension (5 or 20%); and (3) maturation period (36–60 h). The rate of nuclear maturation to metaphase-II (M-II) in the GCOC group (73.0 ± 3.1%) was higher than that in the COC group ( P < 0.05, 60.6 ± 3.5%), but the rates did not differ between the 5 and 20% O 2 tension groups. M-II rate increased ( P < 0.05) to about 70% after 42 h and then remained constant until 60 h of culture. When oocytes were matured under 5% O 2 tension and stimulated, the rate of normal oocyte activation (a female pronucleus formation and emission of the second polar body) was higher ( P < 0.05, 38.5 ± 3.9%) than when oocytes were matured under 20% O 2 tension (24.5 ± 3.9%). On the other hand, the rate of normal activation was not significantly different between the COC and GCOC groups, and the highest ( P < 0.05) normal activation rate was obtained in oocytes cultured for 48 and 54 h (48.4 ± 5.5% and 47.9 ± 8.2%, respectively). When COC and GCOC matured for 48 h under 5 and 20% O 2 tension were stimulated and subsequently cultured in vitro for 6 days, the rate of blastocyst formation did not differ between the oocyte types nor between the O 2 tension groups. However, blastocyst quality, as measured by mean total cell number, was significantly higher in the 5% O 2 group ( P < 0.05, 34.6 ± 2.0 for COC; 33.8 ± 1.8 for GCOC) compared with the 20% O 2 group (25.9 ± 1.8 for COC; 27.0 ± 2.0 for GCOC). In conclusion, low O 2 tension (5%) during in vitro maturation of porcine oocytes promoted their ability to be activated normally and improved the quality of parthenogenetic blastocysts developed in vitro in modified NCSU-37 solutions. This knowledge may be applicable for preparation of in vitro matured oocytes with good quality as recipient oocytes for generating pig clones.

Developmental capacity of bovine cumulus oocyte complexes after transcriptional inhibition of germinal vesicle breakdown

Oocytes cultured in the presence of FSH and the transcriptional inhibitor, 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole (DRB), remain in meiotic arrest at the germinal vesicle (GV) stage. The objectives of this study were to assess the kinetics of maturation and the developmental capacity of bovine cumulus oocyte complexes (COC) following release from prolonged meiotic arrest by DRB. In Experiment I, COC were cultured for 20 h in Tissue culture medium (TCM)-199 supplemented with 10% estrus cow serum (ECS), 5 μg/ml FSH and 1 μg/ml estradiol in the presence of 120 μM DRB. COC were then released from meiotic arrest and cultured for 20 h in DRB-free medium. Control COC were cultured for 20 h in DRB-free medium, with culture initiated concomitant to the release of DRB-treated COC from meiotic arrest. Nuclear maturation was assessed after 0, 5, 10, 15, and 20 h of culture in DRB-free medium. The proportion of DRB-arrested oocytes reaching metaphase II (MII) following 20 h culture in DRB-free medium was not significantly different from controls (96±4% versus 99±4%). In Experiment II, COC were cultured for 20 h in TCM-199 supplemented with 10% ECS, 10 μg/ml LH, 5 μg/ml FSH, and 1 μg/ml estradiol in the presence or absence of 120 μM DRB. COC in the DRB-treated group were then washed and matured coincident with a second group of control COC for 20 h in DRB-free medium. COC in both groups were fertilized and then randomly assigned to one of two culture systems: TCM-199+10%ECS or mSOF+0.6% fatty acid-free BSA. Development was assessed at 72 h post insemination (hpi), 168 hpi (Day 7) and 216 hpi (Day 9). In this experiment, culture with DRB-arrested oocyte maturation at the GV stage (DRB, 85±3% GV; Control, 2±3% GV; P<0.001). Following release from arrest, maturation and fertilization, the proportion of COC that cleaved by 72 hpi was decreased by treatment with DRB (DRB: 78±3% versus Control: 90±3%; P<0.05). However, no effect of DRB was found on the proportion of cleaved zygotes that reached the blastocyst stage on either Day 7 or Day 9 of culture (Day 7: DRB 16±2% versus Control, 21±2%; Day 9: DRB 23±3% versus Control, 31±3%). More embryos reached the blastocyst stage in the TCM-199/serum culture system compared to the mSOF/BSA system on both Days 7 and 9 (Day 7: TCM-199, 23±2% versus mSOF, 13±2%, P<0.05; Day 9: TCM-199, 32±3% versus mSOF, 22±3%, P<0.05). In summary, bovine COC maintained in meiotic arrest for 20 h by culture in the presence of the transcriptional inhibitor DRB retained their capacity to develop to the blastocyst stage after fertilization in vitro.

Toxic effects of in vitro exposure to p-tert-octylphenol on bovine oocyte maturation and developmental competence.

Alkylphenolic compounds are a widespread family of xenoestrogens. High concentrations of these substances are present in sewage sludge that is spread on arable land and pasture as fertilizer. Because of their known endocrine system-disrupting activity, alkylphenols represent a potential risk for the reproductive health of farm animals. In this study, the impact of p-tert-octylphenol (OP) on the developmental competence of bovine oocytes was evaluated. Endocrine activity of OP was investigated for its effect on estrogen receptors alpha and beta (ERalpha and ERbeta) and progesterone receptor (PR) mRNA levels. Cumulus-oocyte complexes (COCs) were exposed during in vitro maturation to serial concentrations of OP (1-0.0001 microg/ml) and were compared with vehicle-treated controls and a group of COCs treated with 17 beta-estradiol (E2). A dose-related decrease in the percentage of oocytes that completed maturation after 24 h and in oocyte fertilization competence was observed at doses of OP as low as 0.01 microg/ml. Groups treated with > or =0.001 microg/ml OP showed impaired embryo development. No adverse effects of E2 were observed. In the E2-treated COCs, ERalpha mRNA was decreased but PR mRNA was upregulated compared with controls. Treatment with 0.001 and 0.0001 microg/ml OP induced a decrease in ERalpha mRNA, but ERbeta and PR mRNA were not affected. Treatment with 0.01 microg/ml OP did not produce changes in the expression of any of the mRNAs studied. OP impairs meiotic progression and developmental competence of bovine oocytes without demonstrating clear estrogen-mimic activity.

