Embryonic chimerism is generally used in basic research and in vivo diagnosis of undifferentiated... more Embryonic chimerism is generally used in basic research and in vivo diagnosis of undifferentiated embryonic stem cells (ESC), mostly using mice embryos, although there have been reports in the literature on using rat, rabbit, sheep, chicken, primate, bovine, goat and pig embryos. Several techniques can currently be used to produce chimeric embryos, including microinjection, co-culture with ESC, fusion and aggregation. Although microinjection is the most commonly used method in mice, the mere aggregation of embryos with ESC may result in viable chimeras and be as efficient as microinjection. In mice, this chimerism technique has been shown to have the advantage of aggregating embryos in different stages of development with different ploidy, in addition to using ESC in the tetraploid complementation assay. Compared to other techniques for producing chimeras, the aggregation technique is a cheaper, faster and easier methodology to be performed. Moreover, aggregation can be simplified b...
Recent work with P-36 demonstrates that the replacement of the last two doses of Follicle-Stimula... more Recent work with P-36 demonstrates that the replacement of the last two doses of Follicle-Stimulating Hormone (FSH) with equine chorionic gonadotropin (eCG) increases embryo yields. However, it is unclear if the positive effect of eCG is related to its FSH-like activity, LH-like activity, or both. This study aimed to verify the replacement of eCG with pLH on the last day of superstimulatory treatment. Twenty-five Nelore cows were allocated to four groups: P-36 (control), P-36/eCG, P-36/LH2, and P-36/LH4. All animals underwent four treatments in a crossover design. The control group cows were superstimulated with decreasing doses of porcine Follicle-Stimulating Hormone (pFSH, 133 mg, im). In the P-36/eCG, P-36/LH2, and P-36/LH4 groups, the last two doses of pFSH were replaced in the former group by two doses of eCG (200 IU each dose, im) and in the latter two groups by two doses of pLH (1 and 2 mg each dose, im), respectively. Donors received fixed-time artificial insemination 12 and...
Bovine follicular growth occurs in a wave pattern of two or three waves per estrous cycle. These ... more Bovine follicular growth occurs in a wave pattern of two or three waves per estrous cycle. These waves are characterized by the growth of a synchronous group of antral follicles from which usually only one becomes dominant. The number of recruited follicles per wave is variable among animals and breeds, but it shows high repeatability within individuals. Ereno et al. (2013) reported that the number of follicles recruited per wave is higher in zebu cattle when compared to taurine. This variation in the ovarian follicular population can interfere with fertility by affecting oocyte competence (Ireland et al., 2007). The objective of this study was to identify Aberdeen Angus cows (Bos t. taurus) of high and low antral follicle count (AFC) recruited per follicular wave and compare their pregnancy rate. We used multiparous cows (272) between 40 and 70 days postpartum and body condition score between 2.5 to 4.0 (5-point scale).The cows were divided into groups according to the antral folli...
In the female reproductive system, angiotensin II (ANG II) is a potential signaling molecule invo... more In the female reproductive system, angiotensin II (ANG II) is a potential signaling molecule involved in ovarian follicle development, which acts through two transmembrane receptors. Within the ovarian follicle, there appear to be species differences in the precise pattern of localization of AGTR2 protein and it has an important role in in vitro maturation of oocytes in mammals. The infusion of ANG II induced ovulation in rabbits and the use of ANG II antagonists inhibited ovulation in rabbits, rats, and cattle. In fetal ovaries, AGTR2 protein was detected in ovigerous cords and preantral follicles throughout porcine and bovine gestation. In the oviduct, ANG II is responsible for the orchestration of the transport of gametes. In the male reproductive system, there is considerable evidence for the local synthesis of components of renin-angiotensin system (RAS) in male reproductive tissues. The roles of RAS in local processes at these sites are still uncertain, although there is evide...
