M. A. N. Dode

Effect of Percoll volume, duration and force of centrifugation, on in vitro production and sex ratio of bovine embryos.

The objective was to evaluate the effect of Percoll volume, and duration and force of centrifugation on sperm quality characteristics, embryo development, and sex ratio of in vitro-produced (IVP) bovine embryos. Frozen-thawed semen from four bulls were submitted to three Percoll procedures: T1-4 mL of Percoll, centrifuged for 20 min at 700 g; T2-800 microL of Percoll, centrifuged for 20 min at 700 g; and T3-800 microL of Percoll, centrifuged for 5 min at 5,000 g. Sperm total motility, morphology and integrity of the sperm acrosome, membrane and chromatin were determined before and after Percoll treatment, and semen was used for in vitro fertilization (IVF) of in vitro-matured oocytes. All Percoll methods increased the proportion of motile sperm (P<0.05). There were no significant effects of treatment for any sperm characteristic; however, for every end point, there were significant differences among bulls. Similarly, rates of cleavage and blastocyst formation were not affected by the Percoll procedure (P>0.05), but were affected by sire (P<0.05). Sex ratio was similar among treatments for Bulls 2 and 3, whereas semen from Bull 1 processed by T1 yielded a greater percentage of male embryos. However, when only treatments were considered, independent of bulls, the proportion of male:female embryos did not differ significantly from an expected 1:1 ratio. In conclusion, decreasing Percoll volume, reducing duration of centrifugation, and using a higher force of centrifugation did not significantly affect sperm quality, embryo development, or sex ratio of in vitro-produced bovine embryos.

Quality assessment of bovine cryopreserved sperm after sexing by flow cytometry and their use in in vitro embryo production.

The objective was to evaluate the structural and functional quality of bull sperm after sexing by flow cytometry. Frozen non-sexed (NS), sexed for X (SX) and sexed for Y (SY) sperm from four bulls was used. Frozen-thawed sperm was analyzed for motility, sperm head agglutination, morphology, capacitation, and integrity of the plasma membrane, acrosome, and chromatin. After Percoll centrifugation (45:60% gradients), the pellet was used for sperm analysis or IVF. Data were analyzed using generalized linear models (P < 0.05) and were reported as least squares means ± standard error (SEM). Based on sperm evaluations, NS sperm had better (P < 0.05) quality than sexed sperm, including higher motility and greater percentages of cells with an intact membrane and acrosome (58.0 ± 3.0, 58.2 ± 3.0, and 60.9 ± 3.3) than SX (29.6 ± 1.3, 36.0 ± 2.9, and 37.1 ± 3.3), and SY (26.2 ± 2.1, 36.4 ± 2.9, and 37.5 ± 3.3). There were no differences (P > 0.05) among groups for fertilization and cleavage rates. Similarly, blastocyst rate on Day 8 (Day 0 = day of insemination) did not differ among groups (22.2 ± 3.2, 18.1 ± 3.3, and 14.8 ± 2.9 for NS, SX, and SY, respectively). Regarding embryo development kinetics, all groups had similar developmental stages from Days 6 to 9. Although the sex-sorting procedure affected sperm characteristics, it did not significantly affect fertilization or embryo development.

Effect of follicle size on mRNA expression in cumulus cells and oocytes of Bos indicus: an approach to identify marker genes for developmental competence

To identify the genes related to oocyte competence, we quantified transcripts for candidate genes in oocytes (H1Foo, H2A, H3A, GHR, GDF9, BMP15, OOSP1) and cumulus cells (FSHR, EGFR, GHR, PTX3, IGFII) using the follicle size model to select oocytes of better developmental quality. Follicles were dissected and distributed into four groups according to diameter as follows: 1.0-3.0, 3.1-6.0, 6.1-8.0 and >or=8.1 mm. Cumulus-oocyte complexes (COCs) were released, classified morphologically, matured, fertilised and cultured in vitro or denuded for measurement of diameter and determination of gene expression. Denuded germinal vesicle oocytes and their cumulus cells were used for gene expression analysis by reverse transcription-polymerase chain reaction. The blastocyst rate was highest for oocytes recovered from follicles>6 mm in diameter. In the oocyte, expression of the H2A transcript only increased gradually according to follicle size, being greater (P<0.05) in oocytes from follicles>or=8.1 mm in diameter than in oocytes from follicles<6.0 mm in diameter. In cumulus cells, expression of FSHR, EGFR and GHR mRNA increased with follicular size. In conclusion, we confirmed the importance of H2A for developmental competence and identified important genes in cumulus cells that may be associated with oocyte competence.

