J. Dobrinsky - Academia.edu (original) (raw)

Papers by J. Dobrinsky

Research paper thumbnail of 160 Effect of Bsa Sources and Types on in Vitro -Produced Livestock Embryo Development

Reproduction, Fertility and Development, 2006

Bovine serum albumin (BSA) is a macromolecule supplement used in embryo and cell culture media. O... more Bovine serum albumin (BSA) is a macromolecule supplement used in embryo and cell culture media. Other chemicals have been used as macromolecule substitutes in embryo culture with variable effectiveness. There are BSA products available that are defined in their disease status, collection method, and manufacturing process. They are compliant for use in raw form or in culture medium in the USA and EU. It was the purpose of this study to compare the effectiveness of highly defined and internationally compliant BSA with typically used BSA on in vitro-produced pig and cow embryo development. Pig oocyte-cumulus complexes were matured in two stages for 44 h in vitro. Semen from one boar of known high fertility was used across the study to fertilize mature oocytes. After 6-h co-incubation with sperm (50 motile sperm/oocyte), presumptive zygotes were cultured for 120 h in modified NCSU-23 containing 4 mg/mL of one of the following heat-shocked BSA fractions: Sigma A-7906 (control), Minitube ...

Research paper thumbnail of 146 Relocation of Swine Genetics Using Embryo Transfer

Reproduction, Fertility and Development, 2009

Swine production requires a stable health status that can be compromised by introduction of live ... more Swine production requires a stable health status that can be compromised by introduction of live animals for genetic change. Our objective was to use embryo transfer to avoid disease transmission during genetic relocation. Forty genotype-specific (GS) donor females were scheduled for 3 sessions of embryo recovery at 6-week intervals using Altrenogest (Matrix®, Intervet, Millsboro, DE), 1250 IU of equine chorionic gonadotropin (eCG/PMSG; Sigma, St. Louis, MO) and 750 IU of human chorionic gonadotropin (hCG; Chorulon®, Intervet). Single-sire GS matings were made 34 h after Chorulon® injection. To accomplish single-sire transfers, color specific (CS) supplemental embryos were used to assist in maintenance of recipient pregnancy. The CS embryo donors and GS embryo recipients were synchronized with Matrix®, P.G. 600® (200 IU hCG, 400 IU PMSG, Intervet) and Chorulon®. Embryos from GS donors were surgically recovered on Day 5 post-insemination, washed per IETS recommendations using a zwitt...

Research paper thumbnail of Transgenic expression of human CD47 markedly increases engraftment in a murine model of pig-to-human hematopoietic cell transplantation

American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 2014

Mixed chimerism approaches for induction of tolerance of solid organ transplants have been applie... more Mixed chimerism approaches for induction of tolerance of solid organ transplants have been applied successfully in animal models and in the clinic. However, in xenogeneic models (pig-to-primate), host macrophages participate in the rapid clearance of porcine hematopoietic progenitor cells, hindering the ability to achieve mixed chimerism. CD47 is a cell-surface molecule that interacts in a species-specific manner with SIRPα receptors on macrophages to inhibit phagocytosis and expression of human CD47 (hCD47) on porcine cells has been shown to inhibit phagocytosis by primate macrophages. We report here the generation of hCD47 transgenic GalT-KO miniature swine that express hCD47 in all blood cell lineages. The effect of hCD47 expression on xenogeneic hematopoietic engraftment was tested in an in vivo mouse model of human hematopoietic cell engraftment. High-level porcine chimerism was observed in the bone marrow of hCD47 progenitor cell recipients and smaller but readily measurable c...

Research paper thumbnail of Paul Valery-Montpellier III University, Montpellier, France

Research paper thumbnail of Miniature Swine Expressing Human CD47 to Enhance Bone Marrow Engraftment in Non-Human Primates