In the female reproductive system, angiotensin II (ANG II) is a potential signaling molecule invo... more In the female reproductive system, angiotensin II (ANG II) is a potential signaling molecule involved in ovarian follicle development, which acts through two transmembrane receptors. Within the ovarian follicle, there appear to be species differences in the precise pattern of localization of AGTR2 protein and it has an important role in in vitro maturation of oocytes in mammals. The infusion of ANG II induced ovulation in rabbits and the use of ANG II antagonists inhibited ovulation in rabbits, rats, and cattle. In fetal ovaries, AGTR2 protein was detected in ovigerous cords and preantral follicles throughout porcine and bovine gestation. In the oviduct, ANG II is responsible for the orchestration of the transport of gametes. In the male reproductive system, there is considerable evidence for the local synthesis of components of renin-angiotensin system (RAS) in male reproductive tissues. The roles of RAS in local processes at these sites are still uncertain, although there is evide...
MicroRNAs (miRNAs) are small noncoding RNA molecules, composed by 19-25 base pair that control th... more MicroRNAs (miRNAs) are small noncoding RNA molecules, composed by 19-25 base pair that control the expression of important genes in physiological processes, including reproduction. Among these miRNAs is found miR-222 that is related to cellular development, reproductive system development and function and possibly in regulating of luteinizing hormone receptor (LHR) in cattle (Hossain et al., 2009) .Superovulation with exogenous gonadotropins affects oocyte and embryo quality as well granulosa cells differentiation. The LHR expression in bovine granulosa and theca cells is crucial for the transition of FSH/LH-dependency in antral follicles and previous data showed that ovarian superstimulation with FSH increases LHR mRNA abundance in bovine granulosa cells. Thus, the aim of present study was investigate the effects of superstimulation with FSH or FHS combined with eCG on the Bos taurus miR-222 (bta-mir-222) abundance on granulosa and theca cells from Nelore cows (Bos taurus indicus)....
The follicular growth in cattle occurs in a wave pattern of 2 to 3 waves per estrous cycle, and i... more The follicular growth in cattle occurs in a wave pattern of 2 to 3 waves per estrous cycle, and is characterized by synchronous growth of a cohort of antral follicles, from which usually only one of these will become dominant. The amount of recruited follicles per wave is variable among animals and breeds, but is highly repeatable among individuals. This variation in ovarian follicular population can interfere with fertility by influencing oocyte competence (Ireland et al., 2007). We aimed to identify Nelore (Bos taurus indicus) cows with high and low antral follicle count (AFC) and to compare their pregnancy rates. We evaluated 268 multiparous Nelore cows from 40 to 70 days postpartum and body condition score between 3.5 to 4.5 (5-point scale). The cows were divided into groups according to the antral follicle count (≥ 3 mm in diameter). Hence, 33% of animals with the greater AFC were enrolled in high population group (HG, n=89, >38 follicles), while the intermediate animals (33...
Oocytes resume meiosis spontaneously when subjected to in vitro maturation (IVM). Cyclic adenosin... more Oocytes resume meiosis spontaneously when subjected to in vitro maturation (IVM). Cyclic adenosine monophosphate (cAMP) inductors have been used for artificial blocking of meiotic resumption to improve developmental competence of IVM oocytes. We tested a prematuration system that uses forskolin (FSK) and 3- isobutyl-1-methylxanthine (IBMX) to maintain higher concentrations of cAMP in the oocyte and sustain transzonal projections with gap junction communication within the cumulus-oocyte complex (COC). This system has been proved to prevent spontaneous meiosis resumption that usually occurs in the conventional IVM systems. Lipid accumulation in oocytes and IVP-embryos has been related to lower developmental potential and cryopreservation inability, especially when compared to their in vivo counterparts. We aimed to evaluate the effect of the in vitro prematuration system in the rates of blastocyst and on embryo quality by the semi-quantitative lipid content in oocytes and blastocysts ...
