Pyruvate Lactate Research Articles

22 The zona pellucida is required for normal development of invitro-produced cat embryos

Limited information have been reported regarding to the role of the zona pellucida during embryo development in the domestic cat. In the present research we compared invitro and invivo development of cat embryos generated with versus without the zona pellucida. Embryos were produced by IVF and cultured up to blastocyst stage accordingly to experimental group: 1) with intact zona pellucida (ZI) and 2) without zona pellucida (ZF). Ovaries of domestic cats were collected by ovariohysterectomy and cumulus-oocyte complexes (COCs) were recovered by slicing. For IVM, COCs were cultured in supplemented medium-199 for 26-28h in a 5% CO2, 5% O2, and 90% N2 atmosphere at 38.5°C. For IVF, 1.5-2.5×106 epididymal spermatozoa/mL were incubated with 20-30 COCs in supplemented Tyrode's albumin lactate pyruvate medium for 18h in a 5% CO2 atmosphere at 38.5°C. The zona pellucida of the presumed zygotes was removed by 2-4min of incubation in 2mgmL−1 pronase. In ZI and ZF groups, embryo culture was done in 4-well dishes; in the ZF group the well of the well system was used. The IVC was done in supplemented synthetic oviductal fluid medium, in a 5% CO2, 5% O2, and 90% N2 atmosphere at 38.5°C for 8 days. The frequencies of cleavage and development to the morula and blastocyst stages were determined. The relative expression of the pluripotency markers OCT4, SOX2, and NANOG was evaluated by RT-qPCR in the blastocysts using the standard curve method. The SDHA gene was used as internal control. Additionally, the invivo development was evaluated. For this, cat recipients were synchronized with 200IU equine chorionic gonadotrophin followed by 100IU human chorionic gonadotrophin 4 days later, and embryo transfers (ET) were made by mid-ventral laparotomy. The Wilcoxon nonparametric test was used to evaluate the developmental competence and the gene expression analysis. Nine and six replicates were performed in the ZI and ZF groups, respectively. No differences were observed between the ZI and ZF groups in the cleavage rate: 155/239 (64.9%) and 116/177 (65.5%), morulae rate: 115/155 (74.2%) and 68/116 (58.6%), and blastocysts rate: 51/155 (32.9%) and 36/116 (31.0), respectively (P>0.05). No differences were observed in the expression of OCT4 (P>0.05). However, the expression of SOX2 and NANOG was lower in blastocysts from the ZF group than in those from the ZI group (P<0.05). Finally, three ET were done at the blastocyst stage in both groups. In the ZI group, 8-16 Day 7 blastocysts were transferred per cat, two pregnancies were obtained with one gestational sac in each cat (2/3, 66.6%), and one live kitten was born at Day 64 of pregnancy (1/3, 33.3%). In the ZF group, 7-14 were transferred per cat, and no pregnancies were obtained after ET. In conclusion, in the domestic cat, zona pellucida removal at the presumed zygote stage did not affect invitro development to blastocyst but affect negatively the expression of SOX2 and NANOG. However, the low survival after ET of ZI embryos is a possible indicator that embryo quality might be affecting survival rates. Nonetheless, these results add credence to previous ET studies that provide indirect evidence of a crucial role of the zona pellucida for successful implantation of cat embryos.

83 Validation of propidium iodide dye for live-dead staining of bovine blastocysts: Preliminary results

A widely used criterion routine laboratories utilise for the selection and classification of oocytes and embryos is morphological appearance. In order to make an objective validation of produced embryos, it is necessary to have a method that can help to distinguish viable cells. One simple and easy-to-perform method is the live-dead staining protocol. Usually, ethidium homodimer (EthD) is used to stain the dead cells, in conjunction with Hoechst 33342 for total cell numbers (Stinshoff et al. 2011). However, EthD is a harmful, toxic, and expensive staining dye. Therefore, the aim of this study was to evaluate propidium iodide (PI) as a viable alternative to EthD. To assess the suitability of PI, groups of 20 cumulus-oocyte complexes were collected from abattoir-derived bovine ovaries and matured in tissue culture medium containing bovine serum albumin and equine chorionic gonadotropin/human chorionic gonadotropin for 24h with 5% CO2. The cumulus-oocyte complexes were then fertilised in Fert-TALP (Tyrode's albumin lactate pyruvate) for 19h with 5% CO2 and finally cultured (n=968) in SOFaa (synthetic oviductal fluid with amino acids) with 5% CO2 and 5% O2 with all steps being performed at 39°C. The development rate on Day 8 was on average 25.4%. Expanded Day 8 blastocysts were then utilised to validate the two different staining protocols. Expanded blastocysts were collected from culture drops and washed in PBS containing polyvinyl alcohol. The blastocysts were then stained with the selective dye and finally counterstained to determine live and dead cells. Four alternative parameters were evaluated: (A) PI 1:5 for 15min, (B) PI 1:10 for 15min, (C) PI 1:5 for 5min, and (D) EthD 1:10 for 15min (control). Counterstaining with Hoechst 33342 was performed for 3min in all groups tested. A minimum number of 10 replicate stainings ((A) n=13, (B) n=13, (C) n=12, (D) n=10) was carried out. The evaluation was performed under a fluorescence microscope (Nikon Eclipse Ci) with the NIS-Elements imaging software (Nikon). The statistical analysis was assessed using R. All data were analysed for normal distribution using a Shapiro-Wilk test. Evaluation of the data was made through the application of t-tests. The total cell numbers observed were in the known range for expanded blastocysts: (A) 106.3±15.3, (B) 109.5±18.8, (C) 119.3±15.0, and (D) 113.1±21.8. The number of dead cells did not differ significantly from the control: (A) 13.3±7.7, (B) 8.5±5.0, (C) 11.8±8.7, and (D) 9.3±2.3. The study indicates that PI could be used as an adequate alternative for EthD. Furthermore, it is possible that the concentration or the incubation time could be reduced to generate similar results to EthD. Further replicates are currently being examined in order to corroborate these results. We gratefully acknowledge our colleague M. Vopel.