Transplantation Journal, 2012

Background: Despite being an effective immunosuppressive agent, the calcineurin inhibitors (ciclo... more Background: Despite being an effective immunosuppressive agent, the calcineurin inhibitors (ciclosporin and tacrolimus) are associated with nephrotoxicity, chronic allograft nephropathy and long term graft loss. The authors proposed that the early replacement of calcineurin inhibitors with everolimus (a mammalian-target-of-rapamycin inhibitor) may be an effective strategy of immunosuppression following renal transplantation, and may improve renal function without compromising efficacy. We present our experience of early conversion to everolimus after 6 months of renal transplantation. Material and methods: Patients who underwent renal transplantation from 2009 to 2011 were enrolled in the study. In is prospective open label study. Patients received triple maintenance immunosuppression; prenisolone 20mg/day, tacrolimus 0.1mg/kg/day, Mycophenolate mofitil 2.0g/day. Basiliximab induction (20 mg, intravenously, on day 0 and on day 4) was given to patients with spousal donors. After 6 months of renal transplantation tacrolimus was replaced by everolimus. (trough concentrations of 6-10 ng/mL] Results: Totally 33 renal transplant recipients were converted to everolimus during this period. Female to male ratio was 1: 5.6. The mean age was 35.8 years. In 3 patients (9.09%), tacrolimus was changed to everolimus at 3 months due to tacrolimus toxicity, another 3 patients (9.09%), mycophenolate mofitil was convered to everolimus due to drug induces bone marrow depression and in 27 patients (81.81%) everolimus was started after 6 months of renal transplantation as a protocol. Ten patients (30.3%) received basileximab, nine (27.27%) were spousal transplantation and one (3.03%) was deseased donor transplantation. Kidney donor was mother in 10 patients (30.3%), father in 3 (9.09%), brother in 5(15.15%) and sister in 4 patients (12.12%). Mean serum creatinine before start of everlimus was 1.34mg/Dl, and GFR was 54ml/ min. At 6 months and 1 year follow-up, patient and graft survivals were 100%. None of the 33 patients suffered rejection. Mean serum creatinine and GFR at 6 months and 1 year were 1. 26 mg/dl, 59 ml/min and 1.21mg/dl, 61/min respectively. Mean 24 hours urinary proteiuria before conversion to everolimus, at 6 months and at 1 year after conversion to everlimus were 342 mg, 397mg and 413 mg. Five patients (15.15%) suffered CMV infection after convertion to everolimus and one (3.03%) patient suffered B K Virus nephropathy. Eighteen patients (54.54%) developed hyperlipidemia after conversion to everolimus. Conclusion: Early elimination of calcineurin inhibitor by use of everolimus-based immunosuppression improved renal function at 12 months while maintaining efficacy and safety, indicating that this strategy may facilitate improved long-term outcomes in selected patients.

Research paper thumbnail of Advancements in cryopreservation of domestic animal embryos

Research paper thumbnail of Semen extender analysis: Eliciting fertility differences by leveraging reduced sperm concentrations used for AI

Research paper thumbnail of Development of vitrified rabbit embryos

Research paper thumbnail of In vivo and in vitro development of embryos from superovulated beef heifers

Research paper thumbnail of Development rate, esterase activity and death of cells in in vitro and in vivo produced bovine embryos

Research paper thumbnail of The effect of protein supplements, phosphate and glucose on the in vitro development of IVM-IVF bovine oocytes

Research paper thumbnail of Cryopreservation of bovine embryos by vitrification

Research paper thumbnail of Staining Sperm for Viability Assessment

Reproduction in Domestic Animals, 1995

Research paper thumbnail of Effect of follicle-stimulating hormone and luteinizing hormone during bovine in vitro maturation on development following in vitro fertilization and nuclear transfer

Molecular Reproduction and Development, 1993

The effects of luteinizing hormone (LH) (0, 100, 10,000 IU/ml) and follicle-stimulating hormone (... more The effects of luteinizing hormone (LH) (0, 100, 10,000 IU/ml) and follicle-stimulating hormone (FSH) (20 micrograms/ml) supplementation during in vitro maturation of slaughterhouse-derived oocytes on polar body formation and embryo development subsequent to in vitro fertilization and nuclear transfer were evaluated. Gonadotropin supplementation of maturation medium in the presence of serum neither enhanced the proportion of oocytes forming a polar body nor significantly affected development following in vitro fertilization or nuclear transfer, except at the highest LH concentration. A very high concentration of LH (10,000 IU/ml) significantly decreased polar body formation, initial cleavage, and blastocyst development (P < 0.05).

Research paper thumbnail of Development of vitrified embryos from different mouse strains

Research paper thumbnail of 149 Laparoscopic Embryo Transfer in Pigs

Reproduction, Fertility and Development, 2008

In an effort to optimize the number of live offspring from cloned and transgenic pig embryos, emb... more In an effort to optimize the number of live offspring from cloned and transgenic pig embryos, embryos are surgically transferred into the isthmus region of the oviduct soon after micromanipulation. Surgical embryo transfer in pigs is successful but still an invasive process. Laparoscopic embryo transfer (Besenfelder et al. 1997 Theriogenology 47, 1051–1060) is much less invasive than surgery and is more adaptable to a variety of commercial embryo transfer conditions. Our goal was to develop the use the laparoscope as an alternative method of embryo transfer for micromanipulated embryos. Naturally cycling maternal white line donor and recipient females were used for laparoscopic embryo transfer. Donors were selected to be in estrus 0 to 24 h before recipients. Two- to four-cell embryos were surgically recovered via mid-ventral laparotomy and immediately prepared for laparoscopic transfer into the oviduct through the infundibulum or through puncture of the oviduct into the ampulla. Si...