Follicular growth in cattle occurs in a wave pattern varying from 2 to 3 waves during each estrou... more Follicular growth in cattle occurs in a wave pattern varying from 2 to 3 waves during each estrous cycle, and is characterized by the initial development of a synchronous group of antral follicles, from which only one will become dominant. The number of recruited follicles is variable among individuals and breeds; however, it is highly repeatable in individuals. Literature reports indicate that the number of follicles recruited in each wave is higher in zebu breeds compared with taurine. A relationship between the follicular population and the oocyte competence has been suggested, and ultimately the follicular population could influence the fertility of the animal (Ireland et al., 2007, Human Reproduction 22:1687-1695). The objectives of this study were: i) to identify females in Nelore (Bos indicus) and Aberdeen Angus (Bos taurus) breeds that have high and low population of recruited antral follicles per wave and ii) to characterize, in both breeds, the differences in lipid profile...
Reproduction in mammals depends on the pulsatile release of the gonadotropins LH and FSH, which a... more Reproduction in mammals depends on the pulsatile release of the gonadotropins LH and FSH, which act together to regulate the function of the gonads. In the ovaries, LH influences follicular development until ovulation and this depends on the interaction with its receptor (LHR). However, there is no consensus regarding the use of LH during the in vitro maturation (IVM) of cumulus-oocyte complexes (COC) in cattle. Reports in the literature differ on mRNA expression of LHR in oocytes and cumulus oophorus cells (CC). Thus, the aim of this study was to characterize the expression of the LHR gene in CC and oocytes from bovine follicles at different diameters and after IVM. Ovaries were collected at a commercial abattoir and follicles were dissected and separated into two groups for experiment 1, G1: 3 to 6mm and G2: >6 to 10mm. On a Petri dish, follicles were individually ruptured with 18G needles in order to obtain their respective COC. Pools of 25 COC were used per experimental group...
Superstimulation can affect cellular and molecular events in ovarian follicular cells, cumulus-oo... more Superstimulation can affect cellular and molecular events in ovarian follicular cells, cumulus-oocyte complexes (COCs), oviduct and embryo, as well as cell-to-cell communication through secretion and uptake of exosomes (Exo). The aim of this study was to investigate the effects of the extracellular vesicles (specifically, Exo), recovered from follicular fluid (FF), on the oocyte meiosis progression and apoptosis; and also on gene expression of COCs and blastocysts. Exo were recovered by ultracentrifugation and filtration of the FF from Nelore cows submitted to ovarian superstimulation with FSH (Exo/FSH); with FSH combined to eCG (Exo/eCG) or from non-superstimulated cows (Exo/NS). The presence of Exo was confirmed by Transmission Electron Microscopy. COCs from slaughterhouse were in vitro matured (IVM) for 22-24h with TCM199 medium enriched with 10% Exo from each group (Exo/FSH, Exo/eCG or Exo/NS) or without Exo (Control). IVM-oocytes were assessed for meiosis progression (Hoescht-3...
The Insulin-like Growth Factor (IGF) system has been demonstrated as an important factor to cumul... more The Insulin-like Growth Factor (IGF) system has been demonstrated as an important factor to cumulus oocyte complex (COC) maturation and early embryo development. Studies demonstrate benefits of IGF-I addition during the in vitro maturation of oocytes in different species, including cattle. The IGF availability is controlled by two mechanisms: IGF Binding Proteins (IGFBPs), which inhibit IGF actions; and Pregnancy-Associated Plasma ProteinA (PAPP-A), responsible to cleavage the IGFBP and to release free IGF. The aim of this study was to investigate the effects of PAPP-A on cellular aspects of in vitro-matured COCs and further, to explore their impacts in the blastocysts’ yield and pattern of transcription. Bovine COCs (5 replicates/20 COC per replicate) from ovaries obtained in a local abattoir, predominantly from Nelore cows, were aspirated and matured in serum free medium in the absence (control group) or presence of 1 ng/mL (P1 group), 10 ng/mL (P10 group) or 100 ng/mL (P100 group...
