Grazing systems remain a major source of greenhouse gases (GHG) to the livestock sector. However, these systems stand as the most cost-efficient source of nutrients for dairy cows, and they also provide additional benefits for animal welfare . The promotion of sustainable grazing practices is among the interventions that might reduce GHG emissions per unit of product (carbon footprint) without sacrificing the associated benefits of grazing systems . It is well known the effect of greater nutrient quality and digestibility of forages is positively correlated with reduced methane (CH4) emissions. Pasture characteristics that determine forage quality, such as botanical composition   , regrowth period of pasture    and season , affect herbage digestibility, daily intake, milk production and enteric CH4 emissions. However, the effects of pre-grazing herbage mass (HM) on the dairy industry’s carbon footprint have not been studied. Reduced herbage mass and/or sward height, are important extrinsic factors limiting herbage intake on dairy cows, because of their effect on the ease of prehension of the herbage  . The difficulty to access the most easily harvestable material forces the cow to graze the deeper strata of the swards. This, demands a greater strength for prehension and cutting, leading to smaller bite mass, resulting in a decrease of the herbage instantaneous intake. Cattle will try to compensate this reduction in bite mass by increasing biting rate and grazing time   . Higher grazing activity increases energy requirement for maintenance and could reduce efficiency of production of dairy cows in grazing systems . On a low height ryegrass sward,  registered 59 bites per minute, which approaches the upper biting rate limit reported for grazing cattle . In these conditions, the increment in energy expenditure, regarding the same animals at rest, was 52%, during the grazing period .
Although production eﬃciency is essential to maintain proﬁtability and reduce the environmental impacts of grazing dairy production system, information on the effect of herbage mass on energy expenditure and methane emission per litter of milk produced is limited for our country. The aim of the present study was to quantify the effect of the level of pre-grazing herbage mass at the same daily herbage allowance, on herbage intake, grazing behaviour, milk production, energy expenditure, and daily methane emission of grazing dairy cows.
2. Materials and Methods
2.1. Experimental Treatments and Design
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.
2.2. Pasture and Grazing Management
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.
2.3. Pasture Measurements
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.
2.4. Animal Measurements
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 . 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  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 . 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 . 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  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 , 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  and adapted by , for 5-day collection period. Methane sampling equipment and procedures were as reported by . 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.
2.5. Chemical Analysis
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 , 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 . 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.
2.6. Statistical Analysis
Sward characteristics and herbage chemical composition were analyzed with ANOVA including the fixed effects of treatment and period:
where μ was the overall mean, Ti is the fixed effect of treatment (i = 1 to 2), Pj is the fixed effect of period (j = 1 to 2) and εij 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:
where μ was the overall mean,Ti is the fixed effect of treatment (i = 1 to 2), Pj is the fixed effect of period (j = 1 to 2), Ak is the random effect of animal (k = 1 to 8) and εijk 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 . 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 . 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.
According to INUMET (National Institute of Meteorology), total rainfall over all the experimental period was similar to the 10-year average for the region (77 vs 79 mm), while daily mean temperature (19˚C) was 1˚C lower than the 10-year average (2005-2014). However, Period 2 (6-11-2015 to 15-11-2015) was characterized by a higher daily mean temperature (+2˚C) and higher precipitation (+21 mm) compared to Period 1 (27-10-2015 to 05-11-2015).
3.1. Sward Characteristics and Defoliation
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).
3.2. Herbage Intake
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).
3.3. Grazing Behavior
No differences were found between treatments for grazing time (324 min, on
Table 1. Pre-grazing pasture characterization of high herbage mass (HHM) and low herbage mass (LHM) swards.
†measured as extended tiller height; ǂexpressed as percentage of grasses; #above 5 cm.
Table 2. Depth of defoliation and chemical composition of the herbage defoliated by dairy cows grazing high herbage mass (HHM) and low herbage mass (LHM) treatments.
†measured as extended tiller height; ǂexpressed as percentage of grasses; #above 5 cm.
Table 3. Effect of herbage mass on faecal output, herbage OM digestibility and daily intake by dairy cows grazing high herbage mass (HHM) and low herbage mass (LHM) treatments.
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).
3.4. Milk Production and Milk Composition
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).
3.5. Energy Expenditure
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 , for animals that are not subject
Table 4. Grazing behavior of dairy cows on high herbage mass (HHM) and low herbage mass (LHM) treatments during the observation period, from 9:00 to 18:00 h.
Table 5. Milk yield and milk composition by grazing dairy cows on high herbage mass (HHM) and low herbage mass (LHM) treatments.
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).
3.6. Methane Emission
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 (Ym = 6.7% on average) were different (Table 7).
Table 6. Heart rate (HR) and energy expenditure (HP) of cows grazing high herbage mass (HHM) and low herbage mass (LHM) treatments.
MW = metabolic weight (117.8 kg for HHM and 118.2 kg for LHM, P = 0.699).
Figure 1. Evolution of heart rate and the ingestive behavior during the observation period (9:00 to 18:00) for (a) HHM sward and (b) LHM sward.
Table 7. Daily methane emission and methane yield by grazing dairy cows on high herbage mass (HHM) and low herbage mass (LHM) treatments.
The aim of this study was to investigate if increasing pre-grazing herbage mass without changes in nutritive value during late spring offered at the same herbage allowance could reduce enteric methane emission intensity (g CH4/kg FPCM) by lowering energy expenditure of grazing dairy cows.
4.1. Sward Characteristics
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 . Studies assessing the effect of the available HM, while maintaining the same chemical composition of the forage are scarce . Several authors report variations in DMI, milk production and methane yield between treatments, but the pastures also differed in quality  . 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.
4.2. Herbage Intake and Grazing Behavior
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   , 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    . In this regard,  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  . 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  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).
4.3. Energy Expenditure
Energy expenditure analysis was made based on the technique developed by  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 , 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  and . 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  and , associated to an increase in ingested DM throughout the day. In fact, as reported by , 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.
4.4. Methane Emission
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    and national literature , 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  and with values reported in the meta-analysis presented by  for grazing dairy cows on temperate pastures.
The ranges of pre-grazing biomass evaluated in this experiment did not affect forage intake, milk production or methane emission from grazing dairy cows. Although intake was not affected, the cows that grazed the sward with lower biomass adapted their behaviour by means of a higher biting rate. This was not accompanied by an increase in the grazing time during the observation period in this study. The energy cost of prehension bites is very low in temperate pastures with herbage with high digestibility, which could explain why a greater number of bites in LHM treatment did not result in significant differences in terms of energy expenditure due to extra activity of grazing.
It is possible that, at lower daily herbage allowance, animals express behavior adaptations to reach higher intakes that are mediated by the HM and pasture height, with consequences in harvest efficiency and in energy expenditure by extra activity.
The authors would like to show their gratitude to Amabelia del Pino, Leticia Martinez, Shirley Furtado and Gabriela Arias for chemical analyses. The authors gratefully acknowledge Cristina Cabrera and Carlos Mantero for sharing facilities and technical advice.
This research was funded by the “Comisión Sectorial de Investigación Científica” (CSIC I+D 2014, Project 258) of the Universidad de la República (Uruguay).
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