1. Introduction
Although most dairy farms in Victoria, Australia, rely on grazed pasture as their main feed source, pasture alone cannot fully meet the nutritional requirements of a high-producing dairy cow [
1]. Both dry matter intake (DMI) and metabolizable energy limit milk production on a pasture-only diet [
2]. Due to this, grazing dairy cows are typically fed supplementary nutrients, commonly cereal grains or pelleted concentrates offered twice daily in the dairy during milking and, at certain times, conserved fodder fed in the paddock. In Australia, wheat and barley grains are the most commonly used grains and are typically fed at an average rate of 1.6 t/cow year
−1 [
3]. The amount and type of concentrates fed at different stages of lactation can be altered to reflect the nutrients supplied from pasture and the energy requirements of the cows, known as stepped flat rate feeding [
4]. A sudden introduction or increase in the amount of starch offered during stepped flat rate feeding can cause dramatic changes to the ruminal environment, including a rapid increase in acid production as a result of fermentation, to which ruminal microbes require time to adapt. If large quantities of concentrates are introduced abruptly to unadapted cows, the ruminal environment may not be able to cope with the increased acid load, leading to metabolic issues such as acute acidosis or sub-acute ruminal acidosis [
5]. Therefore, adaptation processes are typically implemented over several weeks with the amount of concentrates being offered, gradually increasing.
A ruminal fluid pH below 6.0 for extended periods of time can severely inhibit fibre digestion [
6]; hence, a lower threshold of pH 6.0 is typically used to identify optimal ruminal function. While it is commonly the feeding of concentrates that causes reductions in ruminal fluid pH, the responses in the rumen to different forages are not always equal. For example, Williams et al. [
7] reported a ruminal fluid pH consistently below 6.0 when dairy cows were consuming highly digestible fresh Persian clover (
Trifolium resupinatum) or grazing perennial ryegrass (
Lolium perenne L.). In contrast, Leddin et al. [
8] reported a ruminal fluid pH that remained consistently above 6.0 when lactating dairy cows were consuming a diet of solely perennial ryegrass hay. Furthermore, the rate at which ruminal fluid pH declines can be greater for cows fed legumes compared to cows fed grass [
9]. Ruminal responses to increasing amounts of crushed wheat grain also vary depending on forage type [
8,
10]. Eating behaviour and intake rates vary with forage type, and both impact ruminal fluid pH, mainly through saliva production [
11,
12]. Introducing or increasing concentrate supplements in a forage-based diet also alters eating behaviour, with both the amount of time spent eating and ruminating decreasing as the proportion of wheat in the diet increases [
13].
The process of gradually adapting cows to large amounts of concentrates can come at a cost of production, convenience and efficiency. It is therefore desirable to accelerate the process while still optimizing rumen function and milk production. This experiment investigated the effects of different forages during the abrupt introduction of wheat grain, with the aim of providing some insight into the possibility of using forages for improving concentrate adaptation processes. The hypotheses tested were that (1) the amount of time per day that ruminal fluid pH was below 6.0 would be greater for fresh forages compared to hays; (2) there would be no difference in the time per day that ruminal fluid pH was below 6.0 for the two fresh forages, nor between the two hays; (3) the minimum ruminal fluid pH would be lowest for cows fed fresh cut perennial ryegrass herbage compared to hays; and (4) the minimum ruminal fluid pH would not differ between the two fresh forage treatments, nor between the two hays.
2. Materials and Methods
The experiment was conducted at the Agriculture Victoria Research Centre, Ellinbank, Victoria, Australia (38°14′ S, 145°56′ E), in September 2017. All procedures were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes [
14]. Approval to proceed was obtained from the Agricultural Research and Extension Animal Ethics Committee, application number 2017-06, and was contingent on having thresholds for minimum ruminal fluid pH for removal of cows (pH 5.0).
Sixteen rumen-fistulated Holstein Friesian dairy cows in their third to ninth lactation were used. While all cows were seasonally calving, a combination of both fresh and carryover cows was used, either having calved between July and October 2016 or 2017 (230 ± 163.1 DIM; mean ± SD). Milking occurred twice daily at ~0600 and 1500 h. Twenty-one days prior to the experiment, concentrates being offered to the cows were gradually reduced, and for the final seven days prior to the experiment, they were fed a forage-only diet. The experiment then ran for 24 days comprising a 3-day covariate period, a 17-day adaptation period and a 4-day measurement period. During the covariate period, all cows grazed perennial ryegrass as a single cohort and received no concentrates. Following the covariate period, four treatments were randomly allocated to cows, such that the treatment groups were balanced for mean ruminal fluid pH (6.4 ± 0.20 pH; mean ± SD), milk yield (milk yield, 27.0 ± 8.63 kg/cow day−1), body weight (617 ± 47.1 kg), DIM (230 ± 163.1 DIM) and age (8.1 ± 2.11 years), as recorded during the covariate period.
