Jo Bijttebier - Academia.edu (original) (raw)

Papers by Jo Bijttebier

Animal Genetics, 2008

Myostatin (MSTN), a transforming growth factor b superfamily member, is an essential factor for t... more Myostatin (MSTN), a transforming growth factor b superfamily member, is an essential factor for the growth and development of muscle mass. The protein functions as a negative regulator of muscle growth and is related to the so-called double-muscling phenotype in cattle, where a series of mutations renders the gene inactive. One particular breed of pigs, the Belgian Piétrain, also shows a heavily muscled phenotype. The similarity of muscular phenotypes between the double-muscled cattle and Piétrain pigs indicated that MSTN may be a candidate gene for muscular hypertrophy in pigs. In this study, we sequenced and analysed the complete MSTN gene from 45 pigs of five different breeds, including the heavily muscled Piétrain breed at one extreme and the Meishan and Wild boar breeds at the other extreme. In total, 7626 bp of the porcine MSTN gene were sequenced, including the 5¢ and 3¢ UTR. Fifteen polymorphic loci were found, three of which were located in the promoter region, five in intron 1 and seven in intron 2. Most mutations were found when comparing the obtained MSTN sequence with porcine MSTN sequences already published. However, one polymorphism located at position 447 of the porcine MSTN promoter had a very high allele frequency in the Piétrain pig breed and disrupted a putative myocyte enhancer factor 3 binding site. Real-time PCR using Sybr Green showed that this mutation was associated with expression levels of the MSTN gene in m. longissimus dorsi at an age of 4 weeks.

A Bird's-Eye View of Veterinary Medicine, 2012

The complex series of molecular interactions between male and female gametes required for success... more The complex series of molecular interactions between male and female gametes required for successful fertilization has captured the interest of several research groups for many years (Benoff 1997). Even though substantial insights into these interactions have been ...

Theriogenology, 2008

Porcine follicular fluid (pFF), as a supplement of maturation media, has been shown several times... more Porcine follicular fluid (pFF), as a supplement of maturation media, has been shown several times to improve the in vitro production (IVP) of porcine embryos. As a transudate of serum, pFF contains locally produced factors in addition to the ones derived from serum. The objective of this study was to determine the additional positive effects of these pFF specific factors on the nuclear and cytoplasmic maturation of porcine oocytes. Follicular fluid and autologous serum were collected from sows in the preovulatory phase of the estrous cycle. Subsequently, oocytes from prepubertal gilts were matured in NCSU23 supplemented with either 10% pFF or 10% autologous serum derived from the same sow. Oocytes were then fertilized and the putative zygotes were cultured for 7 days. Nuclear maturation and cumulus expansion were assessed after the maturation culture. For evaluation of cytoplasmic maturation, oocyte glutathione (GSH) content, fertilization parameters and embryonic development were evaluated. After in vitro maturation (IVM) of the oocytes, both cumulus expansion rate and oocyte GSH content were increased for oocytes matured in pFF (P<0.05). More monospermic penetration was found when cumulus-intact oocytes had been matured in 10% pFF but this effect was lost after fertilization of cumulus denuded oocytes indicating that the pFF was acting through the cumulus. We speculate that the increased cumulus expansion and increased glutathione content, which were prevalent after IVM in pFF, are responsible for the positive effects on fertilization and the pre-implantation development of the embryos.

