The experiment was carried out in “Bernardo Rosengurtt” research station, Universidad de la República, Uruguay (32˚22'S, 54˚26'W), in mid-spring (October-November 2015). The effect of the pre-grazing herbage mass at the same herbage allowance on the performance of grazing dairy cows was examined in a repeated 2 × 2 Latin Square design. Animal procedures were approved by the Honorary Commission on Animal Experimentation of Universidad de la República (Exp. # 021130-001143-14). Treatments consisted of two target levels of herbage mass: 1000 kg of DM/ha above 5 cm (low herbage mass, LHM) and 1500 kg of DM/ha above 5 cm (high herbage mass, HHM). Eight multiparous Holstein Friesian dairy cows with an initial mean body mass of 537 ± 12.9 kg were used in this study. The cows were first blocked by pre-experimental milk yield (21.1 ± 0.5 kg/d) into two groups. Within each group, cows were paired based on their calving dates (190 ± 12.4 DIM) and within pairs randomly allocated to one of both treatments. Cows on both treatments were offered the same total herbage allowance (30 kg DM/cow/d, 5 cm above ground level). Similar herbage allowances per animal were obtained by adjusting the grazing area of each treatment according to their pre-grazing herbage mass. Each grazing period lasted 10 days, 5 days of adaptation, followed by 5 days of animal data collection. Period length was minimized in order to prevent large changes in the herbage conditions within and between periods. For this study it was more important that the chemical composition did not change so as not to confuse possible differences in consumption related to pre-grazing herbage mass (and more specifically its canopy structure), without differences in quality.

The study was conducted during the Spring (from October 27 to November 15, 2015) on a total area of 8 ha, 1 km away from the milking parlor. The forage mixture used was composed of 85% cocksfoot (Dactylis glomerata) and 15% lucerne (Medicago sativa). The area had been sown three years earlier and was subdivided into four equal paddocks. Two paddocks, corresponding to the HHM treatment were cut 35 days (5 cm height above ground level) prior to the beginning of Period 1. An additional cut on the other two paddocks was done 15 days before Period 1, in order to reach the target herbage mass for the LHM treatment. The same procedure was followed on the other two paddocks to prepare the grazing area for Period 2.

Before the experimental periods, the cows grazed a non-experimental pasture (composed of the same grass/legume mixture) as a single herd for two weeks, to allow previous adaptation to a diet based only on pastures. During the experiment, both swards were strip-grazed, at the same herbage allowance (30 kg DM/cow/d, 5 cm above ground level). After morning milking, the cows were offered a fresh strip once a day, using temporary electric fences. The area of each strip was calculated based on the pre-grazing herbage mass and the established daily allowance.

Pre-grazing herbage mass and mean sward height were measured before each plot was grazed (days 1, 5, 7 and 9). Herbage mass was measured by harvesting at random, three diagonal strips (10 m × 0.5 m), using a motor scythe at a cutting height of 5 cm above ground level. Herbage samples were weighed fresh and a bulked sample of 500 g was dried in an oven, at 60˚C for dry matter determination and chemical analysis. The extended tiller height of 50 tillers taken randomly on the same area used for the determination of pre-grazing herbage mass, were measured to estimate the mean sward height (measured from ground level to the uppermost extended point). The same procedure was followed for the determination of the post-grazing herbage mass and sward height after grazing (days 2, 6, 8 and 10). The differences between the pre-grazing and post-grazing height values were used to calculate the mean depth of defoliation for each treatment.

The botanical composition of the pasture was determined three times per treatment and per period, (days 5, 7 and 9). Next to each pre-grazing herbage strip cut for the herbage mass determination, three handfuls of herbage were randomly selected and cut with scissors at ground level. Samples were bulked and arranged in plastic bags in order to keep the herbage structure unaltered and stored at −20˚C. For botanical analysis, a subsample with its original structure still preserved, was used to determine the proportion of grass and legume (on DM basis) in the laboratory. A second subsample was cut at the height corresponding to the mean post-grazing sward height and the upper portion, considered as representative of the defoliated herbage, was dried before chemical analysis.