38 Ram sperm longevity after cryopreservation in extender containing L-carnitine

The cryopreservation process causes oxidative stress to the sperm cell, and the addition of antioxidants to the extender for semen freezing helps sperm protection. This study assessed the effect of L-carnitine (LC) concentrations (0, 5, or 10mM LC) on two ram semen extenders (Tris-egg yolk or the commercial optiXcell IMV medium (IMV Technologies)) for semen cryopreservation. Four Santa Inês rams were used during the breeding season. After semen collection, macroscopic and microscopic evaluations were performed, and a pool of semen was formed. The semen was diluted, and the final concentration was 100×106 per 0.25-mL straw. Cryopreservation was performed with a cooling rate of 0.25°C min−1 until 5°C, and the freezing rate used was 20°C min−1 from 5 to −120°C. After the freezing-thawing process and throughout incubation (38°C in 5% CO2) in Fert-Tyrode's albumin lactate pyruvate medium, every 1h for up to 3h, several parameters were evaluated: sperm kinetics, hypo-osmotic test, plasma membrane integrity, capacitation status, and lipid peroxidation level. We did not find any protective effect of LC on plasma membrane integrity, hypo-osmotic test, and capacitation status. The sperm kinetics values throughout incubation showed that Tris extender promoted better indices of staight-line velocity, linearity, wobble, and straightness than IMV extender along incubation, regardless of the presence of LC. There were no benefits of the LC addition throughout the incubation, and 10mM was deleterious to few parameters (amplitude of lateral head displacement, linearity, and wobble) compared with the control (0mM) in the Tris extender group. The plasma membrane integrity analysis revealed no differences (P>0.05) among the groups. The average number of intact cells (hypo-osmotic) was higher in Tris extender groups supplemented with 10mM LC at 1h and 5mM LC at 2h compared to the respective extender IMV groups. Regarding capacitation status, the Tris 5mM LC group had more acrosome-reacted cells when compared with the IMV 5mM LC group at 2h. At 3h, the percentage of acrosome-reacted cells was higher in the Tris 0-mM group when compared with the IMV 0-mM group. Regardless the presence of LC, IMV had higher (P<0.05) lipoperoxidation than the Tris treatments. In conclusion, LC supplementation in semen extender had no beneficial effect on freezing-thawing ram sperm and throughout incubation for up to 3h, with no difference in each time point evaluated. Under the conditions of this study, the use of Tris extender was superior to IMV extender for ram sperm. Financial support for this work came from the Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (Young Scientist Program of Our State; E-26/203.168/2017).

146 Tributyltin chloride exposure alters ejaculated bull sperm function and embryo development

Male exposure to environmental toxicants can disrupt spermatogenesis and impair sperm function. However, the consequences of environmentally relevant levels of toxicants to ejaculated mammalian spermatozoa on sperm function and male fertility are not well studied. Tributyltin chloride (TBT) is an organotin with historical use as an antifouling agent in paints and is a contaminant of soil and groundwater in the United States. Tributyltin chloride is an endocrine disruptor, is detectable in human cord blood, and has negative effects on female reproduction. We hypothesised that TBT could affect sperm function and thereby affect male fertility. To test our hypothesis, we exposed frozen-thawed bull sperm to environmentally relevant doses of TBT (0, 0.1, 1.0, 10, and 100nM) for 90min and then measured sperm motility parameters, fertilisation, and embryo development by IVF. Briefly, frozen-thawed sperm from two bulls were isolated through a 45:90 Percoll gradient, pooled, and then maintained in noncapacitating conditions at 37°C in Tyrode's albumin lactate pyruvate medium devoid of bovine serum albumin and HCO3 − for 90min. Vehicle control (VC) samples consisted of 0.1% MeOH. Sperm motility kinematics were objectively measured after the addition of treatment and every 30min thereafter using computer-aided sperm analysis (IVOS System, Hamilton Thorne). Five replicates were evaluated, and differences in motility kinematics were analysed by analysis of variance using SAS statistical software (SAS Institute Inc.). Sperm treated with 100nM TBT displayed decreased total motility (88 vs. 79%), progressive motility (80 vs. 70%), curvilinear velocity (100 vs. 88 µ/s), and beat-cross frequency (38 vs. 34Hz) over 90min compared with the VC samples (P<0.05). No differences (P>0.05) were detected among any other treatments. Following 90min of exposure to TBT 100nM, sperm were washed twice by centrifugation and re-extended in fertilisation medium. Abattoir-derived bovine oocytes were fertilised with 100nM TBT and VC-exposed sperm. Embryo cleavage and 8- to 16-cell embryos were quantified at 48 and 72h, respectively, in three replicates, and results were assessed using chi-square analysis. Embryos fertilised by TBT-exposed sperm had reduced cleavage to 2-cell (80 vs. 62%) and 8- to 16-cell morulae stages (56 vs. 24%, respectively; P<0.05). In summary, although sperm kinematics were decreased in TBT-exposed sperm, gross motility parameters remained within acceptable ranges for IVF, suggesting that sperm motility alone is not a sufficient measure of sperm function or indicator of male fertility. In conclusion, ejaculated bull sperm exposed to environmentally relevant levels of TBT for 90min had reduced sperm motility parameters, impaired sperm function, and reduced embryo development potential. Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under award number T32HD087166. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

