2.1. Forage Production
In early Spring 2014, an existing 15.4 ha alfalfa field was split into 2 parcels of 9.3 ha for CON and 6.1 ha for the SCA agronomy program for growing alfalfa. The South Dakota State University (SDSU; Brookings, SD, USA) CON agronomy alfalfa protocol was followed, but no spring fertilization, according to soil tests, was required due to the pH being 7.8 and there being adequate P (145 ppm and 102 ppm for the alfalfa and corn fields, respectively) and K (186 and 205 ppm) concentrations. A foliar feed with boron and a generic insecticide were applied after the 1st, 2nd, and 3rd cutting. On 3 May 2014, for the SCA agronomy program, gypsum was applied at 981 kg/ha, potash at 112 kg/ha, and S at 75 kg/ha. On 14 May 2014, following alfalfa green-up, a foliar application of Kick-Off at 3.4 kg/ha, GroZyme at 1096 mL/ha, and Blitz at 439 mL/ha was applied (Ag Spectrum, Dewitt, IA, USA).
First-cutting alfalfa was harvested as alfalfa haylage (New Holland Chopper, CNH Industrial, New Holland, PA, USA) on 28 May 2014, with each loaded forage wagon weighed on a platform scale (SDSU Feed Mill, Brookings, SD, USA) and ensiled in a 2.4 m × 76.2 m Ag Bag (Ag-Bag by RCI, Mayville, WI, USA) with Silo-King (a silage inoculant containing lactic acid bacteria, enzymes, antioxidants, and a mold inhibitor; Agri-King, Inc. Fulton, IL, USA) applied at a rate of 0.5 kg/t on an “As Is” basis.
After each alfalfa cutting upon green-up, foliar applications were applied on 10 June, 8 July, and 4 August 2014. Second-cutting alfalfa haylage was harvested on 25 June 2014, treated with Silo-King at 0.5 kg/t, and ensiled in another Ag-Bag (RCI, Mayville, WI, USA). Due to the 1st and 2nd cuttings supplying ample tonnage for a lactating dairy cow feeding trial, the 3rd and 4th cuttings were harvested on 22 July 2014 and 26 August 2014 as alfalfa baleage, weighed, and shipped off to other SDSU livestock units. The days between cuttings were 30, 28, and 33, respectively. During this alfalfa growing and harvesting season, no manure was applied to the field between cuttings.
In early Spring 2014, a 13.4 ha field was split into 2 parcels, with 1 being approximately 9.3 ha for CON corn silage production and the 2nd parcel being approximately 5.3 ha for producing corn silage via the SCCA agronomy corn silage program. The field was fully tilled, and the CON agronomy program consisted of no supplemental fertilizer due to soil tests. During this corn silage growing and harvesting season, no manure was applied to the field. After planting, a pre-emergence herbicide, Harness Xtra (Bayer, Crop Science Division, Institute, WV, USA), was applied, and 89 mL of Callisto (Syngenta, Wilmington, DE, USA) and 946 mL of Powermax (Bayer, Crop Science Division, Institute, WV, USA) were sprayed before canopy as post-emergence herbicides. For the SCA agronomy crop production program, preplant gypsum was broadcast at 980 kg/ha, potash at 112 kg/ha, Blitz at 439 mL/ha, and GroZyme at 1096 mL/ha (Ag Spectrum, DeWitt, IA, USA). On 21 May 2014, the seed corn hybrid MC527 (Masters Choice, Anna, IL, USA) was planted at 79,000 kernels/ha, and CleanStart was applied at 32 L/ha, GroZyme at 292 mL/ha, Kick-Off at 5.36 kg/ha (Ag Spectrum, DeWitt, IA, USA), liquid potassium at 18 kg/ha, N at 209 kg/ha, and S at 29 kg/ha, which were side-dressed at planting. On 10 June 2014, a foliar application of GlyCure at 4.7 l/ha, GroZyme at 585 mL/ha, PT-21 at 37 l/ha, and Score at 5.6 kg/ha was applied (Ag Spectrum, DeWitt, IA, USA).
The corn was harvested as corn silage via a New Holland self-propelled chopper with a kernel processor (CNH Industrial, New Holland, PA, USA) on 7 October 2014. The corn silage was weighed, treated with Silo-King (Silage Inoculant, Agri-King, Inc. Fulton, IL, USA) at a rate of 0.5 kg/t on an “As Is” basis using a Gandy applicator (Gandy, Owatonna, MN, USA) mounted on an Ag Bagger, and ensiled in a 2.4 m × 76.2 m Ag Bag (Ag-Bag by RCI, Mayville, WI, USA).
