Phytase

Expression vectors (Table 3) were made by standard molecular biology techniques.
The genes for mature E. coli alkaline phosphatase (PhoA; Arg22-Lys471), mature E. coli phytase (AppA; Gln23-Leu432) and mature DsbC (Asp21-Lys236) were amplified by PCR using a colony of E. coli XL1-Blue as a template. The gene for Erv1p (Met1-Glu189) was amplified using a plasmid kindly provided by Prof Thomas Lisowsky as a template.
Erv1p and mature DsbC were cloned into pET23a. An alternative cloning strategy for Erv1p using pET23d (which replaces the 5' NdeI restriction site with NcoI) was also used. This adds an extra Glycine between Met1 and Lys2 of Erv1p. Both versions of the protein were used in a variety of co- and pre-expression experiments and no significant differences between the two were observed. Mature PhoA and AppA were cloned into a modified version of pET23a which includes an N-terminal his-tag in frame with the cloned gene and an additional SpeI site in the multi-cloning site between the EcoRI and SacI sites. The resulting gene products include the sequence MHHHHHHM- prior to the first amino acid of the protein sequence.
Polycistronic vectors were constructed by taking fragments encoding the folding factors from the pET23 based constructs which include the ribosome binding site e.g. XbaI/X fragments and ligating them into the SpeI/X cut plasmid encoding the protein of interest (where X is an appropriate restriction site found in the multi-cloning site after SpeI and not found in either gene e.g. XhoI). After a single such ligation this generates a plasmid that contains a single transcription initiator/terminator and hence makes a single mRNA, but has two ribosome binding sites and makes two proteins by co-expression from two translation initiation sites. This ligation results in the loss of the original SpeI site. Transfer of a SpeI site after the second gene into the new vector allows a third gene to be cloned by the same method resulting in a tricistronic vector which makes three proteins from a single mRNA.
All plasmid purification was performed using the QIAprep spin miniprep kit (Qiagen) and all purification from agarose gels was performed using the Gel extraction kit (Qiagen), both according to the manufacturers' instructions.
All plasmids generated were sequenced to ensure there were no errors in the cloned genes (see Table 3 for plasmid names and details).

The sample size calculation was based on a two-sided test with a confidence level of 95% and a power of 0.80 to detect a statistically significant difference in ATTD of P in the phytase supplemented treatments compared to CON. The expected effect size was based on published data concerning the variance in P digestibility in lactating dairy cows (Valk et al., 2002; Wu et al., 2003 (link); Kincaid et al., 2005 (link); Knowlton et al. 2007 (link)). The chemical composition of the total rations was calculated based on the chemical composition and intakes of both forages and concentrates. The fecal excretion of DM was calculated for each cow from the daily TiO₂ administration (g/animal) divided by the TiO₂ concentration (g/kg DM) in feces. For this, a fecal recovery of TiO₂ of 100% (Glindemann et al., 2009 (link)) was assumed. The fecal excretion of CP, starch, NDF, P, Ca, and PP was calculated as DM fecal excretion multiplied by the concentration of the respective chemical component in the feces. The ATTD of DM, CP, starch, NDF, P, Ca and PP was computed as ATTD (%) = [(intake – feces excretion)/intake]. For this calculation, the intake and feces excretion of each chemical component was in kilogram per day on a DM basis. The yield of FPCM (kg/d) was calculated on a 4% fat and 3.3% protein basis. The feed efficiency was calculated as FPCM divided by DMI both expressed in kg. Data on somatic cell count (SCC) were log transformed to obtain a normal distribution before statistical analysis. All data were averaged per cow and week for the statistical analysis.
All statistical analyses were performed using Genstat 18th edition (VSN International, Hemel Hempstead, UK). Data were analyzed by ANOVA to identify treatment effects. Treatment means comparisons were carried out using the Tukey test. Data are presented as least squares means and associated pooled SEM values. The statistical analyses for all variables (except P in milk and blood, and ATTD) were carried out using the data of the preperiod as a covariate, using the following model:
where Y_ijk is the response variable, μ is the overall mean, Block_i is the effect of block (i = 1−10), Cov_j is the covariate (response during preperiod), T_rtk is the effect of dietary treatment (k = 1−3), and ε_ijk is the residual error.
For the statistical analysis of P content in milk and blood, and ATTD, the same model was used but without the preperiod as a covariate. In addition, the effect of phytase dose level on nutrient intake ATTD and fecal excretion of nutrients during the fecal collection period was analyzed by polynomial contrasts to determine the linear and quadratic response to increasing phytase dose, with consideration of uneven distribution between phytase dose levels. Statistical significance was declared at P < 0.05. 0.05 ≤ P < 0.1 was considered a tendency.