37 In vitro maturation and fertilization in white-tailed deer (Odocoileus virginianus) oocytes vitrified with trehalose or sucrose

White-tailed deer (WTD) invitro embryo production is not well documented, due to the seasonality of the wildlife species and available hunting permits. The objective of the present study was to conduct IVM and IVF using WTD oocytes vitrified with trehalose (TH) or sucrose (SC). A total of 121 immature oocytes were obtained from ovaries (n=18) using the slicing technique from hunter-slaughtered deer, not more than 2h after death. The WTD oocytes were vitrified using a solid surface vitrification technique in two different groups: 1) TH (n=60) and 2) SC (n=61). Oocyte warming (OW) was done using four concentrations (OW1 1 M, OW2 0.5 M, OW3 0.25 M, and OW4 0 M) in the oocytes after thawing. Then, a sample was used to evaluate viability for TH (n=5) and SC (n=5) (MTT stain (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), 0.5mgmL−1) and nuclear status (NS) for TH (n=4) and SC (n=5) with 4′,6-diamidino-2-phenylindole (1μgmL−1), defining the stage as germinal vesicle (GV), MI, and unable to evaluate (NE). The remaining oocytes (n=88) were used for IVM for 36h in tissue culture medium-199 supplemented with human menopausal hormone (75 UImL−1) and epidermal growth factor (10ngmL−1). The evaluation of NS was defined (GV, MI considered immature and MII matured). The IVF (n=15) took place in Tyrode's albumin lactate pyruvate media supplemented with heparin (10μgmL−1), penicillamine (0.075mgmL−1), hypotaurine (10M), epinephrine (1μM), and bovine serum albumin fraction V (0.4%), with a final semen concentration of 3×106 spermmL−1. Frozen straws of conventional WTD semen were used; the straws were split into 3 parts, with one fraction thawed and capacitated using “swim-up” technique. After 24h of incubation at 38.5°C with 5% CO2, and humidified air, nuclear evaluation was made as fertilized (F), not fertilized (NF), or NE. A Fisher exact test was used (SAS, 9.0 version for Windows), α=0.05. After warming, for the TH group (n=5), viability was 60% and nuclear status (n=4) was 50% GV, 50% MI, and 0% NE. For the SC group (n=5), viability was 40% and nuclear status (n=5) was 60% GV, 20% MI, and 20% NE. After 36h IVM, NS evaluation in the TH group (n=38) was 66% GV, 24% MI, 0% MII, and 10% NE; for the SC group (n=50), 84% GV, 10% MI, 2% MII, and 4% NE, not a statistically significant difference (P>0.05). For IVF after 24h, the NS evaluation in the TH treatment (n=10) was 0% F, 60% NF, and 40% NE versus the SC treatment (n=5) with 20% F, 40% NF, and 40% NE, not a statistically significant difference (P>0.05). A statistically significant difference (P>0.05) was not found between the TH and SC groups with regard to post-thaw viability, IVM, and IVF. Future research with larger numbers of immature or mature oocytes is suggested for further evaluation of both TH and SC for WTD oocyte vitrification for use with invitro-production techniques as a model for other endangered cervid species.

143 Stimulated glycolysis is able to maintain ATP levels and motility of bull spermatozoa submitted to mitochondrial depolarisation

Studies have reported the importance of mitochondria in sperm metabolism. However, for some species, glycolysis appears to be as essential as oxidative phosphorylation in sperm physiology. On the other hand, these mechanisms have not been fully elucidated for bovine spermatozoa. Therefore, the aim of this study was to evaluate the role of mitochondria and glycolysis in ATP synthesis and sperm kinetics of bovine spermatozoa. For this purpose, sperm from seven bovine epididymides (n=7) was collected and diluted to a concentration of 100×106 spermatozoamL−1 in Tyrode's albumin lactate pyruvate medium. Then, each sample was divided into 10 aliquots and evaluated in a 2×5 factorial design, with the first factor being the presence or absence of glucose (5mM) to stimulate glycolysis and the second factor being treatment with the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; 0, 0.1, 0.3, 1, and 3 µM) to deplete mitochondrial ATP. Sperm samples were subjected to measurements of ATP levels using a luminescence technique (CellTiter-Glokit, Promega), with ATP levels measured in duplicate. In addition, sperm samples were subjected to computerised analysis of total and progressive motilities (ISAS, Proiser). Statistical analysis was performed using SAS (SAS Institute Inc.), where the interaction between the factors was analysed using PROC GLM and the comparison between groups was performed using means analysis of variance (least significant difference test). It was considered significant at 5%. Adenosine triphosphate was lower at FCCP concentrations of 0.3 µM (180.3±31.9nM), 1 µM (220.2±40.4nM), and 3 µM (272.3±70.4nM) than at 0 µM (control; 448.6±63.7nM) and 0.1 µM (422.4±41.5nM) in the absence of glucose. However, in the groups treated with FCCP supplemented with glucose, ATP concentrations did not differ among the groups (0 µM: 577.2±70.4 nM; 0.1 µM: 610.8±57.8 nM; 0.3 µM: 606.2±64.2 nM; 1 µM: 670.9±61.9 nM; 3 µM: 696.1±68.5nM). Additionally, total motility was lower in FCCP-treated groups without glucose supplementation. On the other hand, total motility increased in the groups treated with 0.3, 0.1, 1, and 3 µM FCCP supplemented with glucose. A similar effect was verified for progressive motility. Based on these results, we can suggest that glucose supplementation is able to maintain ATP levels and motility in bull sperm undergoing FCCP-induced mitochondrial depolarisation.

