- Email: [email protected]
Glucose uptake and lactate production of equine cumulus-oocyte complexes during in vitro maturation
Abstracts / Journal of Equine Veterinary Science 41 (2016) 51e84
Stables, P.O. Box 77338, Abu Dhabi, UAE; 2 Department of Clinical Sciences, University of California Davis, USA *Corresponding author: [email protected] **Current affiliation: William R. Pritchard Veterinary Medical Teaching Hospital, School of Veterinary Medicine, University of California, Davis, USA The aim of this retrospective study was to compare the number of embryos obtained over two breeding seasons from 33 apparently fertile purebred Arabian mares, which differed in age, the absence or occurrence of laminitis and the presence of normal versus increased ACTH levels in peripheral blood. The mares were flushed on Day 8 after ovulation following insemination with fertile semen. Only 11 of 48 (23%) flushes from mares aged >19 years produced an embryo, compared to 22 of 52 (42%) flushes from mares aged 15-19 years and 30 of 42 (65%) from mares aged < 15 years. Furthermore, only 4 of 22 (18%) mares with chronic laminitis produced an embryo compared to 18 of 30 (60%) of mares not showing laminitis. Only 4 of the 22 (18%) mares, which showed an elevated serum ACTH concentration when flushed, produced an embryo, compared to 8 of 23 (35%)mares flushed when they exhibited normal ACTH levels. Therefore it is concluded that healthy, non-laminitic Arabian mares can produce normal embryos up to 26 years of age although the recovery rate declines with increasing age beyond 15 years. Mares suffering laminitis and/or showing elevated levels of serum ACTH exhibit a marked decline in embryo production. Curiously, the embryos recovered from laminitic mares resulted in pregnancies in every recipient whereas none of the otherwise normal looking embryos from donor mares with elevated ACTH resulted in pregnancies when transferred to well synchronised recipient mares.
Acknowledgements The authors would like to recognize the support of HH Sheik Sultan Bin Zayed Al Nayhan, and his unique vision for caring of the animals in the Middle East.
19 Glucose uptake and lactate production of equine cumulus-oocyte complexes during in vitro maturation Jose Len 1, *, Melanie McDowall 2, Marie Anastasie 2, Dave Kleeman 3 1 School of Animal and Veterinary Sciences, The University of Adelaide, Roseworthy, SA 5371, Australia; 2 Robinson Research Institute, School of Pediatrics and Reproductive Health, The University of Adelaide, Adelaide, SA 5005, Australia; 3 South Australian Research and Development Institute, Turretfield Research Center, SA 5350, Australia *Corresponding author: [email protected] During IVM, bovine and mouse cumulus-oocyte complexes (COC's) utilize glucose as the main energy substrate for final oocyte maturation, and the majority of glucose is metabolized through glycolysis. The uptake and metabolic pathway of glucose during IVM of equine COC's has not been investigated. The objectives of the study were to determine the uptake and metabolic pathway of glucose during IVM of equine COC's. We hypothesized that equine COC's will consume and metabolize glucose via glycolysis during IVM. Ovaries were collected from a local abattoir and transported to the laboratory in a Styrofoam™ box (22 - 25ºC). At arrival to the laboratory, ovaries were rinsed with sterile saline at room temperature (22 e 25 C), dried with gauze and the tunica albuginea trimmed. COC's were collected from each visible follicle by a
59
combination of scraping and aspiration using a 10 mL syringe with an 18 G disposable needle. Recovered COC's were divided into the following groups: COC's with cumulus cells (COC's-CC’s) or COC's with corona radiata only (COC's-corona). The COC's-CC’s group was further divided into compacted (Cp) or expanded (Ex) COC's. COC's were matured in groups of five in a 50 mL drop of sodium bicarbonate-buffered TCM199 supplemented with 10% (v/v) FCS, 1 mL/ mL insulin-transferrin-selenium (ITS; Gibco™; Life Technologies, VIC, AUS), 1 mM sodium pyruvate, 100 mM cysteamine, 0.1 mg/mL FSH (Puregon; Organon, Netherland) and 2% antibiotic-antimycotic (Anti-Anti; Gibco™, Life Technologies, VIC, AUS) covered with mineral oil (Sigma, MO, USA) at 38.6 C in a humidified atmosphere of 5% CO2 in air for 30 h. After maturation COC's were removed, the spent medium recovered from each group and stored at -80ºC until analysis. Spent maturation medium was thawed from each group of COC's then glucose and lactate concentration was determined using a chemical analyzer (COBAS Integra 400 plus, Roche Diagnostics Ltd., Switzerland). Glucose uptake and lactate production (mean ± SEM nmol COC-1) between COC's-CC’s and COC's-corona, and between Cp and Ex treatment groups, were analyzed using student's t-test with statistical significance at p < 0.05. COC's-CC’s glucose uptake (0.78 ± 0.07 nmol COC-1) was higher than COC'scorona (0.41 ± 0.14 nmol/ml COC-1); however lactate production of COC's-CC’s (1.57 ± 0.12 nmol COC-1) was not different than COC'scorona (0.96 ± 0.27 nmol COC-1). Cp glucose uptake (0.93 ± 0.07 nmol COC-1) was higher than Ex (0.65 ± 0.07 nmol COC-1); however Cp lactate production (1.74 ± 0.16 nmol COC-1) was not different than Ex (1.41 ± 0.14 nmol COC-1). Our results confirm our hypothesis that equine COC's consume and metabolize glucose via glycolysis during IVM, as approximately 2 molecules of lactate were produced per molecule of glucose. The presence of CC's increased glucose uptake by COC's. Interesting was the finding that Cp consumed more glucose compared to Ex. The reason for this difference cannot be explained with the results from this study; however we can hypothesized that since most Cp originate.