2.3. Lactation Experiment
The research lactation experiment was conducted at the SDSU Dairy Research and Training Facility (
DRTF; Brookings, SD, USA) from November 2014 to June 2015, following the guidelines that became the 4th edition of the
Guide for the Care and Use of Agricultural Animals in Research and Teaching [
27]. All lactating dairy cows were cared for and managed according to the SDSU Institutional Animal Care and Use Committee recommendations. The cows were housed in a curtain-sided free-stall barn equipped with free access to water, with Calan feeding doors and feed boxes (American Calan Inc., Northwood, NH, USA). A total of 30 peak-lactation (58 days in milk (
DIM) ± 2.9, 38.9 kg milk ± 7.6, and 630 kg body weight (
BW) ± 97.7) Holstein dairy cows (8 primiparous and 22 multiparous), were blocked by milk yield, DIM, and parity, and randomly assigned to 1 of 2 TMR treatments using a randomized complete block design (
RCBD) with a pretreatment covariate. The experimental period was a continuous 13 wk experiment, with the first 7 d for diet adaptation (using a SCON ration fed for the covariate period) and adjustment, followed by 84 d (12 wk) of data collection.
The treatments were as follows: (1) for CON, forages (61.6:38.4 corn silage–alfalfa haylage mixed at 65:35 forage–concentrate in the TMR) were formulated using 1st-cutting alfalfa haylage and corn silage produced via the CON SDSU agronomy program; (2) for SCA, forages (same ratios) were formulated using 1st-cutting SCA alfalfa haylage and corn silage. The ingredient composition of the grain mix was similar between both treatments and was mixed at the SDSU Feed Mill and delivered to the DRTF approximately every 2 wk (
Table 1). All cows were fed the CON TMR during the 7 d covariate period, followed by 12 weeks of data collection when the CON and SCA TMRs were fed. All TMRs were prepared and delivered using a Calan Super Data Ranger (American Calan, Inc., Northwood, NH, USA). The TMR was formulated using NDS Professional (Nutritional Dynamic System, Emilia, Italy), a Cornell Net Carbohydrate and Protein System (
CNCPS)-based platform for ruminant ration formulation and evaluation to predict the lactating dairy cow performance for a 616 kg Holstein cow producing 38.6 kg/d of milk, 3.75% fat, and 3.36% crude (total) protein, consuming approximately 24.2 kg of DM/d. A post hoc power and sample size analysis (SAS version 9.4, Cary, NC, USA) with an SEM of 1.70 indicated that 14 cows/treatment were needed to detect a 7% increase in milk production at greater than 90% power without the use of a covariate.
The cows were milked 3 times daily (at 0600, 1400, and 2100 h) and fed individually (Calan Feeding Doors, American Calan, Inc., Northwood, NH, USA) once daily (at 0700 h) via ad libitum intake with 5 to 10% orts. The total daily feed offerings were adjusted based on the previous 24 h intake to achieve approximately 5% refusals. The amounts fed and orts were recorded daily. The forages were tested weekly for dry matter (DM), and forage adjustments were made for changes in the DM concentrations. The health status of each animal was evaluated daily, and all other bedding, cow monitoring, and manure scraping followed standard DTRF operating procedures supervised by the SDSU attending veterinarian (Dr. Michelle Mucciante).
2.4. Data and Sample Collection
An approximately 500 g aliquot of forage was collected from every load of alfalfa haylage at each cutting or corn silage during harvest and composited into 1 large sample per cutting or crop. These samples were thoroughly mixed, subsampled, dried for 48 h at 55 °C, and ground through a 4 mm screen (Wiley mill, Arthur H. Thomas Co., Philadelphia, PA, USA), followed by grinding through a 1 mm screen using an ultracentrifuge mill (Brinkman Instruments Co., Westbury, NY, USA), and submitted to Analab (Fulton, IL, USA) for nutrient analyses.
Prior to the start of the experiment, the forages were sampled, and the nutrients were analyzed after ensiling, followed by the TMR formulation using actual forage and feed ingredient nutrient concentrations. The dry matter compositions of the forages were determined weekly by drying them in a 105 °C oven (Despatch LEBI-75, Despatch Industries, Minneapolis, MN, USA) for 24 h, and the feed sheets were adjusted accordingly to maintain the ingredients at constant DM concentrations. Samples of the grain mix, individual forages, and TMR were collected and frozen (−20 °C) weekly for future analysis. The daily intake was calculated from the TMR offered and refusals, with the DM intake (DMI) being calculated by correcting the daily intake with the DM concentration. Refusals were assumed to be representative of the TMR fed due to achieving 5 to 10% feed refusals.
