Oestrous Cow Serum Research Articles

Effects of oocyte quality, incubation time and maturation environment on the number of chromosomal abnormalities in IVF-derived early bovine embryos

Chromosomal aberrations are one of the major causes of embryo developmental failures in mammals. The occurrence of these types of abnormalities is higher in in vitro-produced (IVP) embryos. The aim of the present study was to investigate the effect of oocyte morphology and maturation conditions on the rate of chromosomal abnormalities in bovine preimplantational embryos. To this end, 790 early cattle embryos derived from oocytes with different morphologies and matured under different conditions, including maturation period (24 v. 36h) and maturation media (five different serum supplements in TCM-199), were evaluated cytogenetically in three sequential experiments. The rates of normal diploidy and abnormal haploidy, polyploidy and aneuploidy were determined in each embryo. Throughout all the experiments, the rate of chromosomal abnormalities was significantly (P<0.05) affected by oocyte morphology and maturation conditions (maturation time and culture medium). Lower morphological quality was associated with a high rate of chromosome abnormalities (P<0.05). Moreover, polyploidy was associated with increased maturation time (P<0.01), whereas the maturation medium significantly (P<0.05) affected the rates of haploidy and polyploidy. In general, supplementing the maturation medium with oestrous cow serum or fetal calf serum resulted in higher rates of chromosomal aberrations (P<0.05) compared with the other serum supplements tested (bovine steer serum, anoestroues cow serum, bovine amniotic fluid and bovine serum albumin). On the basis of the results of the present study, we conclude that the morphological quality of oocytes and the maturation conditions affect the rate of chromosomal abnormalities in IVP bovine embryos.

137 DIFFERENTIAL GENE EXPRESSION OF IN VITRO-MATURED BOVINE OOCYTES WITH OR WITHOUT A POLAR BODY

Oocyte competence is associated with the amount of transcripts stored in the ooplasm and oocyte ability to extrude polar bodies (PB). To our knowledge, however, no data comparing mRNA levels between bovine oocytes maturated in vitro with or without PB are available. The aim of the present study was to compare the relative abundance of transcripts of glucose transporter 1 (GLUT1), insulin-like growth factor 1 receptor (IGF1R), insulin-like growth factor 2 receptor (IGF2R), growth differentiation factor-9 (GDF9) and aquaporin 3 (AQP3) genes between oocytes with and without PB (PB and NPB groups, respectively) following in vitro maturation. Immature bovine oocytes were obtained by follicular aspiration and matured in TCM-199 (Gibco Life Technologies, New York, NY, USA) containing 10% of oestrus cow serum and 20 μg mL–1 of FSH (Pluset, Serono, Italy) for 24 h under 5% CO2 in air at 38.5°C. Subsequently, oocytes were visually classified according to the presence or absence of PB and then denuded and rapidly frozen in liquid nitrogen. Three pools of 10 oocytes for each group were subjected to total RNA extraction using the RNeasy Micro Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions and treated with DNase. Reverse transcription and cDNA amplification were performed using the TransPlex Complete Whole Transcriptome Amplification Kit (WTA2, Sigma, St. Louis, MO, USA) according to the manufacturer's instructions. Relative abundance of the target transcripts was performed by quantitative RT-PCR (Applied Biosystems Prism 7300 Sequence Detection Systems, Foster City, CA, USA) using a mixture of SYBR® Green PCR Master Mix (Applied Biosystems), 200 ng of cDNA, nuclease-free water and specific primers for each reaction. Expression of the β-actin gene was used as an endogenous reference. Relative gene expression analysis was performed using the software REST© 2005 using the Pair Wise Fixed Reallocation Randomization Test©. The relative expression values are presented as mean ± standard error. The relative abundance of GLUT1 (0.81 ± 0.07), IGF2R (0.72 ± 0.07) and GDF9 (0.82 ± 0.10) genes was lower (P &lt; 0.05) for NPB oocytes. There was no difference (P &gt; 0.05) in relative abundance between PB and NPB groups for the other genes. The results suggest that the amount of some transcripts stored in the matured ooplasm is associated with the presence of PB. The authors acknowledge FAPEMIG, CNPq and Innovation Network Project on Animal Reproduction (01.07.01.002).

Efeito do soro de cadela em estro na maturação in vitro de ovócitos caninos

Avaliou-se o efeito do soro de cadela em estro na maturação in vitro de ovócitos caninos, utilizando-se 92 ovócitos de cadelas, submetidas à cirurgia eletiva de ovarioisterectomia. Os ovócitos foram selecionados e distribuídos em dois tratamentos: T1 (n = 48), ovócitos cultivados in vitro durante 96 horas utilizando meio base - TCM199 + 5µg/mL de LH + 20µg/mL de FSH - mais 10% de soro inativado de vaca em estro e T2 (n = 44), ovócitos cultivados em meio base mais 10% de soro inativado de cadela em estro. O percentual de ovócitos observados em metáfase I não indicou diferenças (P&gt;0,05) entre T1 (2,1%) e T2 (0,0%), porém a taxa de ovócitos maduros (metáfase II) foi diferente (P&lt;0,05), sendo 27,1% em T1 e 47,7% em T2. O mesmo fato ocorreu com a taxa de cromatina condensada (P&lt;0,01), com 14,6 e 0,0%, respectivamente. Nos ovócitos sem configuração cromossômica, não foram observadas diferenças (P&gt;0,05), sendo 56,3% em T1 e 52,3% em T2. Estes resultados indicam que a adição de soro de cadela em estro no meio de cultivo oferece melhores condições para o desenvolvimento in vitro, quando comparado à de soro de vaca em estro.

