Study animals were fed a TMR formulated to meet or exceed requirements of dry or lactating cows, with exception of the P content of the LP dry cow ration (NRC, 2001). The dry cow base ration used in this experiment was based on corn silage, sugar beet pressed pulp silage, hay and straw. Pelleted concentrate, formulated for each treatment, was added to obtain the final TMR with the targeted P content (Supplementary Table S1). The dry cow diet of LP cows contained 0.16% P, while the P content of the TMR for AP cows was 0.35% P in DM. Mono-ammoniumphosphate (Windmill Monamphos FG, Aliphos Rotterdam BV, The Netherlands) was used as P supplement in the AP concentrate to obtain the targeted dietary P content in the AP ration. Urea was added to the LP concentrate to equalize the N content of both diets. The TMR was offered through electronic feeding gates (RIC System, Hokofarm Group, Marknesse, Netherlands) and was available ad libitum for lactating cows, but was restricted to 11.5 kg DM during the dry period for both treatments. Feed restriction in addition to a LP diet was necessary to maintain the daily P intake in LP cows below 20 g P/cow (Cohrs et al., 2018). Studies conducted on dry dairy cows indicated that counter regulation to P deprivation in the form of bone mobilization was consistently induced when dietary P supply did not exceed 20 g per cow and day, equivalent to approximately 80% of the currently recommended dietary P supply to dry cows during the last three weeks of gestation (Cohrs et al., 2022; NASEM, 2021). Water was available ad libitum.
Growth protocol
The study was conducted at the Educational and Research Centre for Animal Husbandry, Hofgut Neumühle, Münchweiler an der Alsenz, Germany. All related procedures were approved by the Animal Welfare and Ethics Committee of the government of Coblenz, Rhineland Palatinate, Germany (permit no 23-177-07/G 19-20-008). The design of the underlying study has been described in detail elsewhere (Wächter et al., 2022a). Briefly, 30 late-pregnant multiparous Holstein-Friesian dairy cows entering their second, third or fourth lactation were randomly assigned to one of the 2 experimental groups, that were fed a dry cow ration with either low (LP, 0.16% P in DM) or adequate P content (AP, 0.35% P in DM) during the last 4 wk of the dry period. The study cows were part of the herd of a research farm with an average 305-d milk yield of 12,500 kg. Dry cows and fresh cows were kept in separated areas of a freestall. Cows with signs of imminent parturition were moved to individual calving pens and if deemed to be clinically healthy proceeded to the lactation group within 24 of calving. From the moment of calving both groups were fed the same standard lactating cow ration with adequate P content. Lactating cows were milked twice daily between 04.30 h and 05.30 h and between 15.30 h and 16.30 h.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using TRIzol reagent (Invitrogen, Karlsruhe) according to the manufacturer’s protocol. RNA quantity and quality were assessed spectrophotometrically using an Infinite 200M microplate reader equipped with a NanoQuant plate (both from Tecan, Mainz). The average RNA concentration and the A260/A280 ratio of all total RNA samples (n = 20, means ± SD) were 424 ± 78 ng/μL and 1.89 ± 0.05, respectively.
Label
Biotin
Label protocol
200 ng of total RNA was used to generate double-stranded cDNA. 12 µg of subsequently synthesized cRNA were purified and reverse transcribed into single-stranded (ss) cDNA, whereat unnatural dUTP residues were incorporated. Purified ss cDNA was fragmented using a combination of uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE 1) followed by a terminal labeling with biotin.
Hybridization protocol
3.8 µg of fragmented and labeled ss cDNA were hybridized to Applied Biosystems GeneChip Clariom S rat arrays for 16 h at 45°C and 60 rpm in an Applied Biosystems GeneChip hybridization oven 640.
Scan protocol
Hybridized arrays were washed and stained in an Applied Biosystems GeneChip Fluidics Station FS450, and the fluorescent signals were measured with an Applied Biosystems GeneChip Scanner 3000 7G System. Fluidics and scan functions were controlled by the Applied Biosystems GeneChip Command Console v4.3.3 software.
Data processing
Summarized probe set signals in log2 scale were calculated by using the GCCN-SST-RMA algorithm with the Applied Biosystems GeneChip Expression Console v1.4 Software. After exporting into Microsoft Excel, average signal values, comparison fold changes (FC) and significance P-values were calculated.