The amounts of basal diet (forages) and concentrates offered and refused were individually weighed and recorded on a daily (forages) or weekly (concentrates) basis. The daily feed intake was calculated as the difference between the offered and refused amount for both basal diet and concentrates. Representative weekly samples of forages were collected, pooled per forage type by mixing equal amounts [on a fresh matter (FM) basis], and sent to the certificated laboratory Eurofins Agro NL (Wageningen, the Netherlands) for chemical analysis based on near infrared spectroscopy (NIRS). The concentrates were produced in one batch each, immediately sampled and analyzed. The chemical composition of these forages and concentrate samples was used to calculate the chemical composition of the total rations. During the final week of the experiment and 2 d prior to the start of fecal sampling, additional samples of the forages and concentrates were collected for the determination of the ATTD of chemical constituents. Forages were sampled daily and concentrates every 2 d. Samples were stored at –20 °C until later analysis. At the end of the experiment, forages were thawed at room temperature, pooled per type of forage by mixing equal amounts on a FM basis, freeze-dried for approximately 96 h in a Zirbus sublimator 3-4-5/20 (Zirbus Technology Benelux B. V., Tiel, the Netherlands) and ground to pass through a 1-mm screen using a Retsch ZM200 grinder (Retsch Benelux, Aartselaar, Belgium). The forage and concentrate samples were analyzed by Schothorst Feed Research (Lelystad, the Netherlands). The DM content was determined by drying at 103 °C to constant weight according to method ISO 6496 (ISO, 1998 ). Crude ash was determined gravimetrically after ashing the samples in a muffler furnace for 3 h at 550 °C, according to method ISO 5984 (ISO, 2002 ). The N content was determined by the Dumas method using a macro determinator (LECO CM928 MLC, LECO, Michigan, USA) according to method ISO 16634 (ISO, 2016 ), and the CP content was calculated as N × 6.25. The starch content (except in grass silage) was determined by the amylo-glucosidase method according to the procedures of Englyst et al. (1992) (link), and sugar content was determined according to the Luff-Schoorl method. Crude fat (CFat) was determined by ether extraction after acid hydrolysis, according to method ISO 11085 (ISO, 2015 ). The NDF content was exclusive of residual ash and a heat-stable α-amylase was added during NDF extraction, according to ISO 16472 (ISO, 2006 ). The ADF content was exclusive of ash and determined according to ISO 13906 (ISO, 2008 ). The P content was determined based on the colorimetric method according to ISO 6491 (ISO, 1998 ) and contents of Ca and TiO₂ were determined based on atomic absorption spectroscopy according to ISO 6869 (ISO, 2000 ). The content of PP in forages and concentrates was analyzed at Danisco Animal Nutrition Research Centre (Brabrand, Denmark) using the HPLC method described by Christensen et al. (2020) (link) modified from Skoglund et al. (1998) (link). Modifications to the analytical procedure were that the extraction of IP₆ from the feces samples was carried out at a concentration of 0.20 g/mL using 1.0M HCl as solvent. The phytase activity in concentrate samples was analyzed by Danisco Animal Nutrition Research Centre (Brabrand, Denmark) according to a modified version of the 2000.12 AOAC method (Engelen et al., 2001 (link)). For this, one FTU was defined as the quantity of enzyme that released 1 µmol of inorganic orthophosphate from a 0.0051 mol/L sodium phytate substrate per minute at pH 5.5 at 37 °C.

The experiment was carried out as a randomized block design with three dietary treatments and 10 blocks (replicates) per treatment. The experiment comprised an 18-d preperiod for the collection of data to facilitate the allocation of cows to the treatments, followed by a 19-d experimental period comprising a 14-d diet adaptation phase, as is recommended for digestibility trials (GfE, 1991 (link)), and 5 d of feces collection. During the preperiod, all cows were fed ad libitum with a mixture of grass silage and corn silage in the same proportions as mentioned above and were supplemented with concentrates (as described above but without supplemental phytase) based on FPCM production. Cows were allocated during the last 3 d of the preperiod to blocks based on parity, DMI, and FPCM production during the preceding week. Within blocks, animals were randomly allocated to treatments.