Research paper thumbnail of 155 Embryo transfer success during concurrent contagious equine metritis infection

Reproduction, Fertility and …, 2007

Contagious Equine Metritis (CEM) is an equine venereal disease caused by the bacterium Taylorella... more Contagious Equine Metritis (CEM) is an equine venereal disease caused by the bacterium Taylorella equigenitalis. CEM reduces fertility by causing acute vaginitis and endometritis in mares bred by infected stallions. Stallions are asymptomatic carriers and mares can pass the bacteria back to stallions. This disease caused massive financial losses in the thoroughbred breeding industry of the United Kingdom and United States in 1977 and 1978. CEM is considered a foreign animal disease in the United States. Fresh or chilled semen from three stallions, unknowingly CEM positive, was used for AI as part of an embryo transfer (ET) program. Positive diagnosis for T. equigenitalis was by culture. Cultures were on carried out on chocolate agar under 5–10% CO2. Test mare breeding per the U.S. Code of Federal Regulations (2006 9 CFR) was performed, and stallions did infect these mares. Real-time PCR was used to distinguish between T. equigenitalis and T. asinigenitalis, with a 97% homology for T. equigenitalis. Kirby-Bauer antibiotic sensitivity testing was performed. Of note, the bacterium was sensitive to gentamicin. Semen was collected using a Minitube artificial vagina (Minitube of America, Inc., Verona, WI, USA), analyzed using CASA (SpermVision™, Minitube), and prepared according to published guidelines. Semen was extended using Minitube EquiPRO� CellGuard™ extender with amikacin and penicillin. Six mares were used for breeding. Donor and recipient mares were examined using transrectal palpation and ultrasound to confirm estrus, follicular size, and ovulation synchrony. Mares were examined for signs of vaginitis, cervicitis, or endometritis. Embryo recovery was performed using EquiPRO recovery media on Day 7. Recovered embryos were washed twice in EquiPRO holding medium at dilution rate of 5 µL recovery media to 3 mL holding media. Media contained gentamicin and kanamycin. No mares were treated with systemic antibiotics. Recovered embryos were transferred to recipients for the purpose of producing live foals. Seventeen embryos were recovered in 19 attempts, yielding an 89% embryo recovery rate. Fourteen of the embryos were transferred to recipient mares. Three embryos were vitrified. Ten of 14 (78%) transfers yielded pregnancies by Day 14 of gestation. Seven live foals were born. After CEM diagnosis in the stallions, the donor and recipient mares were tested. Testing was performed per 2006 9 CFR. The clitoral fossa and sinuses and, in addition, the cervix were cultured. Culture methods were as previously described. No mare sample returned a positive culture. To our knowledge, this is the first report in the United States demonstrating transfer of genetics of CEM-infected stallions without transfer of disease or reduction of fertility since eradication. The use of AI with extended semen is likely the greatest contributor to this success. It is felt that ART (Assisted Reproductive Technology(s)) may be a beneficial tool for propagating genetics of animals confronted with pathogen presence; however, further controlled studies are necessary.

Research paper thumbnail of Patterns of intracellular Ca2+ concentrations in fertilized bovine eggs

Biology of Reproduction

Sperm-induced calcium (Ca2+) changes were examined in zona pellucida-intact, mature bovine eggs i... more Sperm-induced calcium (Ca2+) changes were examined in zona pellucida-intact, mature bovine eggs injected with the fluorescent Ca2+ indicator fura-2 dextran (fura-2 D). Fifty four percent (37/68) of the dye-injected, inseminated bovine eggs were fertilized and 43% (16/37) of the fertilized eggs exhibited Ca2+ elevations during the time of measurement. All (16/16) of the eggs with Ca2+ elevations were fertilized but none of the unfertilized eggs (0/31) showed intracellular Ca2+ elevations. Six of 13 eggs that were later examined and found to be fertilized at the time of the Ca2+ recordings did not show sperm-induced Ca2+ elevations. Fifty percent (8/16) of the eggs with Ca2+ elevations exhibited a single Ca2+ rise as a response to sperm penetration during the 60-min period in which these eggs were monitored. Twelve percent (2/16) of the eggs responded with two Ca2+ elevations spaced by 50- and 51-min intervals and 38% (6/16) of the eggs exhibited multiple elevations with intervals of 15-29 min. In the latter group, one egg was polyspermic. The mean amplitude of the sperm-induced Ca2+ elevations was 564 +/- 58 nM. Eggs with single elevations reached higher peak concentrations than eggs with multiple elevations (p < 0.05). The mean duration of the Ca2+ elevations was 166 +/- 13 sec and was similar among eggs with different Ca2+ patterns. The first elevations detected occurred at a mean of 6.6 +/- 0.5 h after insemination. Fertilization in this study was confirmed by looking at pronuclear formation 16 h post-insemination or by DNA staining immediately after the fluorescence readings. Eggs exhibiting Ca2+ elevations ranged in stage of fertilization from just penetrated to pronuclear. Injection of inositol 1,4,5 trisphosphate (5 microM in the injection pipette) into 6 bovine eggs induced an immediate Ca2+ elevation with a mean peak Ca2+ value of 700 +/- 60 nM and a mean duration of 103 +/- 21 sec. Incubation of bovine eggs with 200 microM thimerosal induced periodic Ca2+ rises. The mean number of Ca2+ elevations observed in 35 min of recordings was 3.0 +/- 0.5 (n = 9, range 1-5). The mean peak Ca2+ value of the first thimerosal-induced Ca2+ elevation was 990 +/- 210 nM. The results of this study indicate that fertilization can evoke intracellular Ca2+ elevations in bovine eggs and that the periodicity of these Ca2+ elevations is different among eggs. Furthermore, both inositol 1,4,5-trisphosphate and thimerosal were able to induce intracellular Ca2+ release in bovine eggs.