Cows were milked twice daily at 08:00 and 18:30 h, individual milk yields were recorded automatically at each milking. Milk composition (fat, protein, lactose) was determined four times per period from samples taken at morning and evening milking, from days 6 to 9. The milk yield and composition reported correspond to the CH₄ measurement (days 6 to 10). Fat Protein Corrected Milk (FPCM) yield was calculated using the equation proposed by [18]. Cows were weighed on the last day of each period (day 10).

Individual herbage OM intake was determined using chromic oxide (Cr₂O₃) to estimate faecal organic matter (OMf) output, and nitrogen (Nf) and acid detergent fiber (ADFf) contents in the faeces (g/kg OM) to estimate OM digestibility (OMd) of ingested herbage, according to the equation established by [19] for herbage-based diets without supplements. However, chromic oxide was offered mixed in concentrate pellets (ca. 20 g Cr₂O₃ per day, in portions of 200 g at each milking). Therefore, for forage intake calculations, OMf output from herbage was estimated by subtracting the indigestible OM attributable to the supplied concentrate containing the chromic oxide (92 g/kg OM) from the total measured faecal OM output. The supply of concentrate pellets containing chromic oxide started on day 1 of the experimental period in order to achieve a ruminal steady state. On days 6 to 10 of each experimental period, faeces were rectal-sampled after morning milking and after evening milking and oven dried at 60˚C during 72 h in order to measure the DM content, the Cr₂O₃ concentration, and the chemical composition.

On day 9, grazing and ruminating time, and biting rate were determined at the grazing session between milking (9:00 to 18:00). The cows were observed in the plot, recording every 5 min the behavior of each animal (ingestion, rumination, rest) and counting (by chronometer) the number of bites per minute during the periods of ingestion as described by [20]. Time spent per activity (min) was calculated assuming that the activity recorded was maintained during the 5 min until the next observation.

Energy expenditure as heat production (HP), was estimated using the heart rate and O₂ pulse method reported by [21]. The heart rate (HR) was recorded by a heart rate Radio transmitter (Polar Electro Oy, Kempele, Finland), fitted to the thorax of each animal, behind the forelegs by specifically designed belts, for 4 days in each experimental period (days 6 to 9). O₂ consumption was measured using an open respiratory system in day 2 of each period, as described by [21] and each cow’s O₂ pulse was calculated as the O₂ consumption per heart beat. Then the HP throughout the day was calculated by multiplying the HR (beats/min) as the mean value of measures throughout the day from days 6 to 9 in each period by the measured O₂ pulse (L/beat) and by the constant value of 20.47 kJ/L O₂ consumed [22], according to the following equation:

Daily HP (MJ/cow/day) = HR × O₂P × 20.47/1000 × 60 × 24 (1)

where: HR is heart rate (beats/min); O₂P is mL of O₂ per beat and 20.47 is the energy consumed by O₂P (kJ/L O₂).

The enteric CH₄ emission was measured using the sulfur hexafluoride (SF₆) tracer gas technique reported by [23] and adapted by [24], for 5-day collection period. Methane sampling equipment and procedures were as reported by [5]. Seven days before the beginning of the experiment, a SF₆ permeation tube was introduced per os into the rumen of each animal. The permeation rate of the tubes containing the SF₆ gas used in this study was 8.5 ± 0.32 mg/d. The breath gas sampling system consisted of two 0.5 L stainless steel collecting vessels per cow, with a ball-bearing inflow restrictor adjusted to accumulate 0.5 bar of air sample during a 5-day period and a short tube used to connect both. Both inflow restrictors were placed above the animal’s nostrils and protected against water and dust. The two collecting vessels were fitted to each animal’s head by means of especially designed halters. This way, it is possible to obtain two repetitions of CH₄ emission per cow and per period. Immediately prior to the sampling period, each collecting vessel was evacuated (<0.5 mb) after cleaning with high purity nitrogen gas (N₂). The breath gas samples were measured over five days in each period (days 6 to 10). Additionally, an identical set as used with the cows was used to collect background air samples during each 5-day period. The breath gas samples collected were analyzed immediately after the end of the experimental period. Daily CH₄ emissions were calculated from SF₆ release rate and the ratio between CH₄ and SF₆ concentrations in breath samples, after correction for background gas concentrations, according to the equation:

CH₄ (g/d) = PRSF₆ × [CH₄]/[SF₆] (2)

where: PRSF₆ is the SF₆ permeation rate from the permeation tube and [CH₄] and [SF₆] are the concentrations of these gases (ppm and ppt, respectively) above atmospheric concentration.