Each treatment group received one of the following forages: lucerne hay, perennial ryegrass hay, fresh perennial ryegrass cultivar Bealey or fresh perennial ryegrass cultivar Base. During the adaptation period, all cows were moved to individual indoor pens for feeding and were offered their allocated forage ad libitum. Both cultivars of perennial ryegrass were harvested to 5 cm above ground level immediately before being offered to the cows. Cows were not given any concentrates during the adaptation period. In between feeding bouts, cows were returned to a bare paddock with no feed but with free access to water. During the measurement period, forage was offered at a rate of 17 kg DM/cow day−1. For the first 2 days of the measurement period, all cows were fed only forage. On days 3 and 4, crushed wheat grain was offered at a rate of 8 kg DM/cow day−1, and forage continued to be offered at a rate of 17 kg DM/cow day−1. Following each milking, cows were moved to individual stalls and given half their ration in the morning and half in the evening. Wheat was offered first, and within 20 min any grain refusals were removed, and forage was offered. All cows were given 4.5 h to consume their forage and had free access to water during this time.
The experiment was designed with four measurement days. However, due to several cows reaching the designated minimum ruminal fluid pH thresholds (pH 5.0), as required by the presiding animal ethics committee, the experiment was concluded 6 h after the morning feed on day 4. No data collected on day 4 were included in the analyses.
All feed offered and refused was weighed, and a representative sample was collected at each feeding. Part of each sample was then dried at 100 °C for 24 h to determine the DM concentration, which facilitated the calculation of individual DMI. The remainder of the samples were then bulked by feed type or, in the case of refusals, by individual cow and stored at 4 °C. At the completion of the experiment, bulked samples were thoroughly mixed and representative sub-samples were freeze dried and ground to pass through a sieve with mesh apertures of 1 mm. The samples were then analysed for crude protein (CP), acid detergent fibre (ADF), neutral detergent fibre (aNDF), lignin, non-fibre carbohydrates (NFC), starch, crude fat (CF), ash and estimated metabolisable energy (ME) by wet chemistry in a commercial laboratory (Dairy One Forage Laboratory, Ithaca, NY, USA). The nutritive characteristics of the feed offered are presented in
Table 1.
Three days prior to the measurement period, all cows were fitted with jaw movement recorders (RumiWatch, ITIN+HOCH GmbH, Liestal, Switzerland) to quantify eating behaviour. The halters remained on the cows for the entire measurement period and enabled the automatic measurement of time spent eating, ruminating and not chewing. The halters collected data via an inbuilt pressure sensor and a triaxial accelerometer.
Milk yield was recorded at each milking throughout the experiment using a DeLaval Alpro milk metering system (DeLaval International; Tumba, Sweden), and a sub-sample was collected for each cow using in-line milk meters (DeLaval International). Samples were analysed for fat, protein and lactose concentrations using an infrared milk analyser (Model 2000, Bentley Instruments, Chaska, MN, USA). Energy-corrected milk (ECM) yield was calculated using the following formula [
15]:
At the commencement of the measurement period, capsules for measuring ruminal fluid pH (KB5; Kahne limited, Auckland, New Zealand) were calibrated and inserted per fistula into the rumen of each cow. The capsules remained in the cows until the end of the measurement period. A 750 g weight was attached to each capsule to ensure it remained on the bottom of the rumen. Ruminal fluid pH was logged every 5 min, and the data were automatically stored in the devices. Capsules were removed once a week for 8 h to recalibrate the pH devices, and a linear interpolation was used to correct for any drift in readings from individual boluses. Following the validation in standard pH buffers (4.01 and 7.01), all data were downloaded, and boluses were recalibrated before re-insertion.