Research paper thumbnail of Patterns of intracellular Ca2+ concentrations in fertilized bovine eggs

Biology of Reproduction

Sperm-induced calcium (Ca2+) changes were examined in zona pellucida-intact, mature bovine eggs i... more Sperm-induced calcium (Ca2+) changes were examined in zona pellucida-intact, mature bovine eggs injected with the fluorescent Ca2+ indicator fura-2 dextran (fura-2 D). Fifty four percent (37/68) of the dye-injected, inseminated bovine eggs were fertilized and 43% (16/37) of the fertilized eggs exhibited Ca2+ elevations during the time of measurement. All (16/16) of the eggs with Ca2+ elevations were fertilized but none of the unfertilized eggs (0/31) showed intracellular Ca2+ elevations. Six of 13 eggs that were later examined and found to be fertilized at the time of the Ca2+ recordings did not show sperm-induced Ca2+ elevations. Fifty percent (8/16) of the eggs with Ca2+ elevations exhibited a single Ca2+ rise as a response to sperm penetration during the 60-min period in which these eggs were monitored. Twelve percent (2/16) of the eggs responded with two Ca2+ elevations spaced by 50- and 51-min intervals and 38% (6/16) of the eggs exhibited multiple elevations with intervals of 15-29 min. In the latter group, one egg was polyspermic. The mean amplitude of the sperm-induced Ca2+ elevations was 564 +/- 58 nM. Eggs with single elevations reached higher peak concentrations than eggs with multiple elevations (p < 0.05). The mean duration of the Ca2+ elevations was 166 +/- 13 sec and was similar among eggs with different Ca2+ patterns. The first elevations detected occurred at a mean of 6.6 +/- 0.5 h after insemination. Fertilization in this study was confirmed by looking at pronuclear formation 16 h post-insemination or by DNA staining immediately after the fluorescence readings. Eggs exhibiting Ca2+ elevations ranged in stage of fertilization from just penetrated to pronuclear. Injection of inositol 1,4,5 trisphosphate (5 microM in the injection pipette) into 6 bovine eggs induced an immediate Ca2+ elevation with a mean peak Ca2+ value of 700 +/- 60 nM and a mean duration of 103 +/- 21 sec. Incubation of bovine eggs with 200 microM thimerosal induced periodic Ca2+ rises. The mean number of Ca2+ elevations observed in 35 min of recordings was 3.0 +/- 0.5 (n = 9, range 1-5). The mean peak Ca2+ value of the first thimerosal-induced Ca2+ elevation was 990 +/- 210 nM. The results of this study indicate that fertilization can evoke intracellular Ca2+ elevations in bovine eggs and that the periodicity of these Ca2+ elevations is different among eggs. Furthermore, both inositol 1,4,5-trisphosphate and thimerosal were able to induce intracellular Ca2+ release in bovine eggs.

Research paper thumbnail of Transgenic expression of human CD47 markedly increases engraftment in a murine model of pig-to-human hematopoietic cell transplantation

American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 2014

Mixed chimerism approaches for induction of tolerance of solid organ transplants have been applie... more Mixed chimerism approaches for induction of tolerance of solid organ transplants have been applied successfully in animal models and in the clinic. However, in xenogeneic models (pig-to-primate), host macrophages participate in the rapid clearance of porcine hematopoietic progenitor cells, hindering the ability to achieve mixed chimerism. CD47 is a cell-surface molecule that interacts in a species-specific manner with SIRPα receptors on macrophages to inhibit phagocytosis and expression of human CD47 (hCD47) on porcine cells has been shown to inhibit phagocytosis by primate macrophages. We report here the generation of hCD47 transgenic GalT-KO miniature swine that express hCD47 in all blood cell lineages. The effect of hCD47 expression on xenogeneic hematopoietic engraftment was tested in an in vivo mouse model of human hematopoietic cell engraftment. High-level porcine chimerism was observed in the bone marrow of hCD47 progenitor cell recipients and smaller but readily measurable c...