Effect of oxygen tension and serum during IVM on developmental competence of bovine oocytes

With an aim to improve the in vitro production of bovine embryos, the present study investigated the effect of serum and oxygen tension during IVM on oocyte developmental competence. Four experimental groups were evaluated: G1, 10% oestrus cow serum (OCS) with 20% O(2); G2, 0.1% polyvinyl alcohol (PVA) with 20% O(2); G3, 10% OCS with 5% O(2); and G4, 0.1% PVA with 5% O(2). The proportion of MII oocytes, blastocyst rates and total cell number were not affected (P > 0.05) when the OCS was replaced with PVA under 5% O(2), whereas a higher (P < 0.05) blastocyst rate and total cell number were found with OCS compared with PVA under 20% O(2). The apoptosis index was lower in blastocysts from oocytes matured with PVA under 5% O(2) (G4) compared with other groups (G1, G2 and G3), but no differences (P > 0.05) were found in maturation and blastocyst rates. Significant differences were found in the amount of specific transcripts in oocytes matured under different conditions. In conclusion maturation with PVA and 5% O(2) provides an efficient in vitro culture condition for the maturation of bovine oocytes.

271 EFFECT OF BULL ON IN VITRO FERTILIZATION OF BOVINE OOCYTES

The main objective of this work was to evaluate the effect of the bull on IVF of bovine oocytes. These oocytes were obtained from ovaries collected in the slaughterhouse and selected for their maturation process in TCM-199 culture medium with 20% estrus cow serum, 10 IU of eCG, and 1 mg mL-1 gentamicin. The oocytes were cultivated for 20 to 24 hours in 39.0°C culture incubator and moist environment with 5% CO2. For the oocyte insemination, frozen/thawed semen of 8 bulls was used by means of swim-up capacity procedure according to Parrhit (1989) and then motility, sperm concentration, and strengh were evaluated. In all cases, 25 to 30 oocytes, previously selected according to cumulus oophorus density were artificially inseminated with 1 × 106 spermatozoids mL-1. The total of selected oocytes by bull for this work was: 5028 (1), 1247 (2), 2888 (3), 650 (4), 1529 (5), 699 (6), 984 (7), and 1250 (8). The results were evaluated according to the average of oocytes matured in vitro and to embryonic division 48 hours after AI. In order to evaluate the effects by bull, the statistical examination of chi-square proportion comparison was used. The average values and standard deviation (SD) motility were 57.0% ± 13.7; 53.2% ± 11.3; 51.2% ± 8.20; 40.9% ± 5.0; 48.1% ± 4.92; 33.5% ± 4.42; 53.5% ± 11.1 and 40.0% ± 8.9 for bulls 1 to 8, respectively. There was a highly significant difference (P &lt; 0.001) in relation to the percentage of division: 46, 42, 34, 18, 30, 31, 30, and 17% among bulls 1 to 8, respectively, in addition to variability among these bulls. The results obtained confirm the effect of the bull on conception rate. The conclusion is that frozen/thawed semen with 50% motility can increase the percentage of embryonic division.

245 EFFECT OF BOVINE OOCYTE MATURATION SYSTEM ON RELATIVE ABUNDANCE OF mRNA

The oocyte cytoplasm contains several transcripts that are important for early pre-implantation embryo development, and alterations on the amount of these stored mRNA can disturb oocyte competence. The aim of this study was to evaluate the relative abundance of specific transcripts in oocytes matured in vivo or in vitro. For in vitro maturation, immature oocytes were obtained by ovum pickup from 4 crossbred cows (group 1: G1) or from ovaries collected at a slaughterhouse (group 2: G2) and matured in TCM-199 containing 10% estrus cow serum and 2 μg FSH for 24 h under 5% CO2 in air at 38.5°C. For in vivo maturation, the same crossbred cows used in G1 received a progesterone intravaginal implant (CIDR®, Eazi-Breed CIDRO, São Paulo, Brazil) and 2 mg of estradiol benzoate (Estrogin®, Farmavet, São Paulo, Brazil) on Day 0. On Day 4, cows were superstimulated with 180 mg FSH (Folltropin®, Bioniche, Canada) injected in 6 decreasing doses every 12 h, and on Day 6, the cows received 0.53 mg of sodium cloprostenol (Ciosin®, Cooper, São Paulo, Brazil). On Day 7, CIDR® was removed and 2.5 mg of gonadorelina (Gestran-Plus®, Tecnopec, São Paulo, Brazil) was injected. Ovum pickup was performed 18 h after gonadorelina injection. Oocytes with expanded cumulus cell were then pooled and used as in vivo-matured oocytes (group 3: G3). Oocytes from all groups were denuded and frozen in liquid nitrogen. Pools of 10 oocytes for each group were subject to RNA extraction and reverse transcription. cDNA was amplified by real-time PCR using the beta-actin gene as the endogenous reference. The transcripts analyzed are encoded by TEA domain 2 (TEAD2), high mobility group N1 (HMGN), zygotic arrest 1 (ZAR1), maternal antigen that embryo requires (MATER), growth differentiation factor-9 (GDF9), and peroxiredoxin 1 (PRDX1) genes. Results were analyzed by REST software v.2 using the pair-wise, fixed reallocation randomization test. Data from G3 were used as calibrator. There was no difference (P &gt; 0.05) on relative abundance of all transcripts between pools of oocytes matured in vitro or in vivo obtained from the same cows (G1 and G3, respectively). However, the relative abundance of GDF9 (0.22 ± 0.04-fold) was less (P &lt; 0.05), whereas the relative abundance of TEAD2 transcripts (4.27 ± 2.14-fold) was greater (P &lt; 0.05) for in vitro-matured oocytes obtained from slaugterhouse ovaries (G2) when compared with in vivo-matured oocytes (G3). No difference (P &gt; 0.05) on relative abundance was found between G2 and G3 for the other genes. These data suggest that in vitro maturation does not alter the relative abundance of some transcripts stored into oocytes when compared with the ones stored in oocytes obtained from the same donors by means of multiple ovulation. Financial support was provided by CNPq and FAPEMIG.