117 Supplementation of IVF medium with nerve growth factor improved bovine embryonic cleavage rates during summer months

Nerve growth factor-β (NGF), a protein originally associated with regulation of neuron development, has been found to play a role in the reproductive system of mammals. Previous research showed that administration of NGF to cows resulted in enhanced conceptus development. Although these effects were speculated to be a result of improved corpus luteum function, whether NGF could act directly on the embryo remained undetermined. Therefore, the direct effects of NGF on fertilization and embryo development warrant investigation to see whether it can be used as a novel tool to improve cleavage and blastocyst rates when producing embryos via IVF during periods of suboptimal oocyte quality, such as with heat stress. The objective of this study was to explore how supplementation of NGF, purified from bull seminal plasma, during IVF may directly affect embryo development in oocytes harvested in the summer. Abattoir-derived bovine ovaries were used for recovery of cumulus-oocyte complexes (COCs) over eight replicates through May and June. On Day −1, COCs were collected and matured for 20h in oocyte maturation medium incubated at 38.5°C. On Day 0, matured oocytes were added to a solution of IVF-Tyrode's albumin lactate pyruvate (TALP) and either phosphate-buffered saline (PBS; control) or 100ngmL−1 NGF. Pooled frozen-thawed semen from two different bulls per replicate were added to the IVF solutions and incubated with COCs for 20h at 38.5°C in a humidified atmosphere of 5% CO2. On Day 1, zygotes were washed in HEPES-TALP, and cumulus cells were removed using 1% hyaluronidase. The zygotes were plated in synthetic oviductal fluid (SOF-BE2) culture medium and incubated at 38.5°C in a tri-gas chamber (5% CO2, 5% O2, and balanced N2). Cleavage rates were recorded at 24 and 48h, calculated by dividing the number of cleaved embryos by the total zygote count. Embryos were incubated until Day 8, when the rate of blastocysts was assessed. This study found that the treatment of IVF medium with NGF increased the cleavage rate of embryos after 48h (Control: 59%; NGF: 66%; P=0.04) and the hatched blastocyst percentage per oocyte on Day 8 (Control: 6.7%; NGF: 9.4%; P=0.01). The treatment did not affect the percentage of blastocysts per cleaved embryos (Control: 21%; NGF: 22%; P=0.16) or the hatched blastocyst rate at Day 8 (Control: 53%; NGF: 55%; P=0.67). These results show that NGF can act directly on the oocyte during fertilization to alter subsequent development, specifically through increased embryonic cleavage rates. Further studies are needed to assess different dosages of NGF in order to mitigate the detrimental effects of heat stress on oocyte competence for use in IVF. Follow-up studies using a whole-animal model are needed to understand the clinical relevance of these findings in the ability of embryos to promote maternal recognition of pregnancy.

Multimodality monitoring in neurocritical care

Monitoring the health of an injured brain is essential to forewarn neurological worsening and to gain insight into the pathophysiology of a complex disorder.Clinical examination remains a cornerstone in monitoring patients with brain injury. The Glasgow coma score (GCS) is widely used but lacks information regarding brain stem functions like pupillary reaction and shows moderate inter-observer reliability. However, despite these shortcomings, GCS remains a robust indicator of the need for surgery and prognosis after cardiac arrest, hypoxic brain injury, and posterior circulation stroke. A new scoring system called Full Outline of Unresponsiveness has been proposed and shows excellent inter-observer reliability and includes points concerning brain stem functions.1 The most commonly used monitoring modality is intracranial pressure (ICP) monitoring. ICP shows threshold physiology where the outcome of the patients changes after a threshold of 20 to 25 mmHg. Refractory ICP is a good predictor of mortality but not of the functional outcome after traumatic brain injury.Brain injury causes varying degrees of disruption to cerebral blood flow and its autoregulation. Studying autoregulation provides a useful strategy for targeting cerebral perfusion pressure close to the autoregulatory range. Transcranial Doppler and ICMplus (Intensive Care Monitoring) are used to study autoregulation. ICMplus is a software-based tool that studies the correlation between slow changes in mean arterial pressure and ICP to evaluate the state of autoregulation throughout the duration of ICP monitoring.2 Brain tissue oxygen measures the partial pressure of oxygen in the extracellular fluid of the neural tissue. Reduction in brain tissue oxygen is a marker of cellular distress. A phase 2 trial on brain tissue oxygen monitoring demonstrated the safety and feasibility of the protocol-based management of brain tissue oxygenation and ICP, and a trend towards lower mortality and improved functional outcome in patients treated with combined oxygen and ICP protocol.3 Microdialysis is a point of care test that monitors substrate delivery and metabolism at the cellular level. The lactate-pyruvate (LP) ratio is an indicator of the redox state of cells and a high LP ratio is associated with an unfavourable outcome.4 The electroencephalogram (EEG) is used in critical care for monitoring sedation and diagnosis of seizure activity. EEG is a complex signal which requires advanced training and skills for interpretation. Novel EEG-based monitors are aimed at simplifying the signal for straightforward interpretation by bedside medical professionals. Cerebral function monitor (CFM) is a compressed single channel amplitude integrated EEG monitor mainly used for the detection of status epilepticus and burst suppression during thiopentone infusion. A novel technique that uses direct electrodes applied on the cortical surface called Electrocorticogram (ECoG) shows spreading depolarisations on the cortical surface that are caused by loss of ionic homeostasis and substrate delivery.5 These depolarisations are a sensitive indicator of impending neuronal death and may serve as a target for novel mechanistically oriented therapies.Detection, prevention, and monitoring of secondary cerebral insults that alter the prognosis from the injury, remains at the centre stage in neurocritical care. In the future, integrated informatics derived from multimodality monitoring will play a pivotal role in clinical decision algorithms.