Research paper thumbnail of 160 Effect of Bsa Sources and Types on in Vitro -Produced Livestock Embryo Development

Reproduction, Fertility and Development, 2006

Bovine serum albumin (BSA) is a macromolecule supplement used in embryo and cell culture media. O... more Bovine serum albumin (BSA) is a macromolecule supplement used in embryo and cell culture media. Other chemicals have been used as macromolecule substitutes in embryo culture with variable effectiveness. There are BSA products available that are defined in their disease status, collection method, and manufacturing process. They are compliant for use in raw form or in culture medium in the USA and EU. It was the purpose of this study to compare the effectiveness of highly defined and internationally compliant BSA with typically used BSA on in vitro-produced pig and cow embryo development. Pig oocyte-cumulus complexes were matured in two stages for 44 h in vitro. Semen from one boar of known high fertility was used across the study to fertilize mature oocytes. After 6-h co-incubation with sperm (50 motile sperm/oocyte), presumptive zygotes were cultured for 120 h in modified NCSU-23 containing 4 mg/mL of one of the following heat-shocked BSA fractions: Sigma A-7906 (control), Minitube ...

Research paper thumbnail of 146 Relocation of Swine Genetics Using Embryo Transfer

Reproduction, Fertility and Development, 2009

Swine production requires a stable health status that can be compromised by introduction of live ... more Swine production requires a stable health status that can be compromised by introduction of live animals for genetic change. Our objective was to use embryo transfer to avoid disease transmission during genetic relocation. Forty genotype-specific (GS) donor females were scheduled for 3 sessions of embryo recovery at 6-week intervals using Altrenogest (Matrix®, Intervet, Millsboro, DE), 1250 IU of equine chorionic gonadotropin (eCG/PMSG; Sigma, St. Louis, MO) and 750 IU of human chorionic gonadotropin (hCG; Chorulon®, Intervet). Single-sire GS matings were made 34 h after Chorulon® injection. To accomplish single-sire transfers, color specific (CS) supplemental embryos were used to assist in maintenance of recipient pregnancy. The CS embryo donors and GS embryo recipients were synchronized with Matrix®, P.G. 600® (200 IU hCG, 400 IU PMSG, Intervet) and Chorulon®. Embryos from GS donors were surgically recovered on Day 5 post-insemination, washed per IETS recommendations using a zwitt...

Research paper thumbnail of Transgenic expression of human CD47 markedly increases engraftment in a murine model of pig-to-human hematopoietic cell transplantation

American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 2014

Mixed chimerism approaches for induction of tolerance of solid organ transplants have been applie... more Mixed chimerism approaches for induction of tolerance of solid organ transplants have been applied successfully in animal models and in the clinic. However, in xenogeneic models (pig-to-primate), host macrophages participate in the rapid clearance of porcine hematopoietic progenitor cells, hindering the ability to achieve mixed chimerism. CD47 is a cell-surface molecule that interacts in a species-specific manner with SIRPα receptors on macrophages to inhibit phagocytosis and expression of human CD47 (hCD47) on porcine cells has been shown to inhibit phagocytosis by primate macrophages. We report here the generation of hCD47 transgenic GalT-KO miniature swine that express hCD47 in all blood cell lineages. The effect of hCD47 expression on xenogeneic hematopoietic engraftment was tested in an in vivo mouse model of human hematopoietic cell engraftment. High-level porcine chimerism was observed in the bone marrow of hCD47 progenitor cell recipients and smaller but readily measurable c...

Research paper thumbnail of Paul Valery-Montpellier III University, Montpellier, France

Research paper thumbnail of Miniature Swine Expressing Human CD47 to Enhance Bone Marrow Engraftment in Non-Human Primates

