Heparin and penicillamine-hypotaurine-epinephrine (PHE) solution during bovine in vitro fertilization procedures impair the quality of spermatozoa but improve normal oocyte fecundation and early embryonic development.

The presence of heparin and a mixture of penicillamine, hypotaurine, and epinephrine (PHE) solution in the in vitro fertilization (IVF) media seem to be a prerequisite when bovine spermatozoa are capacitated in vitro, in order to stimulate sperm motility and acrosome reaction. The present study was designed to determine the effect of the addition of heparin and PHE during IVF on the quality and penetrability of spermatozoa into bovine oocytes and on subsequent embryo development. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes and mitochondrial function, was diminished (P<0.05) in the presence of heparin and PHE. Oocyte penetration and normal pronuclear formation rates, as well as the percentage of zygotes presenting more than two pronuclei, was higher (P<0.05) in the presence of heparin and PHE. No differences were observed in cleavage rates between treatment and control (P>0.05). However, the developmental rate to the blastocyst stage was increased in the presence of heparin and PHE (P>0.05). The quality of embryos that reached the blastocyst stage was evaluated by counting the inner cell mass (ICM) and trophectoderm (TE) cell numbers and total number of cells; the percentage of ICM and TE cells was unaffected (P>0.05) in the presence of heparin and PHE (P<0.05). In conclusion, this study demonstrated that while the supplementation of IVF media with heparin and PHE solution impairs spermatozoa quality, it plays an important role in sperm capacitation, improving pronuclear formation, and early embryonic development.

Efeito das células do cumulus e cisteamina durante o cultivo de maturação in vitro de oócitos bovinos sobre a maturação nuclear e aquisição da competência para desenvolvimento embrionário / Effects of cumulus cells and cysteamine during bovine oocyte in vitro maturation on meiosis progression and acquisition of developmental competence

Complexos cumulus-oócito (COC), oócitos desnudos (DO) e DO cocultivados com células do cumulus em suspensão (DO+CC) foram maturados in vitro (MIV) na presença ou ausência de cisteamina (50mM). Observou-se efeito benéfico da cisteamina durante o cultivo de MIV, pois a maturação nuclear no grupo COC cisteamina foi maior do que a do COC controle (P<0,05). No grupo sem a adição de cisteamina, foi observado que a ausência de CC durante o cultivo de MIV prejudicou a maturação nuclear em DO, em relação ao COC (P<0,05), todavia a cisteamina restaurou a capacidade de progressão da meiose em DO, tornando-os semelhantes aos COC (P>0,05). O acoplamento entre oócitos e CC durante MIV demonstrou ser essencial para aquisição da competência do oócito para suportar o desenvolvimento embrionário inicial, pois COC apresentaram maior porcentagem de blastocistos e eclosão quando comparados a DO e DO+CC (P<0,05). A inclusão de cisteamina no cultivo de MIV não restaurou a aquisição da competência em DO e DO+CC, que permaneceram semelhantes aos do grupo-controle (P>0,05). Conclui-se que a cisteamina no meio de MIV melhora as taxas de maturação nuclear em COC e restaura a capacidade de progressão da meiose em DO. Todavia, na concentração utilizada neste estudo, não promove efeito benéfico no desenvolvimento embrionário.

Cumulus-oocyte complexes (COC), denuded oocytes (DO) and DO co-cultured with cumulus cells in suspension (DO+CC) were in vitro matured (IVM) in the presence or absence of cysteamine (50mM). A beneficial effect of cysteamine was observed during IVM, because the nuclear maturation in the COC cysteamine group was higher than in COC control (P<0.05). In the control group, the absence of CC during IVM impaired nuclear maturation in DO when compared to COC (P<0.05), but cysteamine restored the ability of meiosis progression in DO, making them similar to COC (P>0.05). The coupling between oocytes and CC during IVM proved to be essential for the acquisition of oocyte competence to support early embryonic development, as COC had higher percentages of blastocyst and hatching when compared to DO and DO+DC (P<0.05). However, the inclusion of cysteamine in the IVM culture did not restore the acquisition of competence in DO and DO+DC, which remained similar to the control group (P>0.05). It is concluded that cysteamine in the IVM culture improves the nuclear maturation in COC and restores the progression ability of meiosis in DO. However, in the concentration used in this study, cysteamine does not promote a beneficial effect on embryo development.

Effect of antioxidants during bovine in vitro fertilization procedures on spermatozoa and embryo development.