Abstract 565: Response of the Ischemic Myocardium to Adrenergic Stimulation as Detected by Hyperpolarized [1- 13 C]pyruvate

Metabolic imaging by nuclear methods is widely used to access ischemic myocardium but provide limited information about individual enzyme-catalyzed reactions. 13 C hyperpolarization methods are sensitive to flux in specific key enzymes such as lactate dehydrogenase and pyruvate dehydrogenase. We tested the hypothesis that LDH flux is increased while PDH flux is abolished in low-flow ischemia. Metabolism of HP [1- 13 C]pyruvate was studied in four groups: (1) normal perfusion pressure (NPP), 100 cm H 2 O; (2) NPP with epinephrine; (3) low-perfusion pressure (LPP), 25 cm H 2 O; (4) LPP with epinephrine. Hearts excised from male Sprague-Dawley rats were perfused (37°C) at an initial pressure of 100 cm H 2 O using standard Langendorff method with Krebs-Henseleit buffer containing 0.75% bovine serum albumin, 0.4 mM non-labeled free fatty acid, 5.5 mM [U- 13 C]glucose, 1 mM [3- 13 C]pyruvate, 0.1 mM [3- 13 C]lactate. Perfusion pressure was reduced to 25 cm H 2 O after 30 min of perfusion for ischemia group and epinephrine added at 50 th min of perfusion. HP [1- 13 C]pyruvate was injected to heart and subsequently, 13 C NMR spectra were acquired. The freeze clamped heart tissues were extracted with perchloric acid and analyzed by high-resolution 1 H and 13 C NMR (14.1 T). The 31 P NMR spectroscopy confirmed significant myocardial ischemia under LPP conditions, demonstrated by a marked increase in the [Pi]/[ATP] ratio. Surprisingly, increased LDH flux was observed in the ischemic hearts (+18.4%) while the metabolism of HP [1- 13 C]pyruvate by PDH remained unchanged as indicated by bicarbonate production. To investigate the oxidative capacity of mitochondria, epinephrine was used to stimulate hearts. As anticipated, epinephrine increased heart rate (+23%) and coronary flow (+11%) under NPP. Epinephrine increased heart rate (-60%) but did not alter coronary flow under LPP. We found that epinephrine had no effect on the rate of bicarbonate production under LPP. The study demonstrated that the conversion of pyruvate to lactate increased in ischemic hearts but the degree of pyruvate oxidation was not affected by the low-flow ischemia. Adrenergic stimulation did not increase PDH flux in these ischemic hearts. Translation to human is likely to provide new data from low-flow myocardium.

Abstract 414: Postnatal Cardiac Tissue Harbors Progenitor Cells With Unique Metabolic Profile

Rationale: The developmental cardiac tissue is a proliferative organ capable of regeneration that extends briefly into the postnatal period. Studies show that the heart undergoes alterations in metabolism that coincide with cessation of regenerative processes during postnatal development. Whether cardiac cells in the early postnatal heart exhibit metabolic properties that favor regeneration remains unknown. Objective: To determine whether postnatal cardiac tissue harbors CPCs with unique metabolic profile. Methods and Results: CPCs were isolated from C57BL/6 mice aged 2-day (NCPC), 2-month (adult CPC) and 2-year old (ACPC). Morphological assessment showed NCPCs were smaller and rounded compared to CPCs and ACPCs and expressed putative stem cell markers and were negative for hematopoietic marker CD45. Interestingly, NCPCs expressed elevated levels of pluripotency markers such as LIN28 compared to ACPCs and CPCs. Increased viability, proliferation rate, metabolic activity and reduced doubling times were observed in NCPCs compared to CPCs and ACPCs measured by CyQuant, MTT, and Resazurin assays. NCPCs demonstrated a youthful phenotype and were resistant to H2O2 induced stress compared to ACPCs and CPCs as measured by β-Galactosidase and TUNEL labeling respectively together with a unique expression of paracrine factors as assessed by proteome profiler array. Interestingly, NCPCs demonstrated increased ATP generation, glycolytic activity and reduced oxidative phosphorylation compared to CPCs and ACPCs measured by seahorse assay parallel with enhanced expression of glycolytic enzymes measured by qRT-PCR and western blot. NCPCs showed increased lactate production and pyruvate kinase activity and low mitochondrial membrane potential. Interestingly, mitochondrial fuel dependency assay showed increased glutamine dependency as the fuel for mitochondrial oxidative phosphorylation. RNA-sequencing analysis demonstrated increased expression of metabolic signaling in NCPCs compared to CPCs and ACPCs. Conclusions: Postnatal cardiac tissue possesses progenitor cell population with unique metabolic profile that coincides with enhanced functional properties of NCPCs suggesting their potential therapeutic value for cardiac repair.

Amino Acid Profile and Lactate Pyruvate Ratio: Potential Adjunct Markers for Differentiating Inborn Errors of Metabolism.