Antral follicle count (AFC) was phenotypically and genetically evaluated in a Bos taurus cow popu... more Antral follicle count (AFC) was phenotypically and genetically evaluated in a Bos taurus cow population. A total of 257 Aberdeen Angus cows underwent an ovarian ultrasound evaluation and hormonal synchronization for timed artificial insemination (TAI). These animals were between 40 and 70 d postpartum with a body score condition (BSC) between 2 and 4 (average = 2.7) on a scale of 1–5. The number of antral follicles was assessed 10 d before (D-10) the beginning of TAI. Visible follicles (≥3 mm in diameter) were counted from both ovaries (right and left) to determine the number of total follicles, and animals were allocated into groups according to the number of antral follicles: low AFC was defined as ≤ 15 follicles (n = 93); intermediate AFC as 16 follicles (n = 97); and high AFC as ≥ 25 follicles (n = 67). The comparison between the average AFC and pregnancy rate was performed using the chi-square test in the Minitab® 19.2 statistical software, adopting a P-value ≤ 0.05. Hair sampl...
Aggregation is one of the main techniques used to obtain embryonic chimeras. This procedure can b... more Aggregation is one of the main techniques used to obtain embryonic chimeras. This procedure can be performed with whole or demi-embryos, in different stages of development and produced by in vivo or in vitro systems. However, aggregation efficiency tends to be reduced when using embryos in advanced stages (e.g. morulae and blastocysts). The aim of this work was to evaluate the effect of the agglutinating agent phytohemagglutinin-L (PHA) in the percentage of chimeras produced with in vitro-produced (IVP) bovine embryos. Cumulus–oocyte complexes (COC; 445; quality I and II) were matured in drops of 90 μL of TCM-199 bicarbonate supplemented with 10% of FCS and incubated for 22 to 24 h. Fertilization was performed in TALP-IVF medium for 18 h. Presumptive zygotes were transferred to SOF medium for in vitro culture. Incubation conditions were 38.5°C and 5% CO2 in air. To conduct the manual bisection, embryos were placed into 3-μL microdrops of protein-free HEPES-buffered SOF medium. The bisection was executed with a microblade (Ultra-Sharp Splitting Blade, Bioniche, Bogart, GA, USA) under stereomicroscope (35× magnification). Half-structures were joined and transferred to an embryo reconstruction plate, where they were kept for 3 min in drops containing 500 μg mL–1 phytohemagglutinin-L, before the approximated pairs were transferred to SOF medium in cell aggregation well-of-the-well (WOW) micro-wells to in vitro culture. The structures were randomly allocated and the aggregation was performed between 2 whole (zona free) 8- to 16-cell stage embryos to construct aggregated chimeras in the presence [group (G)1, n = 32] or absence of PHA (G2, n = 34) and between demi-morula and demi-blastocyst with PHA (G3, n = 28) or without (G4, n = 29). The aggregation of structures was evaluated after 24 h. Aggregation rates among the 4 experimental groups and the main effects were analysed by Chi-square or Fisher’s exact test and significance was considered when P < 0.05. Embryo aggregation was higher in group G1 than G2 (75.0 and 50.0%, respectively; P = 0.045). Aggregation rate of demi-embryos was similar either in the presence (G3, 39.3%) or in the absence of PHA (G4, 20.7%; P = 0.16). The presence of PHA significantly increased the aggregation rates of the whole pre-compaction embryos (G1) compared with G3 (75.0 and 39.3%, respectively; P < 0.01). The use of PHA resulted in higher aggregation rates (58.3%) than non-use (36.5%; P = 0.03), whereas the embryonic stage of pre-compaction development (G1+G2) produced a higher rate of aggregation (62.1%) than post-compaction demi-embryos (G3+G4, 29.8%; P < 0.001). We could infer a positive effect of PHA on the aggregation rate of bovine IVP embryos only to the 8- to 16-cell stage of development. Financial support: FAPESP, Brazil (06/06491-2, 07/07705-9, 09/10679-5, and 09/04888-0).