All chemical analyses were conducted at the Animal Nutrition Laboratory (Faculty of Agronomy). All the dried samples were ground through a 1 mm screen before chemical analysis. The dry matter (DM) concentration was determined by drying at 105˚C in an oven for 24 h and ash content was determined by incineration at 600˚C for 4 h for organic matter (OM) calculation.

The total nitrogen was assayed using the Kjeldahl method (Method 984.13; AOAC 2000) and expressed as crude protein (CP, nitrogen × 6.25). Content of neutral detergent fiber (NDFom) and acid detergent fiber (ADFom) were determined as described by [25], except that the samples were weighted into filter bags and treated with neutral detergent solution that included heat-stable amylase, in ANKOM equipment (ANKOM Technology, Macedon NY, USA), and expressed as ash-free residues. Gross Energy (GE) was determined using an adiabatic bomb calorimeter (Gallenkamp Autobomb; Loughborough, Leics, UK).

Milk samples at every milking of each collection period were analyzed for fat, protein, and lactose content with infrared spectroscopy (Milkoscan 203, Foss Electric, Hillerød, Denmark). Chromium (Cr) concentration in faecal samples was determined by atomic absorption spectrophometry (Perkin-Elmer 2380, Norwald, CT, USA), using air and an acetylene flame according to [26]. Cr standards were prepared using pre-trial faecal collections that contained no Cr.

The concentrations of CH₄ and SF₆ were determined by gas chromatography on an AGILENT 7890 chromatograph. The samples were injected at once in two different setups. For CH₄, a 3 mL loop, a HP-PLOT Q column and an FID detector were used. For SF₆, a 10 mL loop, a HP-MOLSIV column, and an ECD detector were used. Each sample was analyzed at least twice, and the average values were used to obtain CH₄ concentration and CH₄ emission. Maximum delay between the collection and the determination of CH₄ and SF₆ concentrations was 15 days.

Sward characteristics and herbage chemical composition were analyzed with ANOVA including the fixed effects of treatment and period:

Y = μ + T i + P j + ε i j (3)

where μ was the overall mean, T_i is the fixed effect of treatment (i = 1 to 2), P_j is the fixed effect of period (j = 1 to 2) and ε_ij is the associated error.

Herbage intake, live weight, energy expenditure and CH₄ emission were analyzed with a mixed model including the fixed effects of treatment and period, and the random effect of the cow:

Y = μ + T i + P j + A k + ε i j k (4)

where μ was the overall mean,T_i is the fixed effect of treatment (i = 1 to 2), P_j is the fixed effect of period (j = 1 to 2), A_k is the random effect of animal (k = 1 to 8) and ε_ijk is the associated error.

Milk yield and composition were analyzed as repeated measures over time, according to an autoregressive model of order one (AR 1) reported by [27]. The milk yield and composition reported correspond to the CH₄ measurement period.

The interaction treatment × period was initially included, but as interaction was not significant, it was excluded in the final models, following the recommendations of [28]. Significance was declared at P < 0.05 and tendencies at 0.05 ≤ P ≤ 0.10. All statistical analyses were performed using SAS program (SAS Institute Inc., Cary, NC). Data are presented as least square means ± pooled standard errors.

Pre-grazing herbage mass above 5 cm was higher for HHM (+412 kg DM/ha; P = 0.050), as well as the pre-grazing sward height (+11 cm; P = 0.037). However, the botanical composition of the pasture, expressed as the proportion of grass (89% of the herbage biomass on average) and its chemical composition above 5 cm, did not show significant differences between treatments (Table 1).

Post-grazing sward height was lower in LHM (−8 cm; P = 0.002). However, the depth of defoliation did not differ between treatments (20.5 cm on average), representing 44% and 54% of the initial height for the HHM and LHM treatment, respectively. The chemical composition of defoliated herbage did not differ between treatments (Table 2).