Involvement of steroid hormones on in vitro maturation of pig oocytes

The purpose of this study was to determine if the addition of steroid hormones into the culture medium could influence the in vitro maturation of pig oocytes. The cumulus-oocyte complexes (COCs). collected from follicles of 2-5 mm diameter, were matured in steroid-free medium supplemented with various concentrations of estradiol-17beta (0-3000 ng/ml), progesterone (0-5000 ng/ml) and testosterone (0-300 ng/ml). The COCs were cultured for 42 h, then fertilized in vitro. We analyzed nuclear and cytoplasmic maturation with lacmoid stain 20 h after in vitro insemination. We observed no significant effect (P > 0.05) on the percentage of oocytes completing nuclear or cytoplasmic maturation or the number of sperm penetrating each oocyte for any concentration of progesterone, estradiol-17beta or testosterone. Similarly, adding a combination of those hormones to the medium did not significantly (P > 0.05) affect any of the criteria. In order to determine if there was a possible secretion of steroids during maturation, we added COCs, denuded oocytes and stripped cumulus cells to drops of a steroid-free medium and cultured them for 42 h, after which we analyzed the medium, before and after culture, for the presence of progesterone, estradiol-17beta and testosterone by radioimmunoassay (RIA) analysis. COCs, as well as cumulus cells alone, secreted similar amounts of estradiol (43.3 and 37.5 pg/ml, respectively) and progesterone (4.24 and 4.79 ng/ml, respectively) into the maturation medium. A small amount of estradiol (28.8 pg/ml) was also detected when oocytes were cultured alone. These results indicate that no steroids need to be added to the maturation medium of pig oocytes and that the COCs secrete steroids during maturation. It is possible that the amounts produced by the COCs fulfill any requirement for steroids if these steroids are required for either nuclear or cytoplasmic oocyte maturation.

Effects of scrotal insulation in Nellore bulls (Bos taurus indicus) on seminal quality and its relationship with in vitro fertilizing ability.

The objectives of this study were to assess the effects of induced testicular degeneration in Bos taurus indicus (Nellore) bulls on changes in seminal characteristics and fertilizing ability of sperm. Four Nellore bulls (30-36-month-old, 500-550 kg) with good seminal quality (>80% motile and morphologically normal sperm) had scrotal insulation applied for 5d. Semen was collected by electroejaculation and cryopreserved at the pre-insulation moment, and 7, 14, and 21 d after insulation was removed. Gross motility, vigor of sperm movement (1-5), acrosome integrity, sperm morphology (phase-contrast microscopy), nuclear vacuoles and abnormal chromatin (Feulgen-stain) were determined after sperm preparations for in vitro fertilization (IVF). Prior to IVF, sperm were separated using a Percoll gradient (45% and 90%). Normal sperm decreased (P<0.05) 14 and 21 d after insulation was removed. On 14 and 21 d, the incidence of head defects (9.7 +/- 0.6 and 17.0 +/- 0.8, respectively; mean +/- S.E.M.) was higher (P<0.05) in agreement with the incidence of nuclear vacuoles (14.0 +/- 5.0 and 12.3 +/- 2.3) and abnormal chromatin (24.4 +/- 7.2 and 30.8 +/- 2.8). Although the frequency of cleaved oocytes decreased only on 21 d (P<0.05), blastocyst rates were lower (P<0.05) than pre-insulation on 14 and 21 d. In regression analyses, only nuclear vacuoles, head defects and intact acrosome accounted for differences in cleavage (R2=0.38, 0.48, and 0.30, respectively) and blastocyst rates (R2=0.35, 0.37, and 0.44). Abnormal chromatin was associated only with blastocyst rates (R2=0.35). In conclusion, blastocyst rate was more sensitive than cleavage rate and the assessment of nuclear integrity is recommended to predict the fertilizing ability of bull sperm.