Embryonic chimerism is generally used in basic research and in vivo diagnosis of undifferentiated... more Embryonic chimerism is generally used in basic research and in vivo diagnosis of undifferentiated embryonic stem cells (ESC), mostly using mice embryos, although there have been reports in the literature on using rat, rabbit, sheep, chicken, primate, bovine, goat and pig embryos. Several techniques can currently be used to produce chimeric embryos, including microinjection, co-culture with ESC, fusion and aggregation. Although microinjection is the most commonly used method in mice, the mere aggregation of embryos with ESC may result in viable chimeras and be as efficient as microinjection. In mice, this chimerism technique has been shown to have the advantage of aggregating embryos in different stages of development with different ploidy, in addition to using ESC in the tetraploid complementation assay. Compared to other techniques for producing chimeras, the aggregation technique is a cheaper, faster and easier methodology to be performed. Moreover, aggregation can be simplified b...
Recent work with P-36 demonstrates that the replacement of the last two doses of Follicle-Stimula... more Recent work with P-36 demonstrates that the replacement of the last two doses of Follicle-Stimulating Hormone (FSH) with equine chorionic gonadotropin (eCG) increases embryo yields. However, it is unclear if the positive effect of eCG is related to its FSH-like activity, LH-like activity, or both. This study aimed to verify the replacement of eCG with pLH on the last day of superstimulatory treatment. Twenty-five Nelore cows were allocated to four groups: P-36 (control), P-36/eCG, P-36/LH2, and P-36/LH4. All animals underwent four treatments in a crossover design. The control group cows were superstimulated with decreasing doses of porcine Follicle-Stimulating Hormone (pFSH, 133 mg, im). In the P-36/eCG, P-36/LH2, and P-36/LH4 groups, the last two doses of pFSH were replaced in the former group by two doses of eCG (200 IU each dose, im) and in the latter two groups by two doses of pLH (1 and 2 mg each dose, im), respectively. Donors received fixed-time artificial insemination 12 and...
Bovine follicular growth occurs in a wave pattern of two or three waves per estrous cycle. These ... more Bovine follicular growth occurs in a wave pattern of two or three waves per estrous cycle. These waves are characterized by the growth of a synchronous group of antral follicles from which usually only one becomes dominant. The number of recruited follicles per wave is variable among animals and breeds, but it shows high repeatability within individuals. Ereno et al. (2013) reported that the number of follicles recruited per wave is higher in zebu cattle when compared to taurine. This variation in the ovarian follicular population can interfere with fertility by affecting oocyte competence (Ireland et al., 2007). The objective of this study was to identify Aberdeen Angus cows (Bos t. taurus) of high and low antral follicle count (AFC) recruited per follicular wave and compare their pregnancy rate. We used multiparous cows (272) between 40 and 70 days postpartum and body condition score between 2.5 to 4.0 (5-point scale).The cows were divided into groups according to the antral folli...
In the female reproductive system, angiotensin II (ANG II) is a potential signaling molecule invo... more In the female reproductive system, angiotensin II (ANG II) is a potential signaling molecule involved in ovarian follicle development, which acts through two transmembrane receptors. Within the ovarian follicle, there appear to be species differences in the precise pattern of localization of AGTR2 protein and it has an important role in in vitro maturation of oocytes in mammals. The infusion of ANG II induced ovulation in rabbits and the use of ANG II antagonists inhibited ovulation in rabbits, rats, and cattle. In fetal ovaries, AGTR2 protein was detected in ovigerous cords and preantral follicles throughout porcine and bovine gestation. In the oviduct, ANG II is responsible for the orchestration of the transport of gametes. In the male reproductive system, there is considerable evidence for the local synthesis of components of renin-angiotensin system (RAS) in male reproductive tissues. The roles of RAS in local processes at these sites are still uncertain, although there is evide...