20 Thawed stallion sperm refrozen without cryoprotectants activates cow oocytes after intracytoplasmic sperm injection (ICSI) Jose Len 1, *, Dave Kleeman 2, Jen Kelly 2 1 School of Animal and Veterinary Sciences, The University of Adelaide, Roseworthy, SA 5371, Australia; 2 South Australian Research and Development Institute, Turretfield Research Center, SA 5350, Australia *Corresponding author: [email protected] Refreezing thawed stallion semen for further assisted reproductive techniques such as ICSI has proven a viable option to produce embryos and efficiently use frozen stallion semen; however the capacity of stallion sperm refrozen without cryoprotectant to activate oocytes after ICSI is unknown. This project was performed to evaluate the capacity of stallion sperm refrozen without cryoprotectants to activate bovine oocytes. We hypothesize that stallion sperm refrozen without cryoprotectants will have the capacity to activate bovine oocytes. Abattoir cow cumulus-oocyte complexes (COC's) were matured in vitro for 22 h and oocytes in metaphase II (MII) selected for ICSI. A semen straw (0.5 mL) from a fertile stallion frozen with cryoprotectants (SFrozen-thawed) was thawed and subjected to swim up sperm selection. One mL of the selected sperm suspension was immediately used for ICSI and the rest aspirated into a 0.5 mL straw, frozen without cryoprotectants (SSnap-thawed) and stored in liquid nitrogen for at least a week before use for injection. During each Piezo-driven ICSI session, treatment groups were a) oocytes
Stables, P.O. Box 77338, Abu Dhabi, UAE; 2 Department of Clinical Sciences, University of California Davis, USA *Corresponding author: [email protected] **Current affiliation: William R. Pritchard Veterinary Medical Teaching Hospital, School of Veterinary Medicine, University of California, Davis, USA The aim of this retrospective study was to compare the number of embryos obtained over two breeding seasons from 33 apparently fertile purebred Arabian mares, which differed in age, the absence or occurrence of laminitis and the presence of normal versus increased ACTH levels in peripheral blood. The mares were flushed on Day 8 after ovulation following insemination with fertile semen. Only 11 of 48 (23%) flushes from mares aged >19 years produced an embryo, compared to 22 of 52 (42%) flushes from mares aged 15-19 years and 30 of 42 (65%) from mares aged < 15 years. Furthermore, only 4 of 22 (18%) mares with chronic laminitis produced an embryo compared to 18 of 30 (60%) of mares not showing laminitis. Only 4 of the 22 (18%) mares, which showed an elevated serum ACTH concentration when flushed, produced an embryo, compared to 8 of 23 (35%)mares flushed when they exhibited normal ACTH levels. Therefore it is concluded that healthy, non-laminitic Arabian mares can produce normal embryos up to 26 years of age although the recovery rate declines with increasing age beyond 15 years. Mares suffering laminitis and/or showing elevated levels of serum ACTH exhibit a marked decline in embryo production. Curiously, the embryos recovered from laminitic mares resulted in pregnancies in every recipient whereas none of the otherwise normal looking embryos from donor mares with elevated ACTH resulted in pregnancies when transferred to well synchronised recipient mares.
Acknowledgements The authors would like to recognize the support of HH Sheik Sultan Bin Zayed Al Nayhan, and his unique vision for caring of the animals in the Middle East.
