Each animal (6 commercial milking cycling Bos taurus Holstein cows, 4 years old on average, 3.04+/-0.2 years old on average at calving) was exposed in a randomized scheme to the 4 conditions with at least one complete regular sexual cycle between 2 treatments and served as its own control. Each animal was treated during luteal phase to prevent spontaneous ovulation. Hormonal synchronisation was not performed; FSH application and OPU were done using natural di-estrus. We performed P4 serum assays showing no significant differences between coasting durations. The P4 level permitted to determine that there was a corpus luteum. We performed E2 serum assays; there was no significant difference between traitments in term of E2/P4. We could indirectly confirm that LH level was basal. The mean duration between calving and OPUs is 271.8+/-28.9 days. There isn’t correlation between order of OPUs and efficiency; there aren’t significant differences between cows in term of duration between 2 OPUs. There aren’t correlations between blastocyst rate and duration between calving and OPUs, or between coasting duration and duration between calving and OPUs, or between the efficiency to collect COCs and the duration between calving and OPUs. The efficiency to collect COCs (76% on average) is not significantly different from the beginning to the end of the protocol. There isn’t correlation between coasting duration and efficiency. There is no significant difference between cows in term of duration between calving and OPUs. The milk production mean was 8630.17 L +/- 771.1. The energy balance of the cows was positive. The cows were part of the same herd. The dominant follicle was aspirated 36 hours before administration of 229 hormones. Cows were stimulated for 3 days with FSH (6 X 40 mg NIH Folltropin-V, Bioniche Animal Health, Belleville, ON, Canada, given at 12-hour intervals), followed by a coasting (no FSH) period of four different durations (20, 44, 68, or 92 hours). Using transvaginal ultrasonography, follicular diameters were measured and cumulus-oocyte complexes (COCs) were collected by transvaginal puncture (PTV), under epidural, with a 18G needle and COOK aspiration unit (COOK Medical, Bloomington, IN). COCs and granulosa cells were collected in warm HEPES-buffered Tyrode’s medium (TLH) containing Hepalean (10 UI/ml) and transferred to lab for in vitro maturation (half of the COCs) or treated (oocytes, cumulus, granulosa cells) for further studies.
Growth protocol
Cumulus are separated from oocyte with vortexing. Rince 3 times in sterile PBS, centrifuge at 4000 rpm for 4 minutes. Snap freeze in liquid nitrogen and store at -80°C.
Extracted molecule
total RNA
Extraction protocol
Total RNA extraction using the PicoPure RNA Isolation Kit from Molecular Devices. The PicoPure RNA Isolation Kit enables to recover total cellular RNA from pico-scale samples. Researchers can obtain high recoveries of total cellular RNA from as little as a single cell to samples with up to 100 ug of RNA. Total RNA was amplified using the RiboAmp HSPlus RNA Amplification kit (Life Technologies) for 2 rounds according to the recommendations. aRNA was quantify using a Nanodrop (ThermoFisher).
Label
Cy5
Label protocol
The labelling aRNA was done using the Universal Linkage System (ULS) by Kreatech for Gene Expression studies using microarray slides from Agilent. ULS is a non-enzymatic protocol that allows direct labeling of unmodified, amplified RNA. ULS will react with unmodified, amplified RNA and label it by formation of a coordinative bond on the N7 position of guanine. Fluorophore Cy3 and Cy5 are linked via a spacer to the ULS molecule.
Each animal (6 commercial milking cycling Bos taurus Holstein cows, 4 years old on average, 3.04+/-0.2 years old on average at calving) was exposed in a randomized scheme to the 4 conditions with at least one complete regular sexual cycle between 2 treatments and served as its own control. Each animal was treated during luteal phase to prevent spontaneous ovulation. Hormonal synchronisation was not performed; FSH application and OPU were done using natural di-estrus. We performed P4 serum assays showing no significant differences between coasting durations. The P4 level permitted to determine that there was a corpus luteum. We performed E2 serum assays; there was no significant difference between traitments in term of E2/P4. We could indirectly confirm that LH level was basal. The mean duration between calving and OPUs is 271.8+/-28.9 days. There isn’t correlation between order of OPUs and efficiency; there aren’t significant differences between cows in term of duration between 2 OPUs. There aren’t correlations between blastocyst rate and duration between calving and OPUs, or between coasting duration and duration between calving and OPUs, or between the efficiency to collect COCs and the duration between calving and OPUs. The efficiency to collect COCs (76% on average) is not significantly different from the beginning to the end of the protocol. There isn’t correlation between coasting duration and efficiency. There is no significant difference between cows in term of duration between calving and OPUs. The milk production mean was 8630.17 L +/- 771.1. The energy balance of the cows was positive. The cows were part of the same herd. The dominant follicle was aspirated 36 hours before administration of 229 hormones. Cows were stimulated for 3 days with FSH (6 X 40 mg NIH Folltropin-V, Bioniche Animal Health, Belleville, ON, Canada, given at 12-hour intervals), followed by a coasting (no FSH) period of four different durations (20, 44, 68, or 92 hours). Using transvaginal ultrasonography, follicular diameters were measured and cumulus-oocyte complexes (COCs) were collected by transvaginal puncture (PTV), under epidural, with a 18G needle and COOK aspiration unit (COOK Medical, Bloomington, IN). COCs and granulosa cells were collected in warm HEPES-buffered Tyrode’s medium (TLH) containing Hepalean (10 UI/ml) and transferred to lab for in vitro maturation (half of the COCs) or treated (oocytes, cumulus, granulosa cells) for further studies.
Growth protocol
Cumulus are separated from oocyte with vortexing. Rince 3 times in sterile PBS, centrifuge at 4000 rpm for 4 minutes. Snap freeze in liquid nitrogen and store at -80°C.
Extracted molecule
total RNA
Extraction protocol
Total RNA extraction using the PicoPure RNA Isolation Kit from Molecular Devices. The PicoPure RNA Isolation Kit enables to recover total cellular RNA from pico-scale samples. Researchers can obtain high recoveries of total cellular RNA from as little as a single cell to samples with up to 100 ug of RNA. Total RNA was amplified using the RiboAmp HSPlus RNA Amplification kit (Life Technologies) for 2 rounds according to the recommendations. aRNA was quantify using a Nanodrop (ThermoFisher).
Label
Cy3
Label protocol
The labelling aRNA was done using the Universal Linkage System (ULS) by Kreatech for Gene Expression studies using microarray slides from Agilent. ULS is a non-enzymatic protocol that allows direct labeling of unmodified, amplified RNA. ULS will react with unmodified, amplified RNA and label it by formation of a coordinative bond on the N7 position of guanine. Fluorophore Cy3 and Cy5 are linked via a spacer to the ULS molecule.
Hybridization protocol
The hybridization of microarray slides 4x44K printed with oligonucleotides by Agilent for all EmbryoGENE related projects using the expression array protocol.
Scan protocol
Scan on a PowerScanner (Tecan) using the autogain function. Images were quantified using ArrayPro version 6.3 (MediaCybernetics)
Description
Biological replicats 1 of 3. Cumulus cells at 20 hours of coasting compare 44 hours of coasting. Lame072 Array2 Data
Data processing
Background subtracted, Loess and Quantile normalized data obtained from log2 of processed Red signal/processed Green signal. Flexarray software used.