Inborn errors of metabolism (IEM) lead to the physical and mental disability and death of infants, which can be prevented if treated early. Hence it is imperative in diagnosing these disorders at the earliest. The study is planned to differentiate suspected IEM by quantifying the lactate-pyruvate ratio (L/P ratio), Amino acid profiling by HPLC in addition to blood ammonia using the basic methodology in comparison to globally accepted mass spectroscopy technique and specific enzyme activity assays. The objective of this study is not to compete with the existing gold standard methodology rather makes the best of basic diagnostic modalities available. Five cases out of 100 referred cases of IEM suspicion showed the altered pattern of Aminogram correlating with high L/P ratio and Hyperammonemia, which were positive cases, as confirmed by the clinician who referred. Hence study parameters could be used as preliminary protocol to investigate and screen the IEM.

Design paper of the \u201cBlood pressure targets in post-resuscitation care and bedside monitoring of cerebral energy state: a randomized clinical trial\u201d

BackgroundNeurological injuries remain the leading cause of death in comatose patients resuscitated from out-of-hospital cardiac arrest (OHCA). Adequate blood pressure is of paramount importance to optimize cerebral perfusion and to minimize secondary brain injury. Markers measuring global cerebral ischemia caused by cardiac arrest and consecutive resuscitation and reflecting the metabolic variations after successful resuscitation are needed to assist a more individualized post-resuscitation care. Currently, no technique is available for bedside evaluation of global cerebral energy state, and until now blood pressure targets have been based on limited clinical evidence. Recent experimental and clinical studies indicate that it might be possible to evaluate cerebral oxidative metabolism from measuring the lactate-to-pyruvate (LP) ratio of the draining venous blood. In this study, jugular bulb microdialysis and immediate bedside biochemical analysis are introduced as new diagnostic tools to evaluate the effect of higher mean arterial blood pressure on global cerebral metabolism and the degree of cellular damage after OHCA.Methods/designThis is a single-center, randomized, double-blinded, superiority trial. Sixty unconscious patients with sustained return of spontaneous circulation after OHCA will be randomly assigned in a one-to-one fashion to low (63 mm Hg) or high (77 mm Hg) mean arterial blood pressure target. The primary end-point will be a difference in mean LP ratio within 48 h between blood pressure groups. Secondary end-points are (1) association between LP ratio and all-cause intensive care unit (ICU) mortality and (2) association between LP ratio and survival to hospital discharge with poor neurological function.DiscussionMarkers measuring cerebral ischemia caused by cardiac arrest and consecutive resuscitation and reflecting the metabolic changes after successful resuscitation are urgently needed to enable a more personalized post-resuscitation care and prognostication. Jugular bulb microdialysis may provide a reliable global estimate of cerebral metabolic state and can be implemented as an entirely new and less invasive diagnostic tool for ICU patients after OHCA and has implications for early prognosis and treatment.Trial registrationClinicalTrials.gov (ClinicalTrials.gov Identifier: NCT03095742). Registered March 30, 2017.

Individual assessment of bovine embryo development using a homemade chamber reveals kinetic patterns of success and failure to reach blastocyst stage

ABSTRACTMost early developmental data are lost in bovine embryo culture systems. We developed and validated a method for culture of bovine embryos in groups that allow individual assessment. An autoclavable low-cost multiembryo chamber (MEC) was prepared using a polyester mesh fixed to a glass coverslip. Embryonic development was not affected by MEC. Compared to conventional bovine culture system (oil-covered drops, control), cleavage (C, 71.2 ± 7.8%; MEC, 74.3 ± 6.0%), blastocyst rate (C, 29.9 ± 4.4%; MEC, 28.3 ± 5.0%) and blastocyst cell number (C, 94.1 ± 9.7; MEC, 92.9 ± 5.3) were similar. Caspase 3 positive cell index in blastocysts was increased in MEC group, but apoptosis rate was below 5% (C, 2.9 ± 0.5; MEC, 4.6 ± 0.6). Using MEC, we performed a retrospective analysis for ‘failure’ and ‘success’ embryos, based on their ability to reach the blastocyst stage. We detected the majority of ‘success’ embryos displayed 8 cells at 48 h post-insemination (hpi) (48.7%), but blastocysts derived from this pattern presented lower cell numbers (91.3 ± 4.2 vs. 107.9 ± 4.9) and higher apoptosis index (6.2 ± 0.6 vs. 4.4 ± 0.5) than blastocysts from 4-cell embryos at 48 hpi. Most (72.0%) embryos that were at morula stage 120 hpi reached blastocyst stage at 168 hpi. Those blastocysts presented more number of cells than blastocysts derived from embryos exhibiting 16 cells at 120 hpi (108.6 ± 4.1 vs. 83.9 ± 4.8). Combination of embryo kinetics data at 48 and 120 hpi revealed high chances of blastocyst formation for patterns: 8 cells/morula, 4 cells/morula, 8 cells/16 cells and 4 cells/16 cells. Blastocysts formed from 4-cell/morula and 8-cell/morula patterns represented 69% of all 168 hpi blastocysts. Blastocysts derived from 4 cells/16 cells displayed decreased apoptosis (3.1 ± 0.6). Our results suggest that MEC can be used for bovine embryo culture without detrimental effects on development and can help to predict blastocyst formation and quality of in vitro fertilization (IVF) embryos.Abbreviations: BSA: bovine serum albumine; COC: cumulus–oocyte complex; FERT-TALP: Tyrode’s albumin lactate pyruvate fertilization; FBS: fetal bovine serum; IVF: in vitro fertilization; MEC: multiembryo chamber; PBS: phosphate buffered saline; SOF-AA: synthetic oviductal fluid with amino acids medium; TCM: Tissue Culture Medium

Effects of on-pump versus off-pump coronary artery bypass grafting on myocardial metabolism.