The milk production was recorded electronically (DeLaval-ALPRO, Kansas City, MO, USA) at each individual milking and saved daily to a Universal Serial Bus flash drive. Three milk samples were collected at each of the daily milkings, once each wk, from each individual cow. One set of milk samples was composited by day on a weighted basis, proportional to the milk production, and frozen at −20 °C for potential future protein fraction and/or fatty acid composition analyses. The other set of individual milk samples was sent to Dairy Herd Improvement Association Heart of America (Manhattan, KS, USA) for analyses of fat, protein, somatic cell counts (
SCCs), lactose, and milk urea nitrogen (
MUN) using Association of Official Analytical Chemists (
AOAC) International-approved [
28] procedures.
Body weights (
BWs) were electronically collected using a digital livestock scale (AWB-5K-SYS, Triner Scale and Manufacturing Company, Inc., Olive Branch, MS, USA), approximately 3 h after feeding on Thursday of each week. Body condition scores were determined weekly by the same 3 individuals on a scale of 1 to 5, with 1 as emaciated and 5 as obese [
29], using Edmondson et al.’s [
30] scoring chart, approximately 3 h after feeding at the start of this study (covariate) and each week. The scores of the 3 individuals were averaged.
Rumen fluid samples were collected on Thursday of wk 4, 8, and 12 at approximately 3 h after feeding via an esophageal tube attached to a hand-operated pump. The first 100 mL of rumen fluid was discarded to minimize saliva contamination. After collection, the rumen fluid was mixed thoroughly, and the pH was immediately measured using an electronic pH meter (Corning 350, Corning Inc., Corning, NY, USA). If the rumen fluid collected had a pH of >7.0, the rumen fluid was discarded, and additional rumen fluid was collected to ensure minimal saliva contamination. Two 10 mL samples of rumen fluid were collected, where one 10 mL sample was added to a vial containing 200 µL of 50% (vol/vol) H2SO4 for later determination of NH3-N, and the other 10 mL sample was added to a vial containing 2 mL of 25% (wt/vol) meta-phosphoric acid for later volatile fatty acid (VFA) determination. After the sample collection and preparation, the rumen fluid samples were immediately stored at −20 °C.
One 6 mL coccygeal artery blood sample, using a vacutainer tube containing sodium fluoride and a 20 guage 0.9 mm × 25 mm needle (Beckton Dickinson Vacutainer Systems, Rutherford, NJ, USA), was also collected on Thursday of wk 4, 8, and 12 at 3 h after feeding for later analysis of blood glucose concentrations. Fecal grab samples were collected during wk 4, 8, and 12 every 8 h for 3 d, with a forward advancement of 2 h daily to account for diurnal variation. The samples were composited by cow and stored frozen at −20 °C.
2.5. Laboratory Analysis
At the end of the experiment, the feed samples (TMRs, concentrate mixes, and individual ingredients) were thawed and composited by month and dried at 55 °C for 48 h in a forced-air oven (Style V-23, Despatch Oven Co., Minneapolis, MN, USA). The feed sample composites were ground through a 4 mm screen (Wiley mill, Arthur H. Thomas Co., Philadelphia, PA, USA) and then further ground through a 1 mm screen using an ultracentrifuge mill (Brinkman Instruments Co., Westbury, NY, USA) before being sent to Analab (Fulton, IL, USA) for DM and nutrient analyses. The feed samples were analyzed for their nutrient concentrations following standard AOAC International methods [
30], including DM (935.29), crude protein (
CP; 990.03), neutral detergent fiber (
NDF) with amylase (2002.04), acid detergent fiber (
ADF; 973.18), ADF-insoluble nitrogen (
ADIN; 973.18 and 976.06), NDF-insoluble protein (
NDIP; 2002.04 without sulfite and 976.06), lignin (973.18), ash (942.05), Ca (985.01), P (985.01), Mg (985.01), Na (985.01), Cl (915.01), S (923.01), Fe (985.01), Cu (985.01), Zn (985.01), K (985.01), Mn (985.01), and pH (981.12). The remaining nutrient concentrations were measured using the following methods: soluble protein (
SP) [
31], starch [
32], oil [
33], in vitro dry matter digestibility (
IVDMD) (24 h ruminal and 24 h enzymatic digestion using the Kansas State Buffer) [
34], neutral detergent fiber digestibility (
NDFD) ([
35]; incubation for 30 h using the Kansas State Buffer [
31]), ammonia–nitrogen (
NH3-N; the United States Environmental Protection Agency, 1993, method 351.2, and the International Organization for Standardization, 2013, method 11732), lactic acid [
36], acetic acid [
37], non-fiber carbohydrate (
NFC; [
23]), net energy of lactation (
NEL; [
23]), relative forage quality (
RFQ; [
38]), and sugar [
39]. Hemicellulose (
HC) was calculated as HC = NDF – ADF.