Increased amounts of reactive oxygen species (ROS) during in vitro fertilization (IVF) may cause cytotoxic damage to gametes, whereas small amounts of ROS favour sperm capacitation. The aim of this study was to investigate the effect of antioxidants [50 microm beta-mercaptoethanol (beta-ME) and 50 microm cysteamine (Cyst)] or a pro-oxidant (5 mm buthionine sulfoximine) on the quality and penetrability of spermatozoa into bovine oocytes and on the subsequent embryo development and quality when added during IVF. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes, and mitochondrial function, was diminished (p < 0.05) after 4-h culture in the presence of antioxidants. Oocyte penetration rates were similar between treatments (p > 0.05), but antioxidants adversely affected the normal pronuclear formation rates (p < 0.05). The incidence of polyspermy was high for beta-ME (p < 0.05). No differences were observed in cleavage rates between treatments (p > 0.05). However, the developmental rate to the blastocyst stage was adversely affected by Cyst treatment (p < 0.05). The quality of embryos that reached the blastocyst stage, evaluated by total, inner cell mass (ICM) and trophectoderm cell numbers and ICM/total cell ratio was unaffected (p > 0.05) by treatments. The results indicate that ROS play a role in the fertilizing capacity in bovine spermatozoa, as well as in the interaction between the spermatozoa and the oocytes. It can be concluded that supplementation with antioxidants during IVF procedures impairs sperm quality, normal pronuclear formation and embryo development to the blastocyst stage.

The effect of interaction between macromolecule supplement and oxygen tension on bovine oocytes and embryos cultured in vitro.

SummaryAiming to improve in vitro production of bovine embryos and to obtain supplements to replace serum for in vitro maturation (IVM), this study evaluated the effects of macromolecular supplementation of IMV medium (bovine serum albumin - BSA, polyvinyl alcohol - PVA, polyvinyl pyrrolidone - PVP, Ficoll, KnockoutSR, or fetal calf serum - FCS) and oxygen tension [5% CO2 in air (20% O2) or 5% CO2, 5% O2 and 90% N2 (5% O2)] on oocyte maturation and embryo development. Nuclear progression to germinal vesicle breakdown, metaphase I and metaphase II stages were evaluated and overall results revealed that undefined (FCS) and semi-defined (BSA) media gave better results at 20% O2 and defined media (PVA, PVP and Ficoll) at 5% O2. Independent of macromolecule supplement, IVM at 20% O2 was considered optimal for nuclear maturation. To evaluate embryo development, oocytes matured in the previously described conditions were fertilized and cultured at the same oxygen tension used for IVM and assessed for cleavage (43.0 to 74.8%) and development to morulae (16.4 to 33.8%), blastocyst (7.7 to 52.9%) and hatched blastocyst (9.6 to 48.1%). Apart from oxygen tension, all treatments, except Knockout (22.7%), gave similar results for blastocyst development (26.5 to 38.7%). Independently of macromolecule supplement, higher development rates were obtained in an oxygen tension of 20% O2 (67.4% cleavage, 29.2% morulae, 40.8% blastocyst and 34.0% hatched blastocyst) when compared with 5% O2 (52.5, 21.8, 18.2 and 15.6%, respectively). This study indicates that BSA, PVA, PVP and Ficoll can replace serum during IVM and that the optimal atmospheric condition for in vitro production of bovine embryos is 5% CO2 and 20% O2.

Role of roscovitine and IBMX on kinetics of nuclear and cytoplasmic maturation of bovine oocytes in vitro.

The 3-isobutyl-1-methylxanthine (IBMX) is able to prevent resumption of meiosis by maintaining elevated cyclic AMP (cAMP) concentrations in the oocyte, and roscovitine, a purine known to specifically inhibit MPF kinase activity, maintains bovine oocytes at the germinal vesicle (GV) stage. The present study was conducted to analyze whether cytoplasmic maturation (examined by the pattern of cortical granule (CG) distribution) of bovine oocytes is improved during meiotic arrest with IBMX and roscovitine. Oocytes were matured in vitro in a 10% Knockout(SR) supplemented TCM-199 medium (Control) with either 0.5 mM IBMX or 25 microM roscovitine (ROSC). Oocytes were stained with fluorescein isothiocyanate conjugated Lens culinaris agglutinin (FITC-LCA) for CG evaluation and with Hoechst 33342 for nuclear stage assessment. At 16 h of culture, the percentage of oocytes remaining in the GV stage was higher (P < 0.05) in the ROSC group (32.41%) compared with the Control and IBMX groups (8.61% and 9.73%, respectively). At 24 h of culture, progression of meiosis to M II stage was retarded (P < 0.05) in the ROSC group (24.05%) compared to the Control (60.20%), whereas the IBMX group (33.88%) showed no significant difference to the other two groups. At 16 h of maturation, the proportion of oocytes with CG in clusters (immature cytoplasm) was similar between the groups, as was the percentage of peripheral CG (mature) at 24 h of maturation. The results of the present study demonstrated that the meiotic inhibitors IBMX and roscovitine delay the progression of nuclear maturation without affecting cytoplasmic maturation, assessed by the analysis of CG repositioning.