Beginning on day 3 of the measurement period, seven ruminal fluid samples were collected per cow per feed, with the first sample collected immediately prior to feeding and a sample collected every hour thereafter. Samples were collected per fistula using a 100 mL plastic syringe connected to a copper pipe directly inserted into the rumen. Fluid was collected from four different sites within the rumen. A 50 mL sub-sample was immediately poured off and centrifuged (4 °C, 4000×
g, 10 min), while the pH of the remainder was measured using a benchtop pH meter (Orion star A211; Thermo Fisher Scientific, Schwerzenbach, Switzerland). A 0.5 mL aliquot of supernatant was then transferred to a tube containing 4.5 mL of dilute acid (0.1 M HCl) for later analysis of the ammonia concentration. An additional 5 mL aliquot was dispensed into a tube for analysis of VFA and lactate concentrations. Both sub-samples were stored at −20 °C until analyses. Volatile fatty acid concentrations were determined by capillary gas chromatography (Agilent 6890 GC; Agilent Technologies, Santa Clara, CA, USA) using a flame ionisation detector, auto-sampler and auto-injector, and a wide bore capillary column (BP21 column, 12 m × 0.53 mm internal diameter (ID) and 0.5 μm film thickness; SGE International, Ringwood, Victoria, Australia) with a retention gap kit (including a 2 m × 0.53 mm ID guard column). Analyses were conducted following the methodology described by Packer et al. [
16], with 4-methyl-valeric acid (1.58 mmol/L) used as the internal standard. Lactate analyses were conducted with a microplate reader (AMR-100, Allsheng Instruments, Hangzhou, China) using a D/L-lactate kit (K-DLATE; Megazyme, Bray, Ireland). Ammonia concentrations were determined by flow injection (Lachat Quik-Chem 8000; Lachat Instruments, Milwaukee, WI, USA) according to an alkaline phenol-based method (method 12-107-06-1-A; Lachat Instruments, Milwaukee, WI, USA) and analysed against standard ammonia solutions.
All data were analysed using Genstat for Windows (Genstat 18th edition, VSN International Ltd, Indore, India.). For all datasets, days were grouped according to diet, with days 1 and 2 categorised as forage only and day 3 categorised as forage and wheat. As day 4 only consisted of an a.m. period, it was not included in the overall analyses. Comparisons between forage groups hay (lucerne hay and perennial ryegrass hay) and fresh (perennial ryegrass cultivar Bealey and cultivar Base) as well as between forages within these groups, for all variables, were achieved by specifying contrasts on the factor for forage within the treatment structure employed in the ANCOVA. Daily yields (milk, ECM and composition yields) were calculated as the sum of p.m. and a.m. values. Daily milk composition (%) was calculated as the ratio of daily composition yield to milk yield. Milk production and intake data were subject to an analysis of variance (ANOVA) adjusted for data collected during the covariate period. The factorial treatment structure was forage by wheat, with a blocking structure of cow split for period (forage, wheat and forage) split for day.
The pH data from 2 intraruminal capsules were not able to be retrieved, one from a cow in the perennial ryegrass hay treatment and one from a cow in the lucerne hay treatment. Ruminal fluid pH data collected via the intraruminal capsules were summarised daily for each cow as daily mean, minimum, maximum, time under pH 6, area under pH 6 and rate of decline post-feeding. A day was considered from 07:00 h to 07:00 h. To calculate the rate of pH decline following each feeding, each daily set of pH data was also categorised into two ‘peak’ pH intervals and two ‘trough’ pH intervals. These intervals were derived visually from an average ruminal fluid pH (averaged over all cows, at each time) vs. time graph. The daily intervals were peak: from 03:00 to 09:00 h and 14:00 to 18:00 h, and trough: 09:00 to 14:00 h and 18:00 to 03:00 h. The maximum pH within each peak interval and the minimum pH within each trough interval were then identified and the slope (change in pH divided by change in time) was calculated. The data were then summarised as an average daily rate of decline in pH for each cow, the amount of pH decline and the duration of the decline. All summary data for ruminal fluid pH variables were subjected to an ANCOVA with a blocking structure of cow by period (forage, wheat and forage) split for day, with covariate as the corresponding variable measured in the covariate period. The factorial treatment structure was period by forage. Ruminal fluid fermentation profile data consisted of pre-feed and 6 h post-feed measurements for the morning and evening on each of day 2 and day 3. These were subjected to ANOVA with the factorial treatment structure of forage by period by sample (pre- or post-feeding) plus time of day (a.m. or p.m.), and a blocking structure of cow by period (i.e., day) split for time of day split for sample. Lactate data were log transformed prior to analysis. Eating behaviour data were analysed with an ANOVA using the treatment structure forage by wheat and the blocking structure cow by period split for day.