114 DEVELOPMENT OF AN OPTIMIZED WELL IN WELL CULTURE SYSTEM: EFFECT OF MINIWELL CALIBER AND CULTURE DROP VOLUME ON DEVELOPMENTAL COMPETENCE OF BOVINE EMBRYOS

To overcome developmental problems due to the scientific need to track single embryos or due to embryo culture in small groups, the Well in Well culture system has been developed previously. Here we aimed to examine the effects of different MiniWell caliber and different embryo densities on developmental rates. Cumulus oocyte complexes (COC) aspirated from small follicles (2–8 mm) were cultured in modified TCM (TCM199, Sigma, Taufkirchen, Germany) supplemented with 12% heat inactivated oestrus cow serum and 10 μg mL–1 FSH (FSH-p, Sheering, Kenilworth, NJ, USA) for 24 h at 39°C in a humidified atmosphere with 5% CO2 in air. Fertilization was performed in Fert-TALP medium. After 20 h coincubation with sperm cells, presumptive zygotes were allocated into MiniWells of different diameter (0.3, 0.4, 0.5, and 0.7 mm) and depth (0.3, 0.5, and 0.7 mm) to investigate the effect of MiniWell diameter and depth on further development of the embryos. To investigate the effect of embryo density we compared the development of zygotes placed in MiniWells with different total volumes of culture medium per well. For all experiments bovine embryos cultured in groups of 16 and groups of 50 served as controls. Developmental rates of in vitro-produced embryos were analysed by chi-square test. Differences of P &lt; 0.05 were considered to be significant. When embryos were cultured in a 4 × 3 factorial design (n = 240 per treatment group in 16 replicates), MiniWell diameter (0.3 mm v. 0.4 mm v. 0.5 mm v. 0.7 mm) significantly affected developmental rates to the blastocyst stage (21.5% v. 26.9% v. 32.5% v. 31.3%, respectively) and MiniWell depth (0.3 mm v. 0.5 mm v. 0.7 mm) influenced development (24.4% v. 27.4% v. 29.3%, respectively). When embryos (n = 160 per treatment group in 10 replicates) were cultured in different total volumes of culture media per Well (0 μL, v. 150 μL v. 500 μL) development of embryos to the blastocyst stage (3.1% v. 13.1% v. 33.1%, respectively) differed significantly. When a total of 240 embryos cultured in 15 replicates in group of 16 were compared with a total of 750 embryos cultured in 15 replicates in group of 50, embryos cultured in group of 16 reached the blastocyst stage at a significantly lower level than zygotes cultured in the group of 50 (22.2% v. 30.3%), whereas zygotes cultured in MiniWells were able to compensate against low embryo densities reaching a blastocyst rate as high as embryos cultured in group of 50 (31.3 v. 30.3%). The best developmental rate of bovine zygotes to the blastocyst stage was observed in MiniWells with a diameter of 0.7 mm and 0.7 mm depth in 500 μL culture medium per well. In conclusion, we successfully optimized the Well in Well culture system by exploring the most suitable MiniWell properties supporting improved developmental rates to the blastoyst stage by compensating against negative effects of low embryo densities allowing to track each individual embryo over the complete culture period.

Embryos and culture cells: A model for studying the effect of progesterone

A positive association between P4 concentration and initial bovine embryo survival has been reported. The objective of this study was to establish two coculture systems as a model to study the influence of progesterone on the initial bovine embryo development. Granulosa cells (GC) or bovine oviduct epithelial cells (BOECs) were used at the base of embryo culture medium microdroplets (TCM199 and 10% of superovulated oestrus cow serum, (SOCS)) supplemented or not with progesterone (P4, 33.4 ng mL −1) and/or a progesterone receptor antagonist (onapristone, OP, 2.2 × 10 −5 M). Presumptive zygotes were transferred to monolayers after in vitro maturation and fertilization of bovine oocytes with thawed swim-up selected sperm. Embryo development was carried out according to the following groups: experiment 1, BOEC ( n = 378) and BOEC plus OP ( n = 325); experiment 2, GC ( n = 514); GC plus OP ( n = 509); BOEC ( n = 490); BOEC plus P4 ( n = 500); BOEC plus P4 and OP ( n = 502). Embryos were checked for cleavage at day 2 and for stage development between days 8 and 12 of culture. In experiment 1, no differences ( P > 0.05) were identified between BOEC and BOECOP groups for embryo rates of development, quality or developmental stages. Also in experiment 2, no differences were found in embryo rates of development, quality or developmental stages between embryos cultured under the two coculture systems when no supplementation was added. Embryo development rates were not affected by OP presence in GCOP group. However, P4 negatively affected Day 8 (D8) embryo development rates in BOEC system (BOECP4 = 16.8 ± 2.6% vs. BOEC = 23.7 ± 1.7%, P = 0.02). This negative effect was abolished when P4 antagonist (OP) was added to the culture medium. BOEC supplementation with P4 also induced a delay on embryo development at D8 as confirmed by a lower development score (BOECP4 = 3.0 ± 1.4 vs. GC = 3.4 ± 0.1, GCOP = 3.5 ± 0.1, BOEC = 3.4 ± 0.1 and BOECP4OP = 3.5 ± 0.1; P < 0.05). These results demonstrate that OP supplementation had no harmful effect on embryo development either in granulosa, where P4 is naturally synthesised, or in BOEC coculture systems. Also we can not confirm a direct association between high P4 concentrations and embryo survival during early stages, although P4 may influence early embryo development through different mechanisms mediated by the type of cells present.

