Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA090798

Local Sample ID:	1007
Subject ID:	SU001313
Subject Type:	Mammal
Subject Species:	Bos taurus
Taxonomy ID:	9913
Gender:	Female

Select appropriate tab below to view additional metadata details:

Subject:

Subject ID:	SU001313
Subject Type:	Mammal
Subject Species:	Bos taurus
Taxonomy ID:	9913
Gender:	Female

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
1007	SA090798	FL013078	Early	Treatment

Collection:

Collection ID:	CO001307
Collection Summary:	For cows assigned to the day 4 group, upon observation of estrus and a dominant follicle by ultrasound, cows were given an injection of GnRH (Factrel, 100 µg; Zoetis) in order to precisely time ovulation relative to time of collection for these early CL. Cows were slaughtered on day 4 following estrus. For samples collected later than day 4, precise synchrony of ovulation relative to CL collection was not necessary, so no GnRH was given, and CL were collected via colpotomy. For CL of pregnancy, cows were bred by artificial insemination and a uterine flush was performed immediately following CL collection and was examined for embryo fragments to confirm the presence of a viable pregnancy. For all samples, tissue was snap frozen in liquid nitrogen immediately following tissue collection and stored at -80 degrees Celsius thereafter. For in vitro experiments, three to five dairy cows were used in each group, CL were collected on day 10-12 of the estrous cycle, and each treatment was applied to cells from each cow
Sample Type:	Corpus Luteum

Treatment:

Treatment ID:	TR001328
Treatment Summary:	For cows assigned to the day 4 group, upon observation of estrus and a dominant follicle by ultrasound, cows were given an injection of GnRH (Factrel, 100 µg; Zoetis) in order to precisely time ovulation relative to time of collection for these early CL. Cows were slaughtered on day 4 following estrus. For samples collected later than day 4, precise synchrony of ovulation relative to CL collection was not necessary, so no GnRH was given, and CL were collected via colpotomy. For CL of pregnancy, cows were bred by artificial insemination and a uterine flush was performed immediately following CL collection and was examined for embryo fragments to confirm the presence of a viable pregnancy. For all samples, tissue was snap frozen in liquid nitrogen immediately following tissue collection and stored at -80 degrees Celsius thereafter. For in vitro experiments, three to five dairy cows were used in each group, CL were collected on day 10-12 of the estrous cycle, and each treatment was applied to cells from each cow

Treatment ID:

TR001328

Treatment Summary:

For cows assigned to the day 4 group, upon observation of estrus and a dominant follicle by ultrasound, cows were given an injection of GnRH (Factrel, 100 µg; Zoetis) in order to precisely time ovulation relative to time of collection for these early CL. Cows were slaughtered on day 4 following estrus. For samples collected later than day 4, precise synchrony of ovulation relative to CL collection was not necessary, so no GnRH was given, and CL were collected via colpotomy. For CL of pregnancy, cows were bred by artificial insemination and a uterine flush was performed immediately following CL collection and was examined for embryo fragments to confirm the presence of a viable pregnancy. For all samples, tissue was snap frozen in liquid nitrogen immediately following tissue collection and stored at -80 degrees Celsius thereafter. For in vitro experiments, three to five dairy cows were used in each group, CL were collected on day 10-12 of the estrous cycle, and each treatment was applied to cells from each cow

Sample Preparation:

Sampleprep ID:	SP001321
Sampleprep Summary:	Oxylipins and endocannabinoids were isolated using a Waters Ostro™ Sample Preparation Plate. Luteal samples were homogenized and 40 ± 8 mg were added to 2 mL polypropylene tubes spiked with a 5 µL antioxidant solution (0.2 mg/ml solution BHT/EDTA in 1:1 MeOH:water) and 10 μL 1000 nM analytical deuterated surrogates as previously described (Agrawal et al., 2017; La Frano et al., 2017). Samples were then mixed with 35 µL methanol, 550 µL isopropanol w/ 10 mM ammonium formate, 1% formic acid and 100 µL water, and the tube was placed in a Geno/Grinder 2010 (SPEX SamplePrep) for 30 sec and centrifuged at 10,000 x g for 5 min at room temperature. Supernatants were transferred into the Ostro plate wells and captured in glass inserts containing 10 μL of 20% glycerol in methanol by applying 15 mmHg of vacuum for 10 min. The eluent was dried under vacuum and reconstituted with 100 µL, 1:1 MeOH/ACN (v/v) containing 100 nM of 1-cyclohexyl ureido, 3 dodecanoic acid and 1-phenyl ureido, 3-hexanoic acid urea used as internal standards (gifts from Dr. B.D. Hammock, University of California, Davis). The samples were then vortexed and filtered at 0.1µm through PVDF membranes (Millipore) by centrifugation < 4500 x g (rcf) for 3 min at 6 ºC. The filtrate was transferred to inserts in amber glass and stored at -20 ºC for less than 48 hours before analysis by UPLC-MS/MS. Analytes in 5 μL extract aliquot were separated on a 2.1 mm x 150 mm, 1.7 µm Acquity BEH column (Waters) using published protocols for oxylipins and endocannabinoids (Agrawal et al., 2017; Pedersen and Newman, 2018). Samples were held at 10ºC. Separated residues were detected by negative mode electrospray ionization for oxylipins and positive mode electrospray ionization for endocannabinoids using multiple reaction monitoring on an API 6500 QTRAP (AB Sciex). Analytes were quantified using internal standard methods and 5- to 7-point calibration curves (r2 ≥ 0.997). Calibrants and internal standards were either synthesized [10,11-DHN, 10,11-DHHep, 10(11)-EpHep] or purchased from Cayman Chemical, Avanti Polar Lipids Inc., or Larodan Fine Lipids. Data was processed with AB Sciex MultiQuant version 3.0.2. The internal standards were used to quantify recovery of surrogate standards