The milk fat, protein, and lactose were analyzed using near-infrared spectroscopy (Bentley 2000 Mid-Infrared Milk Analyzer, Bentley Instruments, Chaska, MN, USA). The milk urea nitrogen concentrations were determined using a chemical methodology based on a modified Berthelot reaction (ChemSpec 150 Analyzer, Bentley Instruments, Chaska, MN, USA; [
40]). The somatic cell counts were determined using a flow cytometer laser (Somacount 500, Bentley Instruments, Chaska, MN, USA; [
30]). The somatic cell counts were converted to a linear somatic cell score (
SCS) using the following equation: [(ln(SCC/100))/0.693147] + 3, as described by Spaniol et al. [
41]. The fat-corrected milk (
FCM; 3.5%) was calculated using the following equation: (0.432 × kg milk) + (16.216 × kg fat). The energy-corrected milk (
ECM) was calculated using the following equation: (0.327 × kg milk) + (12.95 × kg fat) + (7.65 × kg protein), as described by Orth [
42]. The fat- and protein-corrected milk (
FPCM) was calculated according to the IDF [
43] equation: FPCM = Milk, kg/d × ((0.1226 × fat, %) + (0.0776 × protein, %) + 0.2534).
The rumen fluid samples were thawed and centrifuged at 30,000×
g for 20 min at 20 °C (Eppendorf 5403, Eppendorf North America, Hauppauge, NY, USA). Rumen fluid samples acidified with 50% (vol/vol) H
2SO
4 were analyzed for ruminal NH
3-N using the Chaney and Marbach [
44] procedures. Ruminal fluid samples acidified with 25% (wt/vol) meta-phosphoric acid were prepared according to Erwin et al. [
45] and analyzed for VFA concentrations using an automated gas–liquid chromatograph (model 6890, Hewlett-Packard, Palo Alto, CA, USA) with a flame ionization detector. Once prepared, 1 µL of each prepared sample was injected with a split ratio of 30:1 at the injection port (250 °C). The VFAs were separated in a capillary column (15 m × 0.25 mm i.d.; Nukol, 17926–01C, Supelco Inc., Bellefonte, PA, USA) with a flow rate of 1.3 mL/min of He, using 2-ethylbutyrate as an internal standard. The column and detector temperatures were maintained at 140 °C and 250 °C, respectively. Blood plasma taken 3 h after feeding was analyzed for its glucose concentration via a colorimetric enzymatic kit (Liquid Glucose (Oxidase) Reagent Set; Pointe Scientific, Inc., Canton, MI, USA).
Individual (cow) fecal composite samples were shipped frozen in insulated shippers to Analab Laboratory (Fulton, IL, USA) for nutrient analysis. The fecal samples were analyzed using the following Association of Official Analytical Chemists International [
30] methods: DM (935.29), CP (990.03), ADF (973.18), and NDF (2002.04). Starch was measured using the Hall [
32] method. Acid-insoluble ash (
AIA) was analyzed in both the feed and feces as an internal digestibility marker [
46]. The nutrient digestibility was calculated as follows: digestibility, % = 100 − (100 × (AIA TMR concentration/AIA fecal concentration) × (nutrient fecal concentration/nutrient TMR concentration)).
2.6. Statistical Analysis
All data were checked for normality and outliers using the univariate procedure of SAS (version 9.4, SAS Institute Inc., Cary, NC, USA) before any statistical analyses were conducted. Box and whisker plots and the Shapiro–Wilk test were used to verify that the data were normally distributed (
p > 0.15). All data were then subjected to a least-squares analysis of variance (ANOVA) for an RCBD [
47] with a covariate using the PROC MIXED procedure of SAS (Version 9.4, SAS Institute, Inc., Cary, NC, USA), consisting of 2 treatments and 15 blocks. The statistical model used was as follows:
where Y
ijkl = the dependent variable, µ = the overall mean, Block
i = replication or block, P
j(Block
i) = parity (primiparous vs. multiparous) nested within replication, T
k = the treatment, W
l = the experimental week, T
k × W
l = the treatment-by-week interaction, Cov = the pretreatment covariate for the appropriate variables, Cow
m(T
k) = cow nested within treatment, and e
ijkl = the residual random error. The cows were blocked according to the calving date and parity (4 primiparous vs. 11 multiparous/treatment). Due to parity being nested within Rep and the use of a covariate, Rep was found to be nonsignificant, and, therefore, replication was deleted from the statistical model, but parity remained in the model. The individual cow data from the covariate week (the week before the experimental start) were included in the model. The treatment, week, and treatment × week interactions were considered fixed effects, with cow nested within treatment as a random effect. The experimental week was considered a repeated measurement in time, with an autoregressive covariance structure. The somatic cell counts were log
10 base-transformed prior to the statistical analyses. The least-squares covariate-adjusted means were separated by the PDIFF statement. The PDIFF statement is the least-significant difference method, which was used to compare the treatment means when the ANOVA F-test was significant. All results are reported as least-square means. Differences between treatments were considered significant at
p < 0.05 and trends at 0.05 <
p ≤ 0.10.