In the female reproductive system, angiotensin II (ANG II) is a potential signaling molecule invo... more In the female reproductive system, angiotensin II (ANG II) is a potential signaling molecule involved in ovarian follicle development, which acts through two transmembrane receptors. Within the ovarian follicle, there appear to be species differences in the precise pattern of localization of AGTR2 protein and it has an important role in in vitro maturation of oocytes in mammals. The infusion of ANG II induced ovulation in rabbits and the use of ANG II antagonists inhibited ovulation in rabbits, rats, and cattle. In fetal ovaries, AGTR2 protein was detected in ovigerous cords and preantral follicles throughout porcine and bovine gestation. In the oviduct, ANG II is responsible for the orchestration of the transport of gametes. In the male reproductive system, there is considerable evidence for the local synthesis of components of renin-angiotensin system (RAS) in male reproductive tissues. The roles of RAS in local processes at these sites are still uncertain, although there is evide...
MicroRNAs (miRNAs) are small noncoding RNA molecules, composed by 19-25 base pair that control th... more MicroRNAs (miRNAs) are small noncoding RNA molecules, composed by 19-25 base pair that control the expression of important genes in physiological processes, including reproduction. Among these miRNAs is found miR-222 that is related to cellular development, reproductive system development and function and possibly in regulating of luteinizing hormone receptor (LHR) in cattle (Hossain et al., 2009) .Superovulation with exogenous gonadotropins affects oocyte and embryo quality as well granulosa cells differentiation. The LHR expression in bovine granulosa and theca cells is crucial for the transition of FSH/LH-dependency in antral follicles and previous data showed that ovarian superstimulation with FSH increases LHR mRNA abundance in bovine granulosa cells. Thus, the aim of present study was investigate the effects of superstimulation with FSH or FHS combined with eCG on the Bos taurus miR-222 (bta-mir-222) abundance on granulosa and theca cells from Nelore cows (Bos taurus indicus)....
The follicular growth in cattle occurs in a wave pattern of 2 to 3 waves per estrous cycle, and i... more The follicular growth in cattle occurs in a wave pattern of 2 to 3 waves per estrous cycle, and is characterized by synchronous growth of a cohort of antral follicles, from which usually only one of these will become dominant. The amount of recruited follicles per wave is variable among animals and breeds, but is highly repeatable among individuals. This variation in ovarian follicular population can interfere with fertility by influencing oocyte competence (Ireland et al., 2007). We aimed to identify Nelore (Bos taurus indicus) cows with high and low antral follicle count (AFC) and to compare their pregnancy rates. We evaluated 268 multiparous Nelore cows from 40 to 70 days postpartum and body condition score between 3.5 to 4.5 (5-point scale). The cows were divided into groups according to the antral follicle count (≥ 3 mm in diameter). Hence, 33% of animals with the greater AFC were enrolled in high population group (HG, n=89, >38 follicles), while the intermediate animals (33...
Oocytes resume meiosis spontaneously when subjected to in vitro maturation (IVM). Cyclic adenosin... more Oocytes resume meiosis spontaneously when subjected to in vitro maturation (IVM). Cyclic adenosine monophosphate (cAMP) inductors have been used for artificial blocking of meiotic resumption to improve developmental competence of IVM oocytes. We tested a prematuration system that uses forskolin (FSK) and 3- isobutyl-1-methylxanthine (IBMX) to maintain higher concentrations of cAMP in the oocyte and sustain transzonal projections with gap junction communication within the cumulus-oocyte complex (COC). This system has been proved to prevent spontaneous meiosis resumption that usually occurs in the conventional IVM systems. Lipid accumulation in oocytes and IVP-embryos has been related to lower developmental potential and cryopreservation inability, especially when compared to their in vivo counterparts. We aimed to evaluate the effect of the in vitro prematuration system in the rates of blastocyst and on embryo quality by the semi-quantitative lipid content in oocytes and blastocysts ...
Follicular growth in cattle occurs in a wave pattern varying from 2 to 3 waves during each estrou... more Follicular growth in cattle occurs in a wave pattern varying from 2 to 3 waves during each estrous cycle, and is characterized by the initial development of a synchronous group of antral follicles, from which only one will become dominant. The number of recruited follicles is variable among individuals and breeds; however, it is highly repeatable in individuals. Literature reports indicate that the number of follicles recruited in each wave is higher in zebu breeds compared with taurine. A relationship between the follicular population and the oocyte competence has been suggested, and ultimately the follicular population could influence the fertility of the animal (Ireland et al., 2007, Human Reproduction 22:1687-1695). The objectives of this study were: i) to identify females in Nelore (Bos indicus) and Aberdeen Angus (Bos taurus) breeds that have high and low population of recruited antral follicles per wave and ii) to characterize, in both breeds, the differences in lipid profile...
Reproduction in mammals depends on the pulsatile release of the gonadotropins LH and FSH, which a... more Reproduction in mammals depends on the pulsatile release of the gonadotropins LH and FSH, which act together to regulate the function of the gonads. In the ovaries, LH influences follicular development until ovulation and this depends on the interaction with its receptor (LHR). However, there is no consensus regarding the use of LH during the in vitro maturation (IVM) of cumulus-oocyte complexes (COC) in cattle. Reports in the literature differ on mRNA expression of LHR in oocytes and cumulus oophorus cells (CC). Thus, the aim of this study was to characterize the expression of the LHR gene in CC and oocytes from bovine follicles at different diameters and after IVM. Ovaries were collected at a commercial abattoir and follicles were dissected and separated into two groups for experiment 1, G1: 3 to 6mm and G2: >6 to 10mm. On a Petri dish, follicles were individually ruptured with 18G needles in order to obtain their respective COC. Pools of 25 COC were used per experimental group...
Superstimulation can affect cellular and molecular events in ovarian follicular cells, cumulus-oo... more Superstimulation can affect cellular and molecular events in ovarian follicular cells, cumulus-oocyte complexes (COCs), oviduct and embryo, as well as cell-to-cell communication through secretion and uptake of exosomes (Exo). The aim of this study was to investigate the effects of the extracellular vesicles (specifically, Exo), recovered from follicular fluid (FF), on the oocyte meiosis progression and apoptosis; and also on gene expression of COCs and blastocysts. Exo were recovered by ultracentrifugation and filtration of the FF from Nelore cows submitted to ovarian superstimulation with FSH (Exo/FSH); with FSH combined to eCG (Exo/eCG) or from non-superstimulated cows (Exo/NS). The presence of Exo was confirmed by Transmission Electron Microscopy. COCs from slaughterhouse were in vitro matured (IVM) for 22-24h with TCM199 medium enriched with 10% Exo from each group (Exo/FSH, Exo/eCG or Exo/NS) or without Exo (Control). IVM-oocytes were assessed for meiosis progression (Hoescht-3...
The Insulin-like Growth Factor (IGF) system has been demonstrated as an important factor to cumul... more The Insulin-like Growth Factor (IGF) system has been demonstrated as an important factor to cumulus oocyte complex (COC) maturation and early embryo development. Studies demonstrate benefits of IGF-I addition during the in vitro maturation of oocytes in different species, including cattle. The IGF availability is controlled by two mechanisms: IGF Binding Proteins (IGFBPs), which inhibit IGF actions; and Pregnancy-Associated Plasma ProteinA (PAPP-A), responsible to cleavage the IGFBP and to release free IGF. The aim of this study was to investigate the effects of PAPP-A on cellular aspects of in vitro-matured COCs and further, to explore their impacts in the blastocysts’ yield and pattern of transcription. Bovine COCs (5 replicates/20 COC per replicate) from ovaries obtained in a local abattoir, predominantly from Nelore cows, were aspirated and matured in serum free medium in the absence (control group) or presence of 1 ng/mL (P1 group), 10 ng/mL (P10 group) or 100 ng/mL (P100 group...