Transplantation Journal, 2012

Background: Despite being an effective immunosuppressive agent, the calcineurin inhibitors (ciclo... more Background: Despite being an effective immunosuppressive agent, the calcineurin inhibitors (ciclosporin and tacrolimus) are associated with nephrotoxicity, chronic allograft nephropathy and long term graft loss. The authors proposed that the early replacement of calcineurin inhibitors with everolimus (a mammalian-target-of-rapamycin inhibitor) may be an effective strategy of immunosuppression following renal transplantation, and may improve renal function without compromising efficacy. We present our experience of early conversion to everolimus after 6 months of renal transplantation. Material and methods: Patients who underwent renal transplantation from 2009 to 2011 were enrolled in the study. In is prospective open label study. Patients received triple maintenance immunosuppression; prenisolone 20mg/day, tacrolimus 0.1mg/kg/day, Mycophenolate mofitil 2.0g/day. Basiliximab induction (20 mg, intravenously, on day 0 and on day 4) was given to patients with spousal donors. After 6 months of renal transplantation tacrolimus was replaced by everolimus. (trough concentrations of 6-10 ng/mL] Results: Totally 33 renal transplant recipients were converted to everolimus during this period. Female to male ratio was 1: 5.6. The mean age was 35.8 years. In 3 patients (9.09%), tacrolimus was changed to everolimus at 3 months due to tacrolimus toxicity, another 3 patients (9.09%), mycophenolate mofitil was convered to everolimus due to drug induces bone marrow depression and in 27 patients (81.81%) everolimus was started after 6 months of renal transplantation as a protocol. Ten patients (30.3%) received basileximab, nine (27.27%) were spousal transplantation and one (3.03%) was deseased donor transplantation. Kidney donor was mother in 10 patients (30.3%), father in 3 (9.09%), brother in 5(15.15%) and sister in 4 patients (12.12%). Mean serum creatinine before start of everlimus was 1.34mg/Dl, and GFR was 54ml/ min. At 6 months and 1 year follow-up, patient and graft survivals were 100%. None of the 33 patients suffered rejection. Mean serum creatinine and GFR at 6 months and 1 year were 1. 26 mg/dl, 59 ml/min and 1.21mg/dl, 61/min respectively. Mean 24 hours urinary proteiuria before conversion to everolimus, at 6 months and at 1 year after conversion to everlimus were 342 mg, 397mg and 413 mg. Five patients (15.15%) suffered CMV infection after convertion to everolimus and one (3.03%) patient suffered B K Virus nephropathy. Eighteen patients (54.54%) developed hyperlipidemia after conversion to everolimus. Conclusion: Early elimination of calcineurin inhibitor by use of everolimus-based immunosuppression improved renal function at 12 months while maintaining efficacy and safety, indicating that this strategy may facilitate improved long-term outcomes in selected patients.

Research paper thumbnail of Advancements in cryopreservation of domestic animal embryos

Research paper thumbnail of Semen extender analysis: Eliciting fertility differences by leveraging reduced sperm concentrations used for AI

Research paper thumbnail of Development of vitrified rabbit embryos

Research paper thumbnail of In vivo and in vitro development of embryos from superovulated beef heifers

Research paper thumbnail of Development rate, esterase activity and death of cells in in vitro and in vivo produced bovine embryos

Research paper thumbnail of The effect of protein supplements, phosphate and glucose on the in vitro development of IVM-IVF bovine oocytes

Research paper thumbnail of Cryopreservation of bovine embryos by vitrification

Research paper thumbnail of Staining Sperm for Viability Assessment

Reproduction in Domestic Animals, 1995

Research paper thumbnail of Effect of follicle-stimulating hormone and luteinizing hormone during bovine in vitro maturation on development following in vitro fertilization and nuclear transfer

Molecular Reproduction and Development, 1993

The effects of luteinizing hormone (LH) (0, 100, 10,000 IU/ml) and follicle-stimulating hormone (... more The effects of luteinizing hormone (LH) (0, 100, 10,000 IU/ml) and follicle-stimulating hormone (FSH) (20 micrograms/ml) supplementation during in vitro maturation of slaughterhouse-derived oocytes on polar body formation and embryo development subsequent to in vitro fertilization and nuclear transfer were evaluated. Gonadotropin supplementation of maturation medium in the presence of serum neither enhanced the proportion of oocytes forming a polar body nor significantly affected development following in vitro fertilization or nuclear transfer, except at the highest LH concentration. A very high concentration of LH (10,000 IU/ml) significantly decreased polar body formation, initial cleavage, and blastocyst development (P < 0.05).

Research paper thumbnail of Development of vitrified embryos from different mouse strains

Research paper thumbnail of 149 Laparoscopic Embryo Transfer in Pigs

Reproduction, Fertility and Development, 2008

In an effort to optimize the number of live offspring from cloned and transgenic pig embryos, emb... more In an effort to optimize the number of live offspring from cloned and transgenic pig embryos, embryos are surgically transferred into the isthmus region of the oviduct soon after micromanipulation. Surgical embryo transfer in pigs is successful but still an invasive process. Laparoscopic embryo transfer (Besenfelder et al. 1997 Theriogenology 47, 1051–1060) is much less invasive than surgery and is more adaptable to a variety of commercial embryo transfer conditions. Our goal was to develop the use the laparoscope as an alternative method of embryo transfer for micromanipulated embryos. Naturally cycling maternal white line donor and recipient females were used for laparoscopic embryo transfer. Donors were selected to be in estrus 0 to 24 h before recipients. Two- to four-cell embryos were surgically recovered via mid-ventral laparotomy and immediately prepared for laparoscopic transfer into the oviduct through the infundibulum or through puncture of the oviduct into the ampulla. Si...