Sampleprep ID:

SP001321

Sampleprep Summary:

Oxylipins and endocannabinoids were isolated using a Waters Ostro™ Sample Preparation Plate. Luteal samples were homogenized and 40 ± 8 mg were added to 2 mL polypropylene tubes spiked with a 5 µL antioxidant solution (0.2 mg/ml solution BHT/EDTA in 1:1 MeOH:water) and 10 μL 1000 nM analytical deuterated surrogates as previously described (Agrawal et al., 2017; La Frano et al., 2017). Samples were then mixed with 35 µL methanol, 550 µL isopropanol w/ 10 mM ammonium formate, 1% formic acid and 100 µL water, and the tube was placed in a Geno/Grinder 2010 (SPEX SamplePrep) for 30 sec and centrifuged at 10,000 x g for 5 min at room temperature. Supernatants were transferred into the Ostro plate wells and captured in glass inserts containing 10 μL of 20% glycerol in methanol by applying 15 mmHg of vacuum for 10 min. The eluent was dried under vacuum and reconstituted with 100 µL, 1:1 MeOH/ACN (v/v) containing 100 nM of 1-cyclohexyl ureido, 3 dodecanoic acid and 1-phenyl ureido, 3-hexanoic acid urea used as internal standards (gifts from Dr. B.D. Hammock, University of California, Davis). The samples were then vortexed and filtered at 0.1µm through PVDF membranes (Millipore) by centrifugation < 4500 x g (rcf) for 3 min at 6 ºC. The filtrate was transferred to inserts in amber glass and stored at -20 ºC for less than 48 hours before analysis by UPLC-MS/MS. Analytes in 5 μL extract aliquot were separated on a 2.1 mm x 150 mm, 1.7 µm Acquity BEH column (Waters) using published protocols for oxylipins and endocannabinoids (Agrawal et al., 2017; Pedersen and Newman, 2018). Samples were held at 10ºC. Separated residues were detected by negative mode electrospray ionization for oxylipins and positive mode electrospray ionization for endocannabinoids using multiple reaction monitoring on an API 6500 QTRAP (AB Sciex). Analytes were quantified using internal standard methods and 5- to 7-point calibration curves (r2 ≥ 0.997). Calibrants and internal standards were either synthesized [10,11-DHN, 10,11-DHHep, 10(11)-EpHep] or purchased from Cayman Chemical, Avanti Polar Lipids Inc., or Larodan Fine Lipids. Data was processed with AB Sciex MultiQuant version 3.0.2. The internal standards were used to quantify recovery of surrogate standards

Combined analysis:

Analysis ID	AN002068	AN002069
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Shimadzu Nexera X2	Shimadzu Nexera X2
Column	Waters Acquity BEH C18 (150 x 2mm,1.7um)	Waters Acquity BEH C18 (150 x 2mm,1.7um)
MS Type	ESI	ESI
MS instrument type	Triple quadrupole	Triple quadrupole
MS instrument name	ABI Sciex 6500 QTrap	ABI Sciex 6500 QTrap
Ion Mode	POSITIVE	NEGATIVE
Units	nM	nM

Chromatography:

Chromatography ID:	CH001506
Instrument Name:	Shimadzu Nexera X2
Column Name:	Waters Acquity BEH C18 (150 x 2mm,1.7um)
Column Temperature:	60
Flow Rate:	0.5 mL/min
Chromatography Type:	Reversed phase

MS:

MS ID:	MS001919
Analysis ID:	AN002068
Instrument Name:	ABI Sciex 6500 QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	Multiquant
Ion Mode:	POSITIVE

MS ID:	MS001920
Analysis ID:	AN002069
Instrument Name:	ABI Sciex 6500 QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	Multiquant
Ion Mode:	NEGATIVE