19 Glucose uptake and lactate production of equine cumulus-oocyte complexes during in vitro maturation Jose Len 1, *, Melanie McDowall 2, Marie Anastasie 2, Dave Kleeman 3 1 School of Animal and Veterinary Sciences, The University of Adelaide, Roseworthy, SA 5371, Australia; 2 Robinson Research Institute, School of Pediatrics and Reproductive Health, The University of Adelaide, Adelaide, SA 5005, Australia; 3 South Australian Research and Development Institute, Turretfield Research Center, SA 5350, Australia *Corresponding author: [email protected] During IVM, bovine and mouse cumulus-oocyte complexes (COC's) utilize glucose as the main energy substrate for final oocyte maturation, and the majority of glucose is metabolized through glycolysis. The uptake and metabolic pathway of glucose during IVM of equine COC's has not been investigated. The objectives of the study were to determine the uptake and metabolic pathway of glucose during IVM of equine COC's. We hypothesized that equine COC's will consume and metabolize glucose via glycolysis during IVM. Ovaries were collected from a local abattoir and transported to the laboratory in a Styrofoam™ box (22 - 25ºC). At arrival to the laboratory, ovaries were rinsed with sterile saline at room temperature (22 e 25 C), dried with gauze and the tunica albuginea trimmed. COC's were collected from each visible follicle by a
59
combination of scraping and aspiration using a 10 mL syringe with an 18 G disposable needle. Recovered COC's were divided into the following groups: COC's with cumulus cells (COC's-CC’s) or COC's with corona radiata only (COC's-corona). The COC's-CC’s group was further divided into compacted (Cp) or expanded (Ex) COC's. COC's were matured in groups of five in a 50 mL drop of sodium bicarbonate-buffered TCM199 supplemented with 10% (v/v) FCS, 1 mL/ mL insulin-transferrin-selenium (ITS; Gibco™; Life Technologies, VIC, AUS), 1 mM sodium pyruvate, 100 mM cysteamine, 0.1 mg/mL FSH (Puregon; Organon, Netherland) and 2% antibiotic-antimycotic (Anti-Anti; Gibco™, Life Technologies, VIC, AUS) covered with mineral oil (Sigma, MO, USA) at 38.6 C in a humidified atmosphere of 5% CO2 in air for 30 h. After maturation COC's were removed, the spent medium recovered from each group and stored at -80ºC until analysis. Spent maturation medium was thawed from each group of COC's then glucose and lactate concentration was determined using a chemical analyzer (COBAS Integra 400 plus, Roche Diagnostics Ltd., Switzerland). Glucose uptake and lactate production (mean ± SEM nmol COC-1) between COC's-CC’s and COC's-corona, and between Cp and Ex treatment groups, were analyzed using student's t-test with statistical significance at p < 0.05. COC's-CC’s glucose uptake (0.78 ± 0.07 nmol COC-1) was higher than COC'scorona (0.41 ± 0.14 nmol/ml COC-1); however lactate production of COC's-CC’s (1.57 ± 0.12 nmol COC-1) was not different than COC'scorona (0.96 ± 0.27 nmol COC-1). Cp glucose uptake (0.93 ± 0.07 nmol COC-1) was higher than Ex (0.65 ± 0.07 nmol COC-1); however Cp lactate production (1.74 ± 0.16 nmol COC-1) was not different than Ex (1.41 ± 0.14 nmol COC-1). Our results confirm our hypothesis that equine COC's consume and metabolize glucose via glycolysis during IVM, as approximately 2 molecules of lactate were produced per molecule of glucose. The presence of CC's increased glucose uptake by COC's. Interesting was the finding that Cp consumed more glucose compared to Ex. The reason for this difference cannot be explained with the results from this study; however we can hypothesized that since most Cp originate.
20 Thawed stallion sperm refrozen without cryoprotectants activates cow oocytes after intracytoplasmic sperm injection (ICSI) Jose Len 1, *, Dave Kleeman 2, Jen Kelly 2 1 School of Animal and Veterinary Sciences, The University of Adelaide, Roseworthy, SA 5371, Australia; 2 South Australian Research and Development Institute, Turretfield Research Center, SA 5350, Australia *Corresponding author: [email protected] Refreezing thawed stallion semen for further assisted reproductive techniques such as ICSI has proven a viable option to produce embryos and efficiently use frozen stallion semen; however the capacity of stallion sperm refrozen without cryoprotectant to activate oocytes after ICSI is unknown. This project was performed to evaluate the capacity of stallion sperm refrozen without cryoprotectants to activate bovine oocytes. We hypothesize that stallion sperm refrozen without cryoprotectants will have the capacity to activate bovine oocytes. Abattoir cow cumulus-oocyte complexes (COC's) were matured in vitro for 22 h and oocytes in metaphase II (MII) selected for ICSI. A semen straw (0.5 mL) from a fertile stallion frozen with cryoprotectants (SFrozen-thawed) was thawed and subjected to swim up sperm selection. One mL of the selected sperm suspension was immediately used for ICSI and the rest aspirated into a 0.5 mL straw, frozen without cryoprotectants (SSnap-thawed) and stored in liquid nitrogen for at least a week before use for injection. During each Piezo-driven ICSI session, treatment groups were a) oocytes