Research paper thumbnail of 155 Embryo transfer success during concurrent contagious equine metritis infection

Reproduction, Fertility and …, 2007

Contagious Equine Metritis (CEM) is an equine venereal disease caused by the bacterium Taylorella... more Contagious Equine Metritis (CEM) is an equine venereal disease caused by the bacterium Taylorella equigenitalis. CEM reduces fertility by causing acute vaginitis and endometritis in mares bred by infected stallions. Stallions are asymptomatic carriers and mares can pass the bacteria back to stallions. This disease caused massive financial losses in the thoroughbred breeding industry of the United Kingdom and United States in 1977 and 1978. CEM is considered a foreign animal disease in the United States. Fresh or chilled semen from three stallions, unknowingly CEM positive, was used for AI as part of an embryo transfer (ET) program. Positive diagnosis for T. equigenitalis was by culture. Cultures were on carried out on chocolate agar under 5–10% CO2. Test mare breeding per the U.S. Code of Federal Regulations (2006 9 CFR) was performed, and stallions did infect these mares. Real-time PCR was used to distinguish between T. equigenitalis and T. asinigenitalis, with a 97% homology for T. equigenitalis. Kirby-Bauer antibiotic sensitivity testing was performed. Of note, the bacterium was sensitive to gentamicin. Semen was collected using a Minitube artificial vagina (Minitube of America, Inc., Verona, WI, USA), analyzed using CASA (SpermVision™, Minitube), and prepared according to published guidelines. Semen was extended using Minitube EquiPRO� CellGuard™ extender with amikacin and penicillin. Six mares were used for breeding. Donor and recipient mares were examined using transrectal palpation and ultrasound to confirm estrus, follicular size, and ovulation synchrony. Mares were examined for signs of vaginitis, cervicitis, or endometritis. Embryo recovery was performed using EquiPRO recovery media on Day 7. Recovered embryos were washed twice in EquiPRO holding medium at dilution rate of 5 µL recovery media to 3 mL holding media. Media contained gentamicin and kanamycin. No mares were treated with systemic antibiotics. Recovered embryos were transferred to recipients for the purpose of producing live foals. Seventeen embryos were recovered in 19 attempts, yielding an 89% embryo recovery rate. Fourteen of the embryos were transferred to recipient mares. Three embryos were vitrified. Ten of 14 (78%) transfers yielded pregnancies by Day 14 of gestation. Seven live foals were born. After CEM diagnosis in the stallions, the donor and recipient mares were tested. Testing was performed per 2006 9 CFR. The clitoral fossa and sinuses and, in addition, the cervix were cultured. Culture methods were as previously described. No mare sample returned a positive culture. To our knowledge, this is the first report in the United States demonstrating transfer of genetics of CEM-infected stallions without transfer of disease or reduction of fertility since eradication. The use of AI with extended semen is likely the greatest contributor to this success. It is felt that ART (Assisted Reproductive Technology(s)) may be a beneficial tool for propagating genetics of animals confronted with pathogen presence; however, further controlled studies are necessary.

Research paper thumbnail of Patterns of intracellular Ca2+ concentrations in fertilized bovine eggs

Biology of Reproduction

Sperm-induced calcium (Ca2+) changes were examined in zona pellucida-intact, mature bovine eggs i... more Sperm-induced calcium (Ca2+) changes were examined in zona pellucida-intact, mature bovine eggs injected with the fluorescent Ca2+ indicator fura-2 dextran (fura-2 D). Fifty four percent (37/68) of the dye-injected, inseminated bovine eggs were fertilized and 43% (16/37) of the fertilized eggs exhibited Ca2+ elevations during the time of measurement. All (16/16) of the eggs with Ca2+ elevations were fertilized but none of the unfertilized eggs (0/31) showed intracellular Ca2+ elevations. Six of 13 eggs that were later examined and found to be fertilized at the time of the Ca2+ recordings did not show sperm-induced Ca2+ elevations. Fifty percent (8/16) of the eggs with Ca2+ elevations exhibited a single Ca2+ rise as a response to sperm penetration during the 60-min period in which these eggs were monitored. Twelve percent (2/16) of the eggs responded with two Ca2+ elevations spaced by 50- and 51-min intervals and 38% (6/16) of the eggs exhibited multiple elevations with intervals of 15-29 min. In the latter group, one egg was polyspermic. The mean amplitude of the sperm-induced Ca2+ elevations was 564 +/- 58 nM. Eggs with single elevations reached higher peak concentrations than eggs with multiple elevations (p < 0.05). The mean duration of the Ca2+ elevations was 166 +/- 13 sec and was similar among eggs with different Ca2+ patterns. The first elevations detected occurred at a mean of 6.6 +/- 0.5 h after insemination. Fertilization in this study was confirmed by looking at pronuclear formation 16 h post-insemination or by DNA staining immediately after the fluorescence readings. Eggs exhibiting Ca2+ elevations ranged in stage of fertilization from just penetrated to pronuclear. Injection of inositol 1,4,5 trisphosphate (5 microM in the injection pipette) into 6 bovine eggs induced an immediate Ca2+ elevation with a mean peak Ca2+ value of 700 +/- 60 nM and a mean duration of 103 +/- 21 sec. Incubation of bovine eggs with 200 microM thimerosal induced periodic Ca2+ rises. The mean number of Ca2+ elevations observed in 35 min of recordings was 3.0 +/- 0.5 (n = 9, range 1-5). The mean peak Ca2+ value of the first thimerosal-induced Ca2+ elevation was 990 +/- 210 nM. The results of this study indicate that fertilization can evoke intracellular Ca2+ elevations in bovine eggs and that the periodicity of these Ca2+ elevations is different among eggs. Furthermore, both inositol 1,4,5-trisphosphate and thimerosal were able to induce intracellular Ca2+ release in bovine eggs.