Antral follicle count (AFC) was phenotypically and genetically evaluated in a Bos taurus cow popu... more Antral follicle count (AFC) was phenotypically and genetically evaluated in a Bos taurus cow population. A total of 257 Aberdeen Angus cows underwent an ovarian ultrasound evaluation and hormonal synchronization for timed artificial insemination (TAI). These animals were between 40 and 70 d postpartum with a body score condition (BSC) between 2 and 4 (average = 2.7) on a scale of 1–5. The number of antral follicles was assessed 10 d before (D-10) the beginning of TAI. Visible follicles (≥3 mm in diameter) were counted from both ovaries (right and left) to determine the number of total follicles, and animals were allocated into groups according to the number of antral follicles: low AFC was defined as ≤ 15 follicles (n = 93); intermediate AFC as 16 follicles (n = 97); and high AFC as ≥ 25 follicles (n = 67). The comparison between the average AFC and pregnancy rate was performed using the chi-square test in the Minitab® 19.2 statistical software, adopting a P-value ≤ 0.05. Hair sampl...
Aggregation is one of the main techniques used to obtain embryonic chimeras. This procedure can b... more Aggregation is one of the main techniques used to obtain embryonic chimeras. This procedure can be performed with whole or demi-embryos, in different stages of development and produced by in vivo or in vitro systems. However, aggregation efficiency tends to be reduced when using embryos in advanced stages (e.g. morulae and blastocysts). The aim of this work was to evaluate the effect of the agglutinating agent phytohemagglutinin-L (PHA) in the percentage of chimeras produced with in vitro-produced (IVP) bovine embryos. Cumulus–oocyte complexes (COC; 445; quality I and II) were matured in drops of 90 μL of TCM-199 bicarbonate supplemented with 10% of FCS and incubated for 22 to 24 h. Fertilization was performed in TALP-IVF medium for 18 h. Presumptive zygotes were transferred to SOF medium for in vitro culture. Incubation conditions were 38.5°C and 5% CO2 in air. To conduct the manual bisection, embryos were placed into 3-μL microdrops of protein-free HEPES-buffered SOF medium. The bisection was executed with a microblade (Ultra-Sharp Splitting Blade, Bioniche, Bogart, GA, USA) under stereomicroscope (35× magnification). Half-structures were joined and transferred to an embryo reconstruction plate, where they were kept for 3 min in drops containing 500 μg mL–1 phytohemagglutinin-L, before the approximated pairs were transferred to SOF medium in cell aggregation well-of-the-well (WOW) micro-wells to in vitro culture. The structures were randomly allocated and the aggregation was performed between 2 whole (zona free) 8- to 16-cell stage embryos to construct aggregated chimeras in the presence [group (G)1, n = 32] or absence of PHA (G2, n = 34) and between demi-morula and demi-blastocyst with PHA (G3, n = 28) or without (G4, n = 29). The aggregation of structures was evaluated after 24 h. Aggregation rates among the 4 experimental groups and the main effects were analysed by Chi-square or Fisher’s exact test and significance was considered when P < 0.05. Embryo aggregation was higher in group G1 than G2 (75.0 and 50.0%, respectively; P = 0.045). Aggregation rate of demi-embryos was similar either in the presence (G3, 39.3%) or in the absence of PHA (G4, 20.7%; P = 0.16). The presence of PHA significantly increased the aggregation rates of the whole pre-compaction embryos (G1) compared with G3 (75.0 and 39.3%, respectively; P < 0.01). The use of PHA resulted in higher aggregation rates (58.3%) than non-use (36.5%; P = 0.03), whereas the embryonic stage of pre-compaction development (G1+G2) produced a higher rate of aggregation (62.1%) than post-compaction demi-embryos (G3+G4, 29.8%; P < 0.001). We could infer a positive effect of PHA on the aggregation rate of bovine IVP embryos only to the 8- to 16-cell stage of development. Financial support: FAPESP, Brazil (06/06491-2, 07/07705-9, 09/10679-5, and 09/04888-0).
Uploads
Papers by Eduardo Razza