Effect of Porcine and Ovine FSH on Nuclear Maturation of Pig Oocytes In Vitro

The effect of porcine or ovine FSH on the maturation rate of porcine oocytes and on the time course of meiotic progression was studied. Groups of 20 grade-A cumulus oocyte complexes, aspirated from slaughterhouse cycling-gilt ovaries, were cultured in vitro in 400 mul of Modified Parker's Medium supplemented with oestrous cow serum and porcine FSH (Folltropin(R)-V, 0.50 mg/ml) or ovine FSH (Ovagen(TM), 0.44 iu/ml), in four-well dishes under mineral oil, at 38.5 degrees C, 5% CO(2) in humidified air. At the end of each 3-h interval, from 3 to 42 h of culture, the nuclear status of oocytes was assessed microscopically (1000x), after fixation (methanol/acetic acid: 3/1) and orcein (2%) staining. Oocytes were classified as (i) immature (IMM), i.e. oocytes at germinal vesicle stage, germinal vesicle break down and prophase I, (ii) metaphase I (MI) and (iii) metaphase II (MII), i.e. oocytes at anaphase I, telophase I and metaphase II. Data were analysed using regression analysis, chi-square and t-test. Nuclear status was assessed in 1610 oocytes (porcine FSH: 787, ovine FSH: 823). Most of the oocytes were at MI from 24 to 33 h (porcine FSH 60.27%, ovine FSH 42.80%, p < 0.001) and at MII from 36 to 42 h (porcine FSH 80.38%, ovine FSH 67.45%, p < 0.01) of culture. Significantly higher maturation rate was observed in porcine FSH than in ovine FSH treated oocytes (86.69 +/- 12.97%, 71.34 +/- 9.86%, mean +/- SD, p < 0.05), after 42 h of culture. In conclusion, under the specific culture conditions, porcine FSH seems to support pig oocyte maturation better than ovine FSH.

Expression of Hsp-70 and Bax genes in bovine blastocysts derived of oocytes matured in chemically defined medium supplemented with synthetic macromolecules

Analysis of expression patterns of developmentally important genes essential in early developmental provides a useful tool to assess the normality of produced embryos and to optimize ART. The in vitro production of bovine embryos has been shown to be correlated with significant up- or downregulation of genes critical for undisturbed fetal development. Bax and Hsp-70 genes are strongly transcripts in IVP embryos and their products are involved in processes such as apoptosis and stress adaptation, respectively. This study aimed to quantify Hsp-70 and Bax gene expression in bovine blastocysts produced from oocytes matured in chemically defined IVM system. An experimental study using a bovine model was performed. Immature cumulus oocyte complexes from slaughtered cows were matured for 24 h in three treatments: T1) α-MEM + IGF-I + Insulin + 0.1% PVA; T2) α-MEM + IGF-I + Insulin + 0.1% PVP-40; T3) control: TCM + FSH + 10% oestrous cow serum. Twenty two hours after IVF, zygotes were cultured in CR2aa with 10% fetal calf serum in a humidified atmosphere of 5% CO2 at 38.5°C. Blastocysts on day 8 post-fertilization were frozen in liquid nitrogen and thawed for RNA extraction. Total RNA extraction was performed using Rneasy Micro kit (Qiagen, Valencia, CA, USA) and first strand synthesized using Superscript III First Strand Synthesis kit (Invitrogen, Chicago, IL, USA). Relative quantification was performed in duplicate using Real Time PCR (ABI Prism 7000 Applied Biosystem, Foster City, CA, USA) and reactions consisted of a mixture of iTaq SYBR Green Supermix with ROX (Bio-Rad, Waltham, MA, USA) with cDNA equivalent to 0.8 embryos and gene specific primers. Expression of H2a gene was used as endogenous reference. Calculations of relative quantification were performed by REST © (Relative Expression Software Tool), using the value found in TCM group as calibrator. The blastocyst rates in T1, T2, and T3 were 40%, 48% and 38%, respectively. The relative expression of Hsp-70 and Bax transcripts were, respectively, 0.69 ± 0.69 and 0.53 ± 0.32 for T1, and 1.08 ± 0.75 and 0.7 ± 0.47 for T2 in comparison to the calibrator group (T3), with no significant differences between groups (P > 0.05). These results indicate that oocyte maturation in chemically defined media supplemented with PVA or PVP-40, without FCS and gonadotropins, do not alter the relative abundance of Hsp70–1 and Bax transcripts in resulting early embryos.

Effect of Recombinant Bovine Somatotropin (rbST) on Cytoplasmic Maturation of Bovine Oocytes and Their Developmental Competence In Vitro

