Influence of different sources of zinc and protein supplementation on digestion and rumen fermentation parameters in sheep consuming low-quality hay

Small Ruminant Research 121 (2014) 175–182

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Influence of different sources of zinc and protein supplementation on digestion and rumen fermentation parameters in sheep consuming low-quality hay H.M. Arelovich a,b,c,∗ , M.I. Amela a , M.F. Martínez a , R.D. Bravo a , M.B. Torrea a a b c

Departamento de Agronomía, Universidad Nacional del Sur, 8000 Bahía Blanca, Argentina Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC), 8000 Bahía Blanca, Argentina Centro de Recursos Naturales Renovables de la Zona Semiárida (CERZOS), CCT-CONICET, 8000 Bahía Blanca, Argentina

a r t i c l e

i n f o

Article history: Received 27 December 2013 Received in revised form 14 August 2014 Accepted 18 August 2014 Available online 16 September 2014 Keywords: Zinc Protein Low-quality forage Sheep Rumen fermentation

a b s t r a c t This experiment studied the effect of ZnCl2 or ZnSO4 included into a protein supplement on dry matter intake (DMI), DM digestibility (DMD) and rumen fermentation parameters in sheep consuming low-quality native pasture hay (NPH) as a basal diet. Four ruminally fistulated Corriedale weathers (31 ± 4 kg BW) were randomly assigned to a 4 × 4 latin square design, and received NPH ad libitum in the following treatments: (1) Control (CON) mineral mix only, (2) protein supplement (PROT), (3) PROT + ZnCl2 (CZProt) and (4) PROT + ZnSO4 (SZProt). All supplements were fed at the rate of 150 g animal d−1 . The Zn concentration in CZProt and SZProt was 460 ppm for a total intake of 100 ppm daily as a proportion of total diet. The NPH contained 5.3 and 77.4 of CP and NDF as % of DM respectively. Protein supplements were isonitrogenous (30% CP) with barley grain, wheat middlings and sunflower meal as main components. Mean NPH intake and total DMI were not affected by protein supplementation or Zn addition to the diet. DM digestibility as well as DDMI (digestible DMI) increased 31 and 37% respectively for SZProt compared to CON (P < 0.05). Sampling hour affected most rumen variables with the exception of butyric and total volatile fatty acids (VFA; P < 0.05). A treatment by sampling hour interaction (P < 0.05) was only found for ruminal ammonia-N (NH3 -N) concentration. At time intervals of 2, 4, 8 and 12 h, [NH3 -N] was much lower in CON than in protein supplemental treatments (P < 0.05). A trend was observed for increased NH3 -N concentration in SZProt (39.76 mg dL−1 ) vs. CZProt (29.25 mg dL−1 ) at 4 h (P = 0.0897). The rumen pH was not affected by treatments; the lowest values found 12 h after feeding were 6.2. Zinc, particularly from SZProt, seems to modulate [NH3 -N] by sustaining higher concentrations during the 24 h period. A clear effect on major VFA was not observed. The inclusion of 100 ppm of Zn, and source could differentially affect animal response to protein supplementation and utilization of low quality roughages by affecting some rumen fermentation patterns. © 2014 Elsevier B.V. All rights reserved.

∗ Corresponding author at: Departamento de Agronomía, Universidad Nacional del Sur, 8000 Bahía Blanca, Argentina. Tel.: +54 291 4595102; fax: +54 291 4595127. E-mail address: [email protected] (H.M. Arelovich). http://dx.doi.