Both faecal output (4.4 kg OM/d on average) and digestibility of the defoliated herbage (740 g/kg OM on average) did not show significant differences between treatments. As a consequence, daily OM intake and DM intake did not differ between treatments (16.7 kg OM and 18.3 kg DM/d on average) (Table 3).

No differences were found between treatments for grazing time (324 min, on

^†measured as extended tiller height; ^ǂexpressed as percentage of grasses; ^#above 5 cm.

^†measured as extended tiller height; ^ǂexpressed as percentage of grasses; ^#above 5 cm.

average) during the daytime period observation (9:00 to 18:00). However, ruminating time was higher for animals in HHM (+35 min; P = 0.047), while resting time tended to be longer in LHM (28 min; P = 0.093). The biting rate was higher for the LHM treatment (+8 bites/min, P = 0.030) (Table 4).

The analysis showed no significant effect of pre-grazing HM on milk production. Individual milk yield was 21.1 kg FPCM/d, on average (Table 5). However, significant differences were found in milk composition as fat content (+1.8 g/kg, P = 0.005) and protein content (+0.7 g/kg, P = 0.006) being higher in LHM (Table 5). Regarding the live weight variation, there were no significant differences between treatments, animals gained weight in both treatments (+0.60 kg/d on average).

In this experiment, there were difficulties in measuring the volume of O₂ in P1. However, in P2 the measurements of the O₂ volume were within the expected values for this type of animal. According to [29], for animals that are not subject

to a high heat load or intensive exercise, there is a small variation in O₂P during the day. Hence, measuring the O₂P of an individual animal only once daily on short periods of time (days) could bias the individual energy expenditure (HP) calculations below 5%. Considering that the duration of the experiment was only 20 days, HP was estimated by multiplying the HR measured in each period by the oxygen pulse (O₂P) determined at the beginning of P2 (0.382 mL O₂/MW/ beat). The heart rate (HR) did not differ between treatments (87.0 beats/min on average), and neither did HP (979 kJ/kg MW on average) (Table 6).

Differences between daily HR records were not significant between treatments in the daytime period, with an increase in the heart rate during the sessions of grazing, from values of 77 beats/min at 5:00 to values of 88 (HHM) and 95 (LHM) beats/min at 18:00 (Figure 1).

Pre-grazing herbage mass did not affect enteric CH₄ emission, and it was 363 g/d (equivalent to 551 L per day), on average. Neither emissions expressed per unit of product (18.3 g kg FPCM/d, on average), per unit of DMI (21.0 g/kg DM on average) or as a percentage of GE intake (Y_m = 6.7% on average) were different (Table 7).

MW = metabolic weight (117.8 kg for HHM and 118.2 kg for LHM, P = 0.699).

The pasture management imposed had an effect on the level of pre-grazing herbage mass and on the sward height between HHM and LHM treatments, although the difference was smaller than initially planned. This occurred mainly as a result of the weather conditions (temperature and precipitation) registered during Period 2, that improved herbage growth, principally on LHM sward. It is worth mentioning that the botanical and chemical compositions did not differ between treatments. Cocksfoot, the main species of both swards, blooms relatively late in the season, and may allow high digestibility of the herbage to be maintained in spite of the advance of the Spring [30]. Studies assessing the effect of the available HM, while maintaining the same chemical composition of the forage are scarce [10]. Several authors report variations in DMI, milk production and methane yield between treatments, but the pastures also differed in quality [7] [8]. However, one might expect that the chemical composition of the forage consumed by the cows, would be different from that of the offered forage, since the post-grazing height resulted substantially higher (23 cm on average) than the cutting height of the motor scythe (5 cm on average). In the present study, a herbage subsample, cut at the height corresponding to the mean post-grazing sward height and the upper portion for each sward, was taken as a representative sample of the defoliated forage for quality evaluation. From this analysis, it was possible to estimate that the chemical composition of this fraction did not differ between treatments, and the OM digestibility was high (74% on average). Regarding this, it should be noted that this experiment was carried out with pastures in vegetative stage before initiation of stem elongation and lignification and, consequently, low and high herbage mass were of similar quality with no differences in OM digestibility.