Background:On-pump coronary artery bypass grafting (ON-PCABG) and off-pump coronary artery bypass grafting (OF-PCABG) greatly affect myocardial metabolism (MCMB). However, no study has systematically explored and compared the impacts of ON-PCABG and OF-PCABG on MCMB. This study will aim to explore and to compare the effects of ON-PCABG and OF-PCABG on MCMB systematically.Methods:We will conduct the comprehensive literature search from the following electronic databases from inception to the present: Cochrane Library, EMBASE, MEDILINE, CINAHL, AMED and 4 Chinese databases without language restrictions. This systematic review will only concern randomized controlled trials (RCTs) and case-control studies of ON-PCABG and OF-PCABG on MCMB. The methodological quality of each entered study will be assessed by using Cochrane risk of bias tool.Results:Primary outcomes include myocardial cellular markers, myocardial lactate, oxygen utilization, pyruvate, and intramyocardial concentrations of glucose, urea and lactate. Secondary outcome comprises of glutathione, superoxide dismutase, myeloperoxidase, and oxidative stress and any other complications post surgery.Conclusion:This study will provide a high-quality synthesis and will assess and compare the effects of ON-PCABG and OF-PCABG on MCMB based on the current relevant literature evidence.Dissemination and ethics:The results will be submitted to peer-reviewed journals for publication. This study does not require ethic approval, because it only analyzes the data from published literature.Systematic review registration:PROSPERO CRD42019125381.

Characterization of Testicular Angiotensin-Converting Enzyme before and after Semen Cryopreservation and in the Acrosome Reaction of Spermatozoids of Nelore Bulls

The aim of this study was to characterize the testicular isoform of angio-tensin-converting enzyme (tACE) before and after semen cryopreservation, and in the acrosome reaction of sperm from Nelore bulls in vitro. Ejaculates of 10 sexually mature Nelore bulls were used. After semen was collected, 1.0 mL of the ejaculate was used for the analysis and the rest was subjected to cryopreservation. Fresh semen before freezing, and frozen/thawed semen were centrifuged twice and the pellet was resuspended intyrode’s albumin lactate pyruvate (TALP). Thereafter, 100 μL aliquots containing 100 × 106 spermatozoa were prepared. Aliquots of samples were used for western blot analysis, subjected to capacitation, and thereafter, acrosome reaction assays were performed in vitro. With the help of an anti-ACE monoclonal antibody, a 100 kDa protein band was identified in the spermatozoa of Nelore bulls. Cryopreservation reduced the intensity of the protein bands obtained by western blot assay to less than half of that observed prior to freezing (P P < 0.05), indicating the involvement of ACE in these processes. It is concluded that tACE can be found in the spermatozoa of Nelore bulls, and cryopreservation process decreases the intensity of bands of this enzyme; and that the inactivation of tACE reduces the capacity of spermatozoa to undergo the acrosome reaction.

Characterization of Angiotensin-Converting Enzyme before and after Cryopreservation of Gir Semen

The aim of this study was to characterize the angiotensin-converting enzyme (ACE) in Gir semen before and after cryopreservation. The ejaculate of five sexually mature bulls was used. After collection, one 1-mL aliquot of fresh semen was analyzed immediately, and the rest of the semen was cryopreserved in liquid nitrogen for subsequent analysis. Freshly collected semen and thawed cryopreserved semen were centrifuged twice with Tyrode’s albumin lactate pyruvate medium (TALP) to remove plasma and extender, respectively. Samples were then subjected to western blotting, immunocytochemistry, and enzymatic activity techniques. At least one 100 kDa band was observed in every bull analyzed using western blotting with an anti-ACE monoclonal antibody, and band intensity decreased by 70% (p < 0.05) after cryopreservation. Immunocytochemistry showed periacrosomal ACE localization, and the area stained by the fluorescent antibody significantly decreased (p < 0.05) after cryopreservation. Enzyme activity was evaluated using FAPGG substrate hydrolysis, which was significantly lower (p < 0.05) in cryopreserved semen than in fresh semen. Therefore, the process of cryopreservation decreases ACE band intensity and enzyme activity in Gir bull semen, and reduces the stained area in immunocytochemistry.

Patterns of cerebral tissue oxygen tension and cytoplasmic redox state in bacterial meningitis.

Compromised cerebral energy metabolism is common in patients with bacterial meningitis. In this study, simultaneous measurements of cerebral oxygen tension and lactate/pyruvate ratio were compared to explore whether disturbed energy metabolism was usually caused by insufficient tissue oxygenation or compromised oxidative metabolism of pyruvate indicating mitochondrial dysfunction. Ten consecutive patients with severe streptococcus meningitis were included in this prospective cohort study. Intracranial pressure, brain tissue oxygen tension (PbtO2 ), and energy metabolism (intracerebral microdialysis) were continuously monitored in nine patients. A cerebral lactate/pyruvate (LP) ratio <30 was considered indicating normal oxidative metabolism, LP ratio >30 simultaneously with pyruvate below lower normal level (70µmol/L) was interpreted as biochemical indication of ischemia, and LP ratio >30 simultaneously with a normal or increased level of pyruvate was interpreted as mitochondrial dysfunction. The biochemical variables were compared with PbtO2 simultaneously monitored within the same cerebral region. In two cases, the LP ratio was normal during the whole study period and the simultaneously monitored PbtO2 was 18±6mmHg. In six cases, interpreted as mitochondrial dysfunction, the simultaneously monitored PbtO2 was 20±6mmHg and without correlation with the LP ratio. In one patient, exhibiting a pattern interpreted as ischemia, PbtO2 decreased below 10mmHg and a correlation between LP and PbtO2 was observed. This study demonstrated that compromised cerebral energy metabolism, evidenced by increased LP ratio, was common in patients with severe bacterial meningitis while not related to insufficient tissue oxygenation.