Reproduction, Fertility and Development, 2011

ABSTRACT In pigs, the exact role of the cumulus oöphurus during IVF still needs to be clarified. ... more ABSTRACT In pigs, the exact role of the cumulus oöphurus during IVF still needs to be clarified. Indirect evidence exists that the rate of cumulus expansion is positively correlated with the defense against polyspermy. Epidermal growth factor (EGF) acts synergistically with FSH in the synthesis of hyaluronic acid, the deposition of which in the extracellular matrix is a prerequisite for cumulus expansion. Therefore, we aimed to evaluate the importance of cumulus expansion for fertilization results of porcine cumulus-oocyte complexes (COC) by using different EGF concentrations in the maturation medium. Cumulus-oocyte complexes were matured in vitro in NCSU23 medium supplemented with 10% follicular fluid (FF, obtained from 6- to 10-mm follicles) or 10% serum and 10, 20, or 50ngmL(-1) of EGF (n=480 per experiment). In vitro maturation (IVM) in the presence of 10% FF and 10ngmL(-1) of EGF served as the control group. At 0, 22, 36, and 44h of IVM, 20 COC of each group were selected for evaluation of cumulus expansion by measuring the maximum distance across the cumulus matrix (3 replications). Matured COC were co-incubated with frozen-thawed semen (6000 spermatozoa per oocyte) for 6h. Subsequently, oocytes were cultured for 18h. Zygotes were stained with 10μgmL(-1) of bis-benzimide (Hoechst) to assess the fertilization rate, polyspermy, and sperm penetration index (sp index, mean number of penetrated spermatozoa per fertilized oocyte; 2 replications). Differences in cumulus diameter were analyzed by one-way ANOVA. Fertilization parameters were analyzed by applying a logistic regression model to the results. Cumulus-oocyte complexes selected for IVM had a mean diameter of 240μm. After 22h of IVM in 10% FF, the mean diameter of COC was 336, 313, and 300μm for 10, 20, and 50ngmL(-1) of EGF, respectively. After 44h of IVM, these diameters had increased to 425, 388, and 397μm. Twenty-two hours of IVM in 10% serum resulted in a COC diameter of 296, 305, and 276μm for 10, 20, and 50ngmL(-1) of EGF. After 44h of IVM, these diameters reached 330, 325, and 275μm, respectively. Only 10% serum with 50ngmL(-1) of EGF proved unfavorable for cumulus expansion (P<0.05). In vitro maturation for 44h in 10% serum resulted in a smaller rate of cumulus expansion compared with IVM in 10% FF (P<0.05), irrespective of EGF concentration. Penetration rate fluctuated between 84 and 100%, with no significant differences. Monospermic fertilization was lower in COC matured in the presence of 50ngmL(-1) of EGF compared with the control group (P<0.05). The sp index increased in parallel with EGF concentrations and was higher after IVM in 10% serum than in 10% FF. Oocytes were penetrated by 3.1, 4.3, and 6.0 spermatozoa after IVM in serum with 10, 20, and 50ngmL(-1) of EGF, respectively. Results showed a tendency toward a lower rate of cumulus expansion concomitant with higher EGF concentrations. Follicular fluid was superior to serum in supporting cumulus expansion. Oocytes were penetrated by more spermatozoa when matured in 10% serum and 20 or 50ngmL(-1) of EGF compared with 10ngmL(-1) of EGF. Thus, the degree of cumulus expansion appears to be related to the sp index, confirming that the cumulus matrix may play a role in the polyspermy defense.

Reproduction, 2010

Recent studies have shown that short-term exposure of oocytes to a stressor such as hydrostatic p... more Recent studies have shown that short-term exposure of oocytes to a stressor such as hydrostatic pressure or osmotic stress might induce stress tolerance in embryos. The aim of the present study was to investigate the consequences of short-term hydrogen peroxide (H 2 O 2 ) exposure to bovine in vitro matured cumulus-oocyte complexes (COCs) on subsequent preimplantation embryo development and apoptosis. In the first experiment, mature COCs were incubated in H 2 O 2 at concentrations ranging between 0.01 and 100 mmol/l, and subsequently fertilized and cultured. Oocyte incubation with 50-100 mmol/l of H 2 O 2 resulted in a significantly higher blastocyst yield (47.3%) in comparison with control medium (31.8%), while apoptotic cell ratio was inversely related with H 2 O 2 concentration. In the second experiment, we showed that the stress tolerance after H 2 O 2 exposure was not mediated by increased glutathione content in treated oocytes nor by enhanced fertilization or penetration. Further research should concentrate on the potential role of players that have been associated with stress tolerance in somatic cell lines.