Changes in gene expression profiles of bovine embryos produced in vitro, by natural ovulation, or hormonal superstimulation.

Embryos produced by hormonal superstimulation have been used as an in vivo control in most published research on embryo gene expression. However, it is not known if this is the most appropriate control for gene expression profile studies. We compared the expression of GRB-10, IGF-II, IGF-IIR, MnSOD, GPX-4, catalase, BAX, and interferon-tau genes, in embryos produced in vivo by hormonal superovulation (SOV), by in vitro fertilization (IVF) or in vivo without any hormonal stimulus (NOV). GRB-10 was less expressed in NOV than IVF embryos, whereas no differences were found for the other genes. The genes related to stress response were then grouped and compared; the sum of expression of MnSOD, GPX-4, and catalase genes tended to be greater in IVF than NOV embryos. A correlation analysis was performed; we found a distinct behavior for NOV embryos when compared with SOV and IVF in the expression of GRB-10, IGF-II and IGF-IIR genes. However, the behavior of these genes was similar in SOV and IVF embryos. We conclude that ovarian hormonal stimulation can affect embryos by altering gene expression. Although this conclusion was based on investigation of only a few genes, we suggest that SOV embryos should be used with caution as a control in gene expression studies.

The effect of sperm preparation and co-incubation time on in vitro fertilization of bos indicus oocytes

The objective of the present study was to evaluate the effect of various methods of sperm selection and various sperm-oocyte co-incubation times on in vitro fertilization (IVF) of zebu (Bos indicus) oocytes. Frozen semen from one ejaculate of a single bull was used for all treatments and replicates. After thawed, sperm was subjected to one of the three treatments: 45 and 90% discontinuous Percoll gradient, swim-up and washing by centrifugation. In all treatments, the spermatozoa were incubated with in vitro matured oocytes for 3, 6, 12 and 18h. After co-incubation oocytes were transferred to the culture medium and culture for 44h, when the cleavage was evaluated. The uncleavaged oocytes were fixed and stained to determine penetration, pronucleus formation and polyspermy. The sperm selection method did not influence (P<0.05) polyspermy, pronucleus formation, penetration and cleavage rates. No interaction between method of selection and sperm-oocyte co-incubation time was observed (P>0.05). However, sperm-oocyte co-incubation time affected fertilization. The lower penetration (26.5%) and cleavage rates (13.1%) were obtained at 3-h period. The penetration and cleavage percentages increased (P<0.05) progressively at 6h (63.3 and 54.4%) and 12h (77.6 and 67.6%). No differences (P>0.05) were observed between 12 and 18h of incubation for penetration and cleavage rates. The incidence of polyspermy and pronucleus formation was similar (P>0.05) for all time points. It is concluded that the methods used in this study for sperm selection do not affect fertilization; therefore, they all can be used for bovine IVF. In addition, regardless the method used better fertilization results were obtained when sperm and oocytes were co-incubated for 12h, and the prolongation of that time for up to 18h had no detrimental effect on fertilization.

Nanoscale Differences in the Shape and Size of X and Y Chromosome-Bearing Bovine Sperm Heads Assessed by Atomic Force Microscopy

Sperm dimensions and the question of whether X and Y chromosome-bearing sperm differ in size or shape has been of great interest, especially for the development of alternative methods to sort or classify sperm cells. The aim of the present study was to evaluate possible differences in the shape and size of the sperm head between X and Y chromosome-bearing sperm by atomic force microscopy (AFM). One ejaculate per bull (n = 4) was used. Each ejaculate was separated into four fractions: non-sexed (NS), sexed for X-sperm (SX), sexed for Y-sperm (SY) and a pooling of SX and SY samples (SXY). Using AFM, 400 sperm heads per group were measured. Twenty three structural features were assessed including one-, two- and three-dimensional parameters and shape descriptors. These measurements determine the micro- to nanoscale features of X- and Y-bearing chromosomes in sperm cells. No differences were observed for any individual variables between SX and SY groups. Next, a simultaneous evaluation of all features using statistical discriminant analysis was performed to determine if it was possible to distinguish to which group belong each individual cells. This analysis clearly showed, a distinct separation of NS, SXY, SX and SY groups. The recognition of this structural possibility to distinguish between X and Y sperm cell might improve the understanding of sperm cells biology. These results indicated that the associations of several structural measurements of the sperm cell head are promising candidates for development of a new method of sperm sexing.