DETERMINATION OF AN EFFECTIVE MEDIA AND ITS HORMONE AND PROTEIN SUPPLEMENNTATION FOR IN VITRO MATURATION OF OOCYTES OF INDIGENOUS ZEBU COWS

In vitro maturation (IVM) of oocytes is the first important step for successful in vitro embryo production of any mammalian species. The objectives of the present study were to determine an effective basic medium and its hormone and protein supplementation for IVM of oocytes of indigenous zebu cows. The oocytes were derived from ovaries of locally slaughtered cows after aspiration of follicle. The oocytes were cultured in medium for 24 hrs at 38.5ºC with 5% CO2 in humidified air for maturation. The maturation of oocytes was evaluated by examining the presence of first polar body extrusion in denuded oocytes under inverted microscope. To determine an effective basic medium, the oocytes were cultured in fetal bovine serum (FBS) supplemented tissue culture medium (TCM), modified synthetic oviduct fluid (mSOF) and Tyrodes albumin lactate pyruvate (TALP) medium. The maturation rate was significantly higher (74±4.2) in TCM medium than that of TALP medium (58.2±6.2). To determine an effective hormone supplementation for maturation medium, the oocytes were cultured in either in follicle stimulating hormone (FSH) or gonadotrophin supplemented TCM. The maturation rate of oocytes was significantly (p&gt;0.05) higher (73.3±4.0) in FSH supplemented medium than that of gonadotrophin supplemented counterpart (60.2±6.6). To determine an effective protein supplementation, the oocytes were cultured in FBS, oestrus cow serum (OCS) and bovine serum albumin (BSA) supplemented TCM 199. The maturation rate of oocytes were 73.0±5.9, 71.1±2.8, and 62.5±9.4 in medium supplemented with FBS, OCS and BSA respectively (p&gt;0.05). In conclusions, TCM supplemented with either FBS, OCS or BSA as protein and FSH as hormone may be used as medium for IVM of oocytes of indigenous zebu cows.

Effect of oviduct and follicular fluids on ram sperm capacitation and acrosome reaction in vitro

The present study was designed to study the influence of different concentrations of oviduct fluid (OF) and follicular fluid (FF) on ram sperm capacitation and acrosome reaction in vitro. Forty semen samples were collected from three Barki rams throughout the period of study (10 weeks). Fresh semen was evaluated, layered under S-TALP (sperm Tyrode’s albumin lactate pyruvate) medium and subjected to swim up techniques. Split fractions of semen were incubated in media enriched with different levels of OF (10, 20, 40, 50 and 75 µL/mL) or FF (10, 20, 40, 50, 100, 150 and 200 µL/mL). Best concentrations were compared and used to evaluate the ram sperm functions including progressive motility, hyperactivity and acrosome reaction. The present findings showed a significant increase in individual motility percentage (IM %) when ram spermatozoa were treated with 10 and 50 µL of oviduct fluids as compared to the other treatments after 1 h of incubation. Addition of 50 µL/mL of OF or FF had beneficial effect on sperm hyperactivity after 2 and 1 h incubation respectively. Furthermore, addition of 50 µL/mL oviduct fluid to ram sperm maintained significantly (P < .05) higher total acrosome reaction (AR %) after 3 h of incubations than those observed in other groups. In conclusion, treatment of ram spermatozoa with 50 µL/mL of OF or 40 µL/mL of FF for 3–4 h incubation respectively was considered the best level of oviduct or follicular fluid to be used for IVF.

The relationship between intracranial pressure and lactate/pyruvate ratio in patients with subarachnoid haemorrhage.

The aim of this study was to analyse the relationship between intracranial pressure (intracranial pressure monitoring) and lactate pyruvate ratio (cerebral microdialysis) in patients with ruptured intracranial aneurysms. In a group of fifteen patients, intracranial pressure and lactate/pyruvate ratios were measured and logged in hourly intervals. The relationship between these two variables was subsequently analysed in two ways. 1) Intracranial hypertension (ICP > 20 mmHg) in the presence of energy deprivation (L/P ratio > 30) was noted. 2) The dynamics of L/P ratio changes in relation to immediate ICP and CPP values was analysed. Out of a total of 1873 monitored hours we were able to record lactate/pyruvate ratios higher than 30 in 832 hours (44 %). Of those 832 hours during which lactate/pyruvate ratios were higher than 30, ICP was higher than 20 in 193 hours (23 %). Out of 219 hours of monitoring, in which ICP was higher than 20, a simultaneously increased L/P ratio higher than 30 was recorded in 193 hours (88 %). L/P ratio values above 30 were associated with decreased CPP values (p = 0.04), but not with increased ICP values (p = 0.79). Intracranial hypertension coincides with energetic imbalance in approximately one quarter of cases. This points to the shortcomings of the most common form of neuromonitoring in SAH patients - ICP monitoring. This method may not be reliable enough in detecting hypoxic damage, which is the major cause of morbidity and mortality in SAH patients (Fig. 5, Ref. 11).