The level of pre-grazing herbage mass (1859 vs. 1447 kg DM/ha, P = 0.050) offered at the same daily herbage allowance, had no effect either on the amount of forage ingested by the cows (18.3 kg DM/d on average) or on the milk yield (20.6 kg FPCM/d on average). The high herbage intake is in agreement with that reported by several authors [10] [31] [32], stating that herbage intake was not affected by herbage mass when measured above 5 cm. These authors show that forage intake reaches a maximum when pasture herbage allowance is 25 - 30 kg DM/cow/d above 5 cm or 60 kg DM/cow/d at ground level (similar values to those of pasture allowance expressed at ground level documented in this experiment, not shown here). The aforementioned literature show responses that tend to be asymptotic above these forage allowances, reaching a maximum pasture intake and milk production of approximately 18 kg DM/d and 21 kg/d respectively, similar to the results reported here. Nevertheless, milk composition differed among treatments, which might be most likely related to a milk dilution effect, as differences in milk fat yield (830 g/d on average) and milk protein yield (659 g/d on average) were not significant between treatments.

Time spent grazing did not differ among treatments and it was 324 min on average, 60% approx. of the total observation time (nine hours). However, the biting rate was higher in LHM, which could indicate a compensation mechanism for lower bite weight in this treatment. Intake per bite is the variable most directly influenced by sward conditions, and normally falls as herbage mass or sward height declines [17] [33] [34] [35]. In this regard, [35] found that, at lower pasture height, animals increased both biting rate and grazing time, as a compensation strategy to a lower ingestion rate. Another interpretation, given by several authors, attributes the difference in the bite frequency to a reduction in the number of manipulative jaw movements required on shorter swards, and a consequent increase in the ratio biting/manipulative movements [31] [36]. The longer time required to chew heavier bites explains the increase in time between successive prehension bites. Based on this interpretation, a time of 1.7 seconds per bite could be estimated in HHM compared to 1.4 seconds per bite in LHM. Additionally, when the number of bites of prehension is increased, particle size tends to be shorter [37] and, as chemical composition was similar (NDF content), adaptation is made not on the number of bites during rumination chewing, but on the rumination time (133 vs 98 min, P = 0.047, for HHM and LHM respectively).

Energy expenditure analysis was made based on the technique developed by [21] to evaluate if the level of herbage mass (at the same herbage allowance) could have an effect on the energy cost, indicating a greater physical activity, despite similar herbage intake. This method estimates energy expenditure by heat production (HP) through the O₂P-HR ratio, being HP the sum of HP for maintenance (HPm) and HP for production (HPp). According to [15], there is a negative relationship between the herbage mass and the energy expenditure, that can be explained by the increase in grazing activity (and so in HPm) when the amount of easily harvestable herbage is low. The lack of effect of the amount of pre-grazing mass on energy intake obtained in the present study, confirms that the difference between biomass treatments was beyond the limits that impose extra energy expenditure at grazing. In fact, even if there was an effect of pre-grazing biomass on feeding behavior (bite rate), it was not reflected in a different HP value due to differential activity between treatments. In temperate pastures with non-limiting biomass for herbage intake, the cost of harvest is negligible compared to the energy harvested in each bite according to [38] and [16]. Relating the values of HR to the ingestive behavior (Figure 1), it was observed that HR is lowest at 5:00, and increased after 10:00 and towards evening for both treatments, in agreement with the period of greatest activity of ingestion. Comparable results in dairy cows are reported by [21] and [39], associated to an increase in ingested DM throughout the day. In fact, as reported by [40], the maintenance energy requirement in lactating dairy cow increases at grazing compared to zero-grazing dairy cows, which is at least partly caused by more physical activity.

Finally, no difference in total enteric CH₄ emissions (363 g/d on average) or CH₄ emission intensity (18.4 g/kg FPCM on average) were observed, and the values are in agreement with those reported by previous international [8] [41] [42] and national literature [5], for dairy cows with similar levels of intake and production. This study constitutes the second study of measured enteric CH₄ emissions from grazing dairy cattle in Uruguay. The average CH₄ emission per unit of estimated feed intake (21 g/kg DMI) and CH₄ emission as a percentage of gross energy intake (6.7%) obtained are aligned with previous national findings [5] and with values reported in the meta-analysis presented by [43] for grazing dairy cows on temperate pastures.