Effects of androgens, progesterone and their antagonists on the developmental competence of in vitro matured bovine oocytes

The aim of the present study was to determine whether androgens and progesterone influence the in vitro maturation of bovine oocytes as assessed by cleavage rates and competence to form blastocysts after in vitro fertilization. Bovine cumulus-oocyte complexes were cultured (n = 20 per drop) for 22-24 h at 38.5 degrees C in TCM-199 medium supplemented with 10% oestrous cow serum, eCG (2.5 iu ml(-1)) and a range of treatments that included aromatizable (testosterone; 100 nmol l(-1)) and non-aromatizable (dihydrotestosterone; 100 nmol l(-1)) androgens, an androgen antagonist (flutamide; 36 micromol l(-1)), progesterone (300 nmol l(-1)) and a progesterone antagonist (mifeprisone, RU486; 100 nmol l(-1)). Production of inhibin A, total alpha-subunit, activin A and follistatin by each group of cumulus-oocyte complexes was also measured, since inhibin-related peptides have been implicated as modulators of oocyte maturation and their production may be influenced by steroids and anti-steroids. Both testosterone and dihydrotestosterone increased oocyte cleavage rate (25%; P < 0.01) and dihydrotestosterone also increased (24%; P < 0.05) the proportion of oocytes that reached the >/= eight-cell stage. However, neither androgen affected blastocyst yield, or the proportion of blastocysts that hatched. The stimulatory effect of dihydrotestosterone on cleavage rate was reduced by flutamide but the anti-androgen had no effect when tested alone. Treatment with testosterone, but not dihydrotestosterone, decreased (P < 0.05) endogenous follistatin and increased (P < 0.05) the activin A:follistatin ratio in maturation medium. Concentrations of inhibin A, total alpha-subunit and activin A were not affected significantly by androgen or flutamide. Addition of progesterone or the anti-progestin mifepristone to cumulus-oocyte complexes had no effect on cleavage rate. However, progesterone reduced by approximately 40% (P < 0.05) the proportions of both total oocytes and cleaved oocytes that formed blastocysts. This effect was partially reversed by mifepristone. Neither progesterone nor mifepristone affected inhibin A, activin A or follistatin production. However, total alpha-subunit concentration was significantly greater in the progesterone-treated group than in the controls (50%; P < 0.05), indicating that the negative effect of progesterone on blastocyst yield may be mediated by increased inhibin alpha-subunit expression by cumulus cells.

Quality controls for bovine viral diarrhea virus-free IVF embryos

Introduction of bovine viral diarrhea virus (BVDV) with cumulus-oocyte-complexes (COCs) from the abattoir is a concern in the production of bovine embryos in vitro. Further, International Embryo Transfer Society (IETS) guidelines for washing and trypsin treatment of invivo-derived bovine embryos ensure freedom from a variety of pathogens, but these procedures appear to be less effective when applied to IVF embryos. In this study, COCs were exposed to virus prior to IVM, IVF and IVC. Then, virus isolations from cumulus cells and washed or trypsin-treated nonfertile and degenerated ova were evaluated as quality controls for IVF embryo production. The effect of BVDV on rates of cleavage and development was also examined. All media were analyzed prior to the study for anti-BVDV antibody. Two approximately equal groups of COCs from abattoir-origin ovaries were washed and incubated for 1 h in minimum essential medium (MEM) with 10% equine serum. One group was incubated in 10 7 cell culture infective doses (50% endpoint) of BVDV for 1 h, while the other was incubated without virus. Subsequently, the groups were processed separately with cumulus cells, which were present throughout IVM, IVF and IVC. Cleavage was evaluated at 4 d and development to morulae and blastocysts at 7 d of IVC. After IVC, groups of nonfertile and degenerated ova or morulae and blastocysts were washed or trypsin-treated, sonicated and assayed for virus. Cumulus cells collected at 4 and 7 d were also assayed for virus. Anti-BVDV antibody was found in serum used in IVM and IVC but not in other media. A total of 1,656 unexposed COCs was used to produce 1,284 cleaved embryos (78%), 960 embryos ≥5 cells (58%), and 194 morulae and blastocysts (12%). A total of 1,820 virus-exposed COCs was used to produce 1,350 cleaved embryos (74%), 987 embryos ≥5 cells (54%), and 161 morulae and blastocysts (9%). Rates of cleavage (P=0.021), cleavage to ≥5 cells (P=0.026) and development to morula and blastocyst (P=0.005) were lower in the virus-exposed group (Chi-square test for heterogeneity). No virus was isolated from any samples from the unexposed group. For the exposed group, virus was always isolated from 4- and 7-d cumulus cells, from all washed nonfertile and degenerated ova (n=40) and morulae and blastocysts (n=57) and from all trypsin-treated nonfertile and degenerated ova (n=80) and morulae and blastocysts (n=91). Thus, virus persisted in the system despite the presence of neutralizing antibody in IVM and IVC media, and both washing and trypsin treatment were ineffective for removal of the virus. Presence of virus in 4- and 7-d cumulus cells as well as in nonfertile and degenerated ova were good indicators of virus being associated with morulae and blastocysts.