PROTEOMICS, 2009

Porcine follicular fluid (pFF) constitutes the micro-environment of the maturing oocyte. Although... more Porcine follicular fluid (pFF) constitutes the micro-environment of the maturing oocyte. Although pFF is a transudate of serum, in pigs, it is superior to serum in promoting in vitro expansion of the cumulus cells, a specialized cell population surrounding the oocyte. A comparative proteome analysis of autologous serum and pFF was performed to investigate proteins involved in successful cumulus expansion of porcine oocytes. iTRAQ labeling followed by 2-D LC ESI-Q-TOF MS/MS revealed 63 proteins common to both fluids of which the abundance of 13 proteins was significantly different (p<0.05). Seven proteins were more concentrated in serum whereas six proteins were more abundant in pFF. To investigate the importance of these proteins, the cumulus matrices of COCs were collected after in vitro maturation in media supplemented with either of both biologically fluids and then subjected to 2-D PAGE analysis. alpha(2)-Macroglobulin and CH4 and secrete domains of swine IgM, which were both less abundant in pFF, were absent from cumulus matrix extracts after in vitro maturation in pFF. Although both proteins were incorporated in the matrices of cumulus-oocyte complexes matured in serum, depletion of alpha(2)-macroglobulin from serum could significantly compensate for the impaired cumulus expansion of oocytes matured in serum.

Theriogenology, 2011

The Sperm Quality Analyzer (SQA-Vp) was evaluated for assessing concentration and motility of por... more The Sperm Quality Analyzer (SQA-Vp) was evaluated for assessing concentration and motility of porcine semen. Both fresh and diluted semen from 50 different boars from a commercial artificial insemination (AI) centre were investigated. For the fresh ejaculate, the concentration obtained with SQA-Vp was compared with a photometer and a haemocytometer. For the diluted samples, the concentration and motility were compared with computer assisted semen analysis (CASA) and visual sperm analysis. The agreement between methods was studied with Bland-Altman plots and the repeatability with coefficient of variation (CV) as well as Bland-Altman plots. The sperm concentration (x106/ml) obtained with SQA-Vp (379.3 ± 134.9) for fresh ejaculates agreed well with concentration by the photometer (447.2 ± 154.2; difference= -67.9 x 106/ml; difference + 2SD = 55.3 x 106/ml; difference – 2SD = -191.1 x 106/ml) and with the haemocytometer (332.8 ± 141.11; d = 92.8; d + 2SD = 448.6; d – 2SD = -263). For diluted semen, the agreement between the concentration (x106/ml) assessed with SQA-Vp (20.4 ± 4.3) was good with CASA (23.2 ± 5.8; d = -2.8; d + 2SD = 6.2; d – 2 SD = -11.8) but poor with the haemocytometer (18.8 ± 5.0; d = 1.6; d+ 2SD = 12.2; d – 2SD = -9). The % motile spermatozoa assessed by SQA-Vp (65.8 ± 10.0) in diluted semen agreed well with CASA (72.2 ± 13.7; d = -6.4; d+ 2SD = 20; d – 2SD = -32.8) and with visual assessment (64.1 ± 11.6; d = 1.7; d+ 2SD = 30.9; d – 2SD = -27.5). The SQA-Vp showed a good repeatability (CV; repeatability coefficient) for measuring the concentration of both fresh (3.9%; d = 10.7; d + 2SD = 30.9; d - 2SD = -9.5) and diluted semen (2.6%; d = 1.0; d + 2SD = 2.38; d - 2SD = -0.42) and for motility (3.2%; d = 0.9; d + 2SD = 8.5; d - 2SD = -6.7). The mean SQA-Vp values fell between the other methods′ results for both fresh and diluted semen. Moreover the repeatability was acceptable. Therefore SQA-Vp can be used as a valid device for sperm quality analysis in pigs.