Research paper thumbnail of Patterns of intracellular Ca2+ concentrations in fertilized bovine eggs

Biology of Reproduction

Sperm-induced calcium (Ca2+) changes were examined in zona pellucida-intact, mature bovine eggs i... more Sperm-induced calcium (Ca2+) changes were examined in zona pellucida-intact, mature bovine eggs injected with the fluorescent Ca2+ indicator fura-2 dextran (fura-2 D). Fifty four percent (37/68) of the dye-injected, inseminated bovine eggs were fertilized and 43% (16/37) of the fertilized eggs exhibited Ca2+ elevations during the time of measurement. All (16/16) of the eggs with Ca2+ elevations were fertilized but none of the unfertilized eggs (0/31) showed intracellular Ca2+ elevations. Six of 13 eggs that were later examined and found to be fertilized at the time of the Ca2+ recordings did not show sperm-induced Ca2+ elevations. Fifty percent (8/16) of the eggs with Ca2+ elevations exhibited a single Ca2+ rise as a response to sperm penetration during the 60-min period in which these eggs were monitored. Twelve percent (2/16) of the eggs responded with two Ca2+ elevations spaced by 50- and 51-min intervals and 38% (6/16) of the eggs exhibited multiple elevations with intervals of 15-29 min. In the latter group, one egg was polyspermic. The mean amplitude of the sperm-induced Ca2+ elevations was 564 +/- 58 nM. Eggs with single elevations reached higher peak concentrations than eggs with multiple elevations (p < 0.05). The mean duration of the Ca2+ elevations was 166 +/- 13 sec and was similar among eggs with different Ca2+ patterns. The first elevations detected occurred at a mean of 6.6 +/- 0.5 h after insemination. Fertilization in this study was confirmed by looking at pronuclear formation 16 h post-insemination or by DNA staining immediately after the fluorescence readings. Eggs exhibiting Ca2+ elevations ranged in stage of fertilization from just penetrated to pronuclear. Injection of inositol 1,4,5 trisphosphate (5 microM in the injection pipette) into 6 bovine eggs induced an immediate Ca2+ elevation with a mean peak Ca2+ value of 700 +/- 60 nM and a mean duration of 103 +/- 21 sec. Incubation of bovine eggs with 200 microM thimerosal induced periodic Ca2+ rises. The mean number of Ca2+ elevations observed in 35 min of recordings was 3.0 +/- 0.5 (n = 9, range 1-5). The mean peak Ca2+ value of the first thimerosal-induced Ca2+ elevation was 990 +/- 210 nM. The results of this study indicate that fertilization can evoke intracellular Ca2+ elevations in bovine eggs and that the periodicity of these Ca2+ elevations is different among eggs. Furthermore, both inositol 1,4,5-trisphosphate and thimerosal were able to induce intracellular Ca2+ release in bovine eggs.

Research paper thumbnail of Transgenic expression of human CD47 markedly increases engraftment in a murine model of pig-to-human hematopoietic cell transplantation

American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 2014

Mixed chimerism approaches for induction of tolerance of solid organ transplants have been applie... more Mixed chimerism approaches for induction of tolerance of solid organ transplants have been applied successfully in animal models and in the clinic. However, in xenogeneic models (pig-to-primate), host macrophages participate in the rapid clearance of porcine hematopoietic progenitor cells, hindering the ability to achieve mixed chimerism. CD47 is a cell-surface molecule that interacts in a species-specific manner with SIRPα receptors on macrophages to inhibit phagocytosis and expression of human CD47 (hCD47) on porcine cells has been shown to inhibit phagocytosis by primate macrophages. We report here the generation of hCD47 transgenic GalT-KO miniature swine that express hCD47 in all blood cell lineages. The effect of hCD47 expression on xenogeneic hematopoietic engraftment was tested in an in vivo mouse model of human hematopoietic cell engraftment. High-level porcine chimerism was observed in the bone marrow of hCD47 progenitor cell recipients and smaller but readily measurable c...