Role of estradiol-17β on nuclear and cytoplasmic maturation of pig oocytes

The role of estradiol-17beta on nuclear and cytoplasmic maturation of pig oocytes was investigated in the present study. To determine the estradiol effect, oocytes were cultured for 42 h in a steroid free medium composed of mTCM-199 supplemented with LH, FSH and 10% charcoal extracted follicular fluid. Estradiol receptor (ER), detected by a binding assay, were present in cumulus cells and oocytes during maturation with higher levels observed at 24 h of culture in the oocytes and at 36 h in the cumulus cells. To block estradiol action an antiestrogen (1-p-dimethylaminoethoxyphenyl-1,2-diphenyl-1-butene (tamoxifen)) was added to the maturation medium at various concentrations. The percentage of treated oocytes that underwent nuclear maturation was similar (P>0.05) to the control group. Cytoplasmic maturation, determined by the ability to form female pronucleus (FPN) and male pronucleus (MPN), was not different (P>0.05) among all groups. The presence of 4-hydroxy-4-androstene-3-17-dione (4-OHA) also did not influence nuclear (P>0.05) or cytoplasmic maturation (P>0.05). The results suggest that estradiol is not involved in maturation of pig oocytes. However, the present experiment used pronuclei formation as the endpoint, no studies were done in regard to estradiols effects on the embryonic development.

Developmental capacity of Bos indicus oocytes after inhibition of meiotic resuption by 6-dimethylaminopurine

Several reports have suggested that a treatment before in vitro maturation might improve oocyte competence and increase its developmental potential. Therefore, the objectives of the present study were to establish the kinetics of IVM in Zebu oocytes, to assess the effect of 6-dimethylaminopurine (6-DMAP), a phosphorylation inhibitor, on meiotic resumption, and to verify the developmental potential of the blocked oocytes after removal of the inhibitory conditions. To establish the kinetics of in vitro maturation 1422 oocytes were obtained from Nellore cows ovaries and matured in presence and absence of gonadotropins. Samples of oocytes were taken from culture at 0, 6, 9, 12, 15, 18, 21 and 24h, and the oocytes were fixed, stained and evaluated for nuclear morphology. Germinal vesicle break down (GVBD) occurred between 6 and 12h of culture in both groups. By 21h the majority of the oocytes had reached metaphase II in presence (71%) and absence (62%) of gonadotropins. In order to examine the inhibitory effect of 6-DMAP, 585 oocytes were cultured for 12, 18 and 24h in the presence or absence of 2mM of 6-DMAP. At each time point the oocytes were evaluated for nuclear morphology. To test the reversibility of meiotic inhibition 366 oocytes were incubated for 0, 12, 18 and 24h in the presence of 6-DMAP and then were transferred to the maturation medium and cultured for further 24h. A total of 429 oocytes were used to evaluate the developmental potential after meiotic inhibition. The oocytes were cultured in the presence of 6-DMAP for 0, 12, 18 and 24h, and then were matured, fertilized and cultured in vitro. Culture of bovine oocytes in the presence of 6-DMAP up to 24h completely blocked GVBD with more than 90% of the oocytes at GV stage. The inhibitory effect of 6-DMAP was fully reversible since maturation rates were similar (P>0.05) among all treatment groups. The evaluation of embryo development after various periods of meiotic blockage showed that inhibition, regardless the time period, had no effect (P>0.05) on penetration and cleavage rates. However, the proportion of embryos at blastocyst stage was reduced after inhibition for 12 (20.2%), 18 (20.1%) and 24h (19.0%) compared with the control group (35.6%). 6-DMAP has a reversible effect on maintenance of meiotic arrest, but reduced further embryo development.