Animal Genetics, 2008

Myostatin (MSTN), a transforming growth factor b superfamily member, is an essential factor for t... more Myostatin (MSTN), a transforming growth factor b superfamily member, is an essential factor for the growth and development of muscle mass. The protein functions as a negative regulator of muscle growth and is related to the so-called double-muscling phenotype in cattle, where a series of mutations renders the gene inactive. One particular breed of pigs, the Belgian Piétrain, also shows a heavily muscled phenotype. The similarity of muscular phenotypes between the double-muscled cattle and Piétrain pigs indicated that MSTN may be a candidate gene for muscular hypertrophy in pigs. In this study, we sequenced and analysed the complete MSTN gene from 45 pigs of five different breeds, including the heavily muscled Piétrain breed at one extreme and the Meishan and Wild boar breeds at the other extreme. In total, 7626 bp of the porcine MSTN gene were sequenced, including the 5¢ and 3¢ UTR. Fifteen polymorphic loci were found, three of which were located in the promoter region, five in intron 1 and seven in intron 2. Most mutations were found when comparing the obtained MSTN sequence with porcine MSTN sequences already published. However, one polymorphism located at position 447 of the porcine MSTN promoter had a very high allele frequency in the Piétrain pig breed and disrupted a putative myocyte enhancer factor 3 binding site. Real-time PCR using Sybr Green showed that this mutation was associated with expression levels of the MSTN gene in m. longissimus dorsi at an age of 4 weeks.

A Bird's-Eye View of Veterinary Medicine, 2012

The complex series of molecular interactions between male and female gametes required for success... more The complex series of molecular interactions between male and female gametes required for successful fertilization has captured the interest of several research groups for many years (Benoff 1997). Even though substantial insights into these interactions have been ...

Theriogenology, 2008

Porcine follicular fluid (pFF), as a supplement of maturation media, has been shown several times... more Porcine follicular fluid (pFF), as a supplement of maturation media, has been shown several times to improve the in vitro production (IVP) of porcine embryos. As a transudate of serum, pFF contains locally produced factors in addition to the ones derived from serum. The objective of this study was to determine the additional positive effects of these pFF specific factors on the nuclear and cytoplasmic maturation of porcine oocytes. Follicular fluid and autologous serum were collected from sows in the preovulatory phase of the estrous cycle. Subsequently, oocytes from prepubertal gilts were matured in NCSU23 supplemented with either 10% pFF or 10% autologous serum derived from the same sow. Oocytes were then fertilized and the putative zygotes were cultured for 7 days. Nuclear maturation and cumulus expansion were assessed after the maturation culture. For evaluation of cytoplasmic maturation, oocyte glutathione (GSH) content, fertilization parameters and embryonic development were evaluated. After in vitro maturation (IVM) of the oocytes, both cumulus expansion rate and oocyte GSH content were increased for oocytes matured in pFF (P<0.05). More monospermic penetration was found when cumulus-intact oocytes had been matured in 10% pFF but this effect was lost after fertilization of cumulus denuded oocytes indicating that the pFF was acting through the cumulus. We speculate that the increased cumulus expansion and increased glutathione content, which were prevalent after IVM in pFF, are responsible for the positive effects on fertilization and the pre-implantation development of the embryos.