The objectives of this study were to evaluate the effects of recombinant bovine somatotropin (rbST) on the nuclear and cytoplasmic maturation of bovine oocytes and their further developmental competence to blastocysts in vitro. We analyzed the mitochondrial activity and concentration of intracellular stored calcium ([Ca(2+)](is)) in matured oocytes and the morphology and chromatin status of produced embryos after in vitro fertilization. Cumulus-oocyte complexes were incubated in TCM 199 containing 10% fetal calf serum (control medium 1: CM 1) or 10% estrus cow serum (control medium 2: CM 2). The culture medium of the treatment groups was modified by supplementation of the control medium with 10 ng/ml rbST (CM 1A and CM 2A), 10(6)/ml granulosa cells (CM 1B and CM 2B), or 10 ng/ml rbST plus 10(6)/ml granulosa cells (CM 1C and CM 2C). No differences were observed in the percentages of oocytes reaching metaphase II between the groups. However, the proportion of blastocysts was highest in treatment groups CM 1C and CM 2C (P<0.05). The type of serum did not alter the positive effect of rbST on the developmental competence of embryos. The fluorescence intensity of metabolically active mitochondria measured by intensity per oocyte (Em 570) after MitoTracker CMTM Ros Orange labeling was significantly increased in oocytes matured in the presence of 10 ng/ml rbST and granulosa cells (309.21 vs. 119.97 microA; P<0.01). In parallel, the concentration of [Ca(2+)](is) in oocytes, determined using fluorophore chlortetracycline, was significantly decreased (0.85 +/- 0.02 vs. 0.97 +/- 0.03 AU; P<0.05). Based on these results, we concluded that rbST, in interaction with granulosa cells stimulates the oxidative activity of ooplasmic mitochondria and decreases the content of [Ca(2+)](is) in oocytes. These facts support the hypothesis that somatotropin influences the developmental competence of bovine oocytes during maturation in vitro, and this effect can be modulated by granulosa cells.

Leptin Promotes Meiotic Progression and Developmental Capacity of Bovine Oocytes Via Cumulus Cell-Independent and -Dependent Mechanisms1

Leptin has been shown to exert positive effects during the maturation of bovine oocytes, influencing blastocyst development, apoptosis, and the transcript levels of developmentally important genes. The present study was conducted to characterize further the mechanisms of leptin action on oocytes and the role of cumulus cells (CCs) in this context. In the first series of experiments, cumulus-oocyte complexes (COCs) were matured in serum-free medium that contained 0, 1 or 10 ng/ml leptin or in medium that was supplemented with 10% (v/v) estrus cow serum (ECS). Leptin concentrations of 1 and 10 ng/ml stimulated the meiotic progression of oocytes. Moreover, TUNEL staining demonstrated that these leptin doses reduced the proportion of apoptotic CCs. In the second series of experiments, COCs or denuded oocytes (DOs) were matured in the presence of 0 or 10 ng/ml leptin. The percentages of COCs and DOs with extruded polar bodies were increased by leptin. In contrast, positive effects of leptin on fertilization rates and blastocyst development were only observed after treatment of COCs but not of DOs. Leptin treatment of COCs consistently enhanced blastocyst development even after parthenogenetic activation of oocytes or after the removal of CCs before fertilization. The proportion of polyspermic oocytes was not affected by leptin treatment or oocyte denudation. In the third series of experiments, COCs were matured in the presence of 0, 1 or 10 ng/ml leptin. The transcript levels of specific genes were determined by reverse transcriptase-quantitative PCR (RT-qPCR) analysis of cumulus cells and single oocytes. Leptin treatment increased the levels of FAS, FASLG, and STAT3 transcripts in oocytes, but did not affect the LEPR, BAX, and BIRC4 mRNA concentrations. In cumulus cells, leptin treatment increased the mRNA levels for LEPR, STAT3, BAX, BIRC4, and FAS, but did not alter FASLG mRNA abundance. In conclusion, leptin differentially regulates gene expression in oocytes and cumulus cells. Moreover, leptin enhances both oocyte maturation and developmental capacity via cumulus cell-independent and -dependent mechanisms.

In vitro production of bovine embryos using flow-cytometrically sexed sperm

Abstract. The investigation aimed to compare the effect of fresh and frozen-thawed X and Y fractions of flow-cytometrically sorted bovine spermatozoa on in vitro fertilization of bovine in vitro matured oocytes and subsequent blastocyst development. Sperm cells sorted in MoFloSX cytometer were used either for IVF or frozen and stored in liquid nitrogen. Immature oocytes recovered from ovaries of slaughtered animals and matured in vitro in TCM-199 containing 20% estrus cow serum and additional granulosa cells were fertilized in vitro with fresh or frozen-thawed fractions of sorted sperm. Simultaneously, control, fresh or frozen/thawed sperm was used for IVF. A total number of 2712 IVM oocytes were fertilized with sorted and control sperm of 6 bulls. Embryo cleavage rates were significantly affected by bull (P&lt;0.0001), sperm sexing (P&lt;0.0001) and sperm freezing (P&lt;0.01). Blastocysts development was affected by sperm freezing (P&lt;0.04) and sperm sexing (P&lt;0.01). The significant differences were shown between unsorted and sorted sperm, however no differences in embryo cleavage rates and blastocysts rates were observed between X- and Y-sperm fractions, both fresh and frozen/ thawed. There were significant differences in cleavage rates among fresh, control sperm (52.7%), X fraction (26.8%) and Y fraction (24.7%). Similar differences in cleavage rates were shown for frozen/thawed control sperm (52.8%), X fraction (33.9%) and Y fraction (26.2%). The female blastocysts were frozen for further transfer, while the hatched male blastocysts were analysed by PCR revealing 76.2% accuracy. The results suggest that there were significant differences in cleavage rates and blastocyst rates due to sperm sorting in comparison to unsorted sperm and no differences between effectiveness of X and Y fractions of spermatozoa.

Cultivo de embriões bovinos produzidos in vitro: efeito do número de embriões e da proporção de meio

Na última década, os produtores de leite e carne bovina incrementaram consideravelmente o uso da aspiração folicular (OPU) associada à produção in vitro de embriões (IVP). Oócitos recuperados pela OPU têm qualidade diversificada e seu número reduzido requer condições diferenciadas de cultivo para a IVP. Para determinar o efeito do número de embriões e volume de meio sobre a IVP, 1428 embriões bovinos foram cultivados em grupos de 5, 10 e 20, em 1:1, 1:5 e 1:10mL de meio. Grupos de 20 oócitos foram maturados in vitro em 200mL de TCM-199+ rFSHh+10% de soro de vaca em estro (SVE), por 24h. A fecundação in vitro foi em grupos de 20 oócitos/200mL de meio Fert-Talp, por 18h com 1x10(6) espermatozóides/mL. O cultivo foi em SOFaaci+5% SVE por 8 dias. Considerando-se o dia da fecundação como D0, conduziu-se as avaliações no D2 (clivagem), D7 (blastocistos) e no D9 (eclosão). A taxa de clivagem foi superior quando o cultivo foi com 20 embriões e não foi afetada quando a proporção de meio variou (1:1; 1:5 e 1:10). Com as proporções de meio de cultivo 1:5 ou 1:10, a taxa de embriões em D7 foi superior (P&lt;0.05) à 1:1. O número de embriões cultivados influenciou a produção de blastocistos em D7 (P&lt;0,05) com 20 embriões/gota. Estes resultados também se refletem nos percentuais de blastocistos eclodidos em D9, sendo que 1:5 e 1:10 foram superiores (P&lt;0,05) à 1:1. Da mesma forma, a taxa de eclosão foi superior (P&lt;0,05) com o cultivo de 10 ou 20 embriões/gota quando comparado ao grupo de 5 embriões/gota. A redução do número de embriões e da proporção de volume de meio no cultivo, afeta os índices de desenvolvimento na produção in vitro de embriões bovinos.

Gene Expression and Developmental Competence of Bovine Embryos Produced In Vitro with Different Serum Concentrations

The objective of this study was to determine the developmental rates and relative abundance of Hsp 70.1 and Glut-1 transcripts in in vivo- and in vitro-produced (IVP) bovine embryos in media supplemented with bovine serum albumin (BSA) or different oestrous cow serum concentrations. In experiment 1, in vitro maturation and culture media were supplemented with 0.4% BSA or 1, 5, 10 or 20% of oestrous cow serum (ECS). The analysis of the expression of Hsp 70.1 and Glut-1 was carried out in individual days 7 and 8 embryos by a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay. In experiment 2, in vivo-produced morulae were collected on day 7 of the oestrous cycle and employed for the comparison of the relative abundances of Hsp 70.1 and Glut-1 transcripts with IVP morulae produced using two protein sources (10% ECS or 0.4% BSA). No differences were observed in cleavage rate among groups, but blastocyst formation (27%) and hatching rates (78%) were significantly higher in IVP embryos produced with 20% ECS than the other groups (p<0.05). No significant differences were observed in the relative abundances of Hsp 70.1 and Glut-1 mRNA in days 7 and 8 blastocysts expanded blastocysts between groups. The abundances of mRNA for those genes were similar between IVP and in vivo-produced morulae. In spite of the alterations observed in embryonic development, the presence of serum at distinct concentrations did not appear to alter the relative abundance profiles of Hsp 70.1 and Glut-1 compared with controls or the BSA supplementation to the IVP media.

267 TARGETED SUPPRESSION OF THE EXPRESSION OF MATERNAL AND EMBRYONIC GENES DURING IN VITRO DEVELOPMENT OF BOVINE EMBRYOS

Despite enormous advances in the identification and sequencing of developmentally relevant bovine genes, the function of the majority of these transcripts is not yet known. Here we aimed to apply the RNA interference (RNAi) approach to suppress the expression of the maternal transcript c-mos (AY630920) and embryonic transcripts E-cadherin (AY508164) and Oct-4 (AY490804) during in vitro development of bovine embryos using microinjection of sequence-specific double-stranded RNA (dsRNA). For this 435-, 341- and 341-bp-long dsRNA specific to the coding sequences of c-mos, E-cadherin and Oct-4 transcripts, respectively, were synthesized using Promega RiboMax" T7 system (Promega, Madison, WI, USA), where sense and antisense strands were transcribed from the target DNA template. Slaughterhouse ovaries were used to aspirate bovine oocytes, which were matured in TCM-199 with 12% estrus cow serum (ECS), fertilized in Fert-TALP, and cultured in CR1 medium at 39�C under humidified atmosphere of 5% CO2 in air. In Experiment 1, immature oocytes were categorized into three groups, each containing 50-60 oocytes: those injected with c-mos dsRNA, those injected with RNase-free water, and uninjected controls. In Experiment 2, zygotes were categorized into four groups, each containing 50-60 zygotes: those injected with E-cadherin dsRNA, those injected with Oct-4 dsRNA, those injected with RNase-free water, and uninjected controls. Each experiment was repeated four times. The effect of dsRNA on in vitro development of oocytes or embryos was assessed after microinjection during culture. The level of mRNA and protein expression was investigated using real-time PCR and western blot analysis, respectively. Data were analyzed using SAS, version 8 (SAS Institute Inc., Cary, NC, USA). Microinjection of c-mos dsRNA resulted in a 70% reduction of c-mos transcript abundance after maturation compared to the water-injected and uninjected controls (P &lt; 0.05). Similarly, microinjection of E-cadherin and Oct-4 dsRNA at the zygote stage resulted in 80% and 60% reduction in transcript abundance at the blastocyst stage, respectively, compared to the uninjected controls (P &lt; 0.05). Decreases in the c-mos (39 kDa) and E-cadherin proteins (119 kDa) were observed in the c-mos and E-cadherin dsRNA-injected groups, respectively, compared to the control. A higher proportion of oocytes (75%) showed first polar body extrusion after maturation in c-mos dsRNA-injected groups, compared to 52% in water-injected and 57% in uninjected controls. Only 22% from E-cadherin dsRNA- and 24% from Oct-4 dsRNA-injected zygotes developed to the blastocyst stage compared to 39 and 37% blastocyst rates in water-injected and uninjected control groups, respectively. In conclusion, injection of sequence-specific dsRNA in bovine oocytes and embryos resulted in suppression of mRNA and their protein products, thereby affecting in vitro development of bovine embryos.

236 SPATIAL GENE EXPRESSION PATTERNS OF Dnmt1, Dnmt3a, AND Hdac2 IN PREIMPLANTATION EMBRYOS

In mammals, DNA methylation and the modification of histones account for the major epigenetic alterations. Usually DNA methylation is associated with transcriptional silencing, which is catalyzed by two important classes of DNA methyltransferases. DNA methyltransferase 1 (Dnmt1), a maintenance enzyme, methylates hemi-methylated DNA after DNA replication. Dnmt3a and Dnmt3b are required for de novo methylation in vivo and for establishing new DNA methylation marks during development. In addition to DNA methylation, post-translational modifications of the amino termini of core histones are thought to affect the expression or repression of transcription. Histone deacetylation catalyzsed by histone deacetylases (Hdac) often results in transcriptional repression. Perturbated epigenetic reprogramming of the genome is a likely cause of developmental abnormalities and epigenetic diseases introduced by assisted reproduction technologies. The objective of the present study was to determine the relative abundance of Dnmt1, Dnmt3a, and Hdac2 transcripts in ICM and TE cells of pre-implantation bovine embryos of different origin. Embryos were generated with standard protocols of in vitro production (IVP) and parthenogenetic activation using SOF medium supplemented with BSA (SOF d7/d8, Parth d7/d8) or TCM medium supplemented with estrus cow serum (TCM d7/d8; Wrenzycki et al. 2001 Hum. Reprod. 16, 893–901; Wrenzycki et al. 2002 Biol. Reprod. 66, 127–134). Expanded blastocysts were collected at Days 7 and Day 8 (d7/d8) of culture (Day 0 = IVF). In vivo-generated blastocysts collected from superovulated animals were included as controls (Wrenzycki et al. 1996 J. Reprod. Fert. 108, 17–24). A highly sensitive RT-PCR assay (Wrenzycki et al. 2003 Biol. Reprod. 68, 2073–2080) was used to determine the relative abundance (RA) of gene transcripts in isolated ICM and TE cells. The allocation of ICM and TE cells was analyzed using a differential staining technique (Eckert and Niemann 1998 Mol. Hum. Reprod. 4, 957–965) to calculate the RA of each transcript on a per cell basis. RT-PCR assays were repeated at least six times. Significant differences in the RA of Dnmt1 (In vivo, TCM d7, Parth d7/d8, SOF d8), Dnmt3a (In vivo, SOF d7), and Hdac2 (TCM d7) transcripts were detected between ICM and TE cells. No differences were detected for all transcripts in ICM cells of embryos collected at either Day 7 or Day 8. In TE cells, the RA of Dnmt1 transcripts was significantly reduced or increased in embryos generated in SOF or TCM medium at Day 7, respectively, and the RA of Hdac2 transcripts was significantly higher in embryos generated in TCM medium at Day 8. These results suggest that in vitro culture alters the spatial expression pattern of genes related to epigenetic modifications in the cell lineages critical for a normal embryonic and fetal development. This work was supported by the Deutsche Forschungsgemeinschaft (DFG Ni 256/18-1).

272 DEVELOPMENTAL COMPETENCE OF OOCYTES OBTAINED FROM BOS TAURUS AND BOS INDICUS DAIRY COWS RAISED IN A TROPICAL CLIMATE

The effects of heat stress on Bos taurus reproductive performance in tropical and subtropical regions are well known, and have been associated with lower oocyte developmental capacity. The aim of this study was to evaluate the developmental competence of oocytes from Bos taurus (Holstein) and Bos indicus (Gyr) dairy cows raised in a Brazilian tropical region, located at 21°35′S latitude, 43°51′W longitude, and 435 meters altitude. Cumulus–oocyte complexes (COCs) were recovered by oocyte pickup (OPU) from mature non-lactating Holstein (n = 9) and Gyr (n = 13) donor cows between the end of spring and the beginning of autumn, with at least two OPU sessions/cow. COCs were in vitro-maturated in TCM-199 (GIBCO, Grand Island, NY, USA) with 10% inactivated estrus cow serum for 24 h under 5% CO2 at 38.5°C in air. Bos taurus and Bos indicus semen with similar cleavage rates, previously evaluated by in vitro fertilization with oocytes obtained from slaughterhouse ovaries, were used to reduce bull effect. Holstein and Gyr spermatozoa were obtained through swim-up method and co-incubated with Holstein (n = 390) and Gyr (n = 505) oocytes, respectively, in Fert-TALP medium (Parrish et al. 1988 Biol. Reprod. 38, 1171–1180) supplemented with 10 μg/mL heparin (Sigma-Aldrich, Sao Paulo, Brazil) and 6 mg/mL fatty acid-free bovine albumin (Sigma) for 18 h in 5% CO2 at 38.5°C in air. Presumptive zygotes were co-cultured with their own cumulus cells in CR2aa medium (Wilkinson et al. 1996 Theriogenology 45, 41–49) supplemented with 10% fetal calf serum in humid atmosphere of 5% CO2 at 38.5°C in air. On Day 7 to 8 of co-culture, Gyr and Holstein blastocysts were assessed and those classified as grade 1 (IETS Manual) were transferred to synchronized Bos indicus × Bos taurus crossbred recipients managed under the same nutritional and environmental conditions. Pregnancy diagnosis was performed between 35 and 50 days after estrus. Cleavage, blastocyst, and pregnancy rates were analyzed by chi-square test. Cleavage and blastocyst rates were greater (P &lt; 0.05) in Gyr than in Holstein (66.7% vs. 53.1% for cleavage and 19.6% vs. 10.8% for blastocyst, respectively), but the pregnancy rate was similar (P &gt; 0.05; 44.5% vs. 60% for Gyr and Holstein, respectively). These results show that Gyr oocytes obtained in a tropical region have greater developmental capacity than Holstein oocytes, suggesting an interaction between genotype and environment that influences the success of an in vitro embryo production program; nevertheless, the blastocyst viability after transferring to recipients is similar for both breeds.

Produção in vitro de embriões bovinos com soro de égua ou de vaca em estro com ou sem a adição de LH/FSH

Mil duzentos e setenta e um oócitos foram divididos em 4 tratamentos com a finalidade de se avaliar a influência da adição de LH e FSH na produção in vitro de embriões bovinos com soro de vaca em estro (SVE) ou soro de éguas obtido no 1º dia do estro (SE). Independente do tratamento os oócitos foram maturados com TCM199 + 5,95mg/ml de Hepes, 0,025mg/ml de piruvato de sódio e 2,2mg/ml de bicarbonato de sódio, sendo adicionado 10% de soro de égua (ES), 10% de SVE (VS), 10% de soro de égua + 0,5mg/ml de hormônio luteinizante bovino (LHb) + 0,01UI/ml de hormônio folículo estimulante recombinante humano-rFSHh (EH) e 10% SVE + LHb + rFSHh (VH). Os oócitos assim tratados, foram maturados em estufa com 5% de CO2 a 39ºC sob umidade saturada por 22-24h. Depois, foram fecundados em TALP-FERT por 18-20h e cultivados por 8 dias em meio SOF + 5% de SE (ES e EH) ou SVE (VS e VH). As taxas de clivagem de 72% obtidas no grupo VH (229/316) e 61% no grupo VS (193/315) foram significativamente menores (p&lt;0,05) que as dos grupos ES (80% - 254/317) e EH (80% - 257/323). A produção de embriões (blastocistos iniciais, blastocistos, blastocistos expandidos e eclodidos) no D7 após a inseminação para os grupos ES (32%), EH (28%) e VH (27%), foi significativamente superior aos 20% obtidos no VS (p&lt;0,05). No D9, verificou-se uma diferença significativa (p&lt;0,05) entre os grupos ES (31%) e EH (29%) quando comparados com o grupo VS (22%), mas não houve diferença quando comparamos com o VH (24%). A taxa de eclosão em D9 foi de 11% (ES), 11% (EH), 10% (VS) e 5% (VH). Não houve diferença entre as médias do número de células dos embriões (Blastocistos expandidos e eclodidos) obtidos no D9. Conclui-se, com estes resultados que, para a obtenção de taxas regulares de blastocistos, não seja necessária a adição de hormônios quando se utilize o soro de égua, e que os hormônios devam ser adicionados quando se utilize soro de vaca.

Transporte de oócitos bovinos em meio de maturação sem controle de atmosfera gasosa

Oócitos (n=1177) bovinos obtidos da aspiração de folículos com diâmetro entre 2 e 8mm, de ovários de matadouro foram divididos aleatoriamente em quatro tratamentos com 11 repetições. Os oócitos foram maturados por 24h em TCM-199 Sais de Earle, acrescido de 25mM de bicarbonato de sódio, 25mM de HEPES, rFHS-h, Soro de Vaca em Estro (SVE) e piruvato, em estufa a 39ºC, com 5% de CO2 em ar e umidade saturada (Grupo Controle, n=296) ou, submetidos ao transporte simulado por 6 (T6, n=286), 12 (T12, n=294) ou 18h (T18, n=301) em meio de maturação TCM+HEPES, em banho-maria a 39ºC, com os mesmos componentes utilizados para o Grupo Controle, porém com apenas 1mM de bicarbonato. Decorrido cada período de transporte, os mesmos foram transferidos para placas com meio de maturação, completando o período de 24h em estufa, nas mesmas condições do Grupo Controle. O período de fecundação foi de 18h em condições semelhantes de temperatura e atmosfera gasosa, em FERT-TALP acrescido de heparina, sendo a dose inseminante de 1x106 espermatozóides/mL, selecionados por migração ascendente. Os prováveis zigotos foram cultivados em meio SOF + 5% SVE por 8 dias, em estufa a 39ºC, em bolsas gaseificadas com 5% CO2, 5% O2 e 90% N2. Na avaliação da clivagem, não houve diferença entre os tratamentos. As taxas de desenvolvimento embrionário no dia 7 foram semelhantes para os grupos Controle (20,9%), T6 (19,2%) e T12 (21,4%), com uma redução (P&lt;0,05) observada no grupo T18 (12,3%), em relação aos grupos Controle e T12. No dia 9, o T18 apresentou uma menor (P&lt;0,05) produção de blastocistos expandidos mais eclodidos, em relação aos demais grupos, embora não tenha sido observada diferença (P&gt;0,05) na taxa de eclosão. O número médio de células dos blastocistos eclodidos não diferiu (P&gt;0,05) entre os grupos Controle (136), T6 (125,5) e T12 (126,8). Esses resultados indicam a possibilidade do transporte de oócitos bovinos em meio de maturação TCM+HEPES, sem controle da atmosfera gasosa, a 39ºC, pelo período de até 12h. Esta técnica oferece uma alternativa prática e eficiente para o transporte dos oócitos bovinos destinados à produção in vitro de embriões bovinos (PIV).