Reproduction, Fertility and Development, 2011

ABSTRACT In pigs, the exact role of the cumulus oöphurus during IVF still needs to be clarified. ... more ABSTRACT In pigs, the exact role of the cumulus oöphurus during IVF still needs to be clarified. Indirect evidence exists that the rate of cumulus expansion is positively correlated with the defense against polyspermy. Epidermal growth factor (EGF) acts synergistically with FSH in the synthesis of hyaluronic acid, the deposition of which in the extracellular matrix is a prerequisite for cumulus expansion. Therefore, we aimed to evaluate the importance of cumulus expansion for fertilization results of porcine cumulus-oocyte complexes (COC) by using different EGF concentrations in the maturation medium. Cumulus-oocyte complexes were matured in vitro in NCSU23 medium supplemented with 10% follicular fluid (FF, obtained from 6- to 10-mm follicles) or 10% serum and 10, 20, or 50ngmL(-1) of EGF (n=480 per experiment). In vitro maturation (IVM) in the presence of 10% FF and 10ngmL(-1) of EGF served as the control group. At 0, 22, 36, and 44h of IVM, 20 COC of each group were selected for evaluation of cumulus expansion by measuring the maximum distance across the cumulus matrix (3 replications). Matured COC were co-incubated with frozen-thawed semen (6000 spermatozoa per oocyte) for 6h. Subsequently, oocytes were cultured for 18h. Zygotes were stained with 10μgmL(-1) of bis-benzimide (Hoechst) to assess the fertilization rate, polyspermy, and sperm penetration index (sp index, mean number of penetrated spermatozoa per fertilized oocyte; 2 replications). Differences in cumulus diameter were analyzed by one-way ANOVA. Fertilization parameters were analyzed by applying a logistic regression model to the results. Cumulus-oocyte complexes selected for IVM had a mean diameter of 240μm. After 22h of IVM in 10% FF, the mean diameter of COC was 336, 313, and 300μm for 10, 20, and 50ngmL(-1) of EGF, respectively. After 44h of IVM, these diameters had increased to 425, 388, and 397μm. Twenty-two hours of IVM in 10% serum resulted in a COC diameter of 296, 305, and 276μm for 10, 20, and 50ngmL(-1) of EGF. After 44h of IVM, these diameters reached 330, 325, and 275μm, respectively. Only 10% serum with 50ngmL(-1) of EGF proved unfavorable for cumulus expansion (P<0.05). In vitro maturation for 44h in 10% serum resulted in a smaller rate of cumulus expansion compared with IVM in 10% FF (P<0.05), irrespective of EGF concentration. Penetration rate fluctuated between 84 and 100%, with no significant differences. Monospermic fertilization was lower in COC matured in the presence of 50ngmL(-1) of EGF compared with the control group (P<0.05). The sp index increased in parallel with EGF concentrations and was higher after IVM in 10% serum than in 10% FF. Oocytes were penetrated by 3.1, 4.3, and 6.0 spermatozoa after IVM in serum with 10, 20, and 50ngmL(-1) of EGF, respectively. Results showed a tendency toward a lower rate of cumulus expansion concomitant with higher EGF concentrations. Follicular fluid was superior to serum in supporting cumulus expansion. Oocytes were penetrated by more spermatozoa when matured in 10% serum and 20 or 50ngmL(-1) of EGF compared with 10ngmL(-1) of EGF. Thus, the degree of cumulus expansion appears to be related to the sp index, confirming that the cumulus matrix may play a role in the polyspermy defense.

Reproduction, 2010

Recent studies have shown that short-term exposure of oocytes to a stressor such as hydrostatic p... more Recent studies have shown that short-term exposure of oocytes to a stressor such as hydrostatic pressure or osmotic stress might induce stress tolerance in embryos. The aim of the present study was to investigate the consequences of short-term hydrogen peroxide (H 2 O 2 ) exposure to bovine in vitro matured cumulus-oocyte complexes (COCs) on subsequent preimplantation embryo development and apoptosis. In the first experiment, mature COCs were incubated in H 2 O 2 at concentrations ranging between 0.01 and 100 mmol/l, and subsequently fertilized and cultured. Oocyte incubation with 50-100 mmol/l of H 2 O 2 resulted in a significantly higher blastocyst yield (47.3%) in comparison with control medium (31.8%), while apoptotic cell ratio was inversely related with H 2 O 2 concentration. In the second experiment, we showed that the stress tolerance after H 2 O 2 exposure was not mediated by increased glutathione content in treated oocytes nor by enhanced fertilization or penetration. Further research should concentrate on the potential role of players that have been associated with stress tolerance in somatic cell lines.

PROTEOMICS, 2009

Porcine follicular fluid (pFF) constitutes the micro-environment of the maturing oocyte. Although... more Porcine follicular fluid (pFF) constitutes the micro-environment of the maturing oocyte. Although pFF is a transudate of serum, in pigs, it is superior to serum in promoting in vitro expansion of the cumulus cells, a specialized cell population surrounding the oocyte. A comparative proteome analysis of autologous serum and pFF was performed to investigate proteins involved in successful cumulus expansion of porcine oocytes. iTRAQ labeling followed by 2-D LC ESI-Q-TOF MS/MS revealed 63 proteins common to both fluids of which the abundance of 13 proteins was significantly different (p<0.05). Seven proteins were more concentrated in serum whereas six proteins were more abundant in pFF. To investigate the importance of these proteins, the cumulus matrices of COCs were collected after in vitro maturation in media supplemented with either of both biologically fluids and then subjected to 2-D PAGE analysis. alpha(2)-Macroglobulin and CH4 and secrete domains of swine IgM, which were both less abundant in pFF, were absent from cumulus matrix extracts after in vitro maturation in pFF. Although both proteins were incorporated in the matrices of cumulus-oocyte complexes matured in serum, depletion of alpha(2)-macroglobulin from serum could significantly compensate for the impaired cumulus expansion of oocytes matured in serum.

Theriogenology, 2011

The Sperm Quality Analyzer (SQA-Vp) was evaluated for assessing concentration and motility of por... more The Sperm Quality Analyzer (SQA-Vp) was evaluated for assessing concentration and motility of porcine semen. Both fresh and diluted semen from 50 different boars from a commercial artificial insemination (AI) centre were investigated. For the fresh ejaculate, the concentration obtained with SQA-Vp was compared with a photometer and a haemocytometer. For the diluted samples, the concentration and motility were compared with computer assisted semen analysis (CASA) and visual sperm analysis. The agreement between methods was studied with Bland-Altman plots and the repeatability with coefficient of variation (CV) as well as Bland-Altman plots. The sperm concentration (x106/ml) obtained with SQA-Vp (379.3 ± 134.9) for fresh ejaculates agreed well with concentration by the photometer (447.2 ± 154.2; difference= -67.9 x 106/ml; difference + 2SD = 55.3 x 106/ml; difference – 2SD = -191.1 x 106/ml) and with the haemocytometer (332.8 ± 141.11; d = 92.8; d + 2SD = 448.6; d – 2SD = -263). For diluted semen, the agreement between the concentration (x106/ml) assessed with SQA-Vp (20.4 ± 4.3) was good with CASA (23.2 ± 5.8; d = -2.8; d + 2SD = 6.2; d – 2 SD = -11.8) but poor with the haemocytometer (18.8 ± 5.0; d = 1.6; d+ 2SD = 12.2; d – 2SD = -9). The % motile spermatozoa assessed by SQA-Vp (65.8 ± 10.0) in diluted semen agreed well with CASA (72.2 ± 13.7; d = -6.4; d+ 2SD = 20; d – 2SD = -32.8) and with visual assessment (64.1 ± 11.6; d = 1.7; d+ 2SD = 30.9; d – 2SD = -27.5). The SQA-Vp showed a good repeatability (CV; repeatability coefficient) for measuring the concentration of both fresh (3.9%; d = 10.7; d + 2SD = 30.9; d - 2SD = -9.5) and diluted semen (2.6%; d = 1.0; d + 2SD = 2.38; d - 2SD = -0.42) and for motility (3.2%; d = 0.9; d + 2SD = 8.5; d - 2SD = -6.7). The mean SQA-Vp values fell between the other methods′ results for both fresh and diluted semen. Moreover the repeatability was acceptable. Therefore SQA-Vp can be used as a valid device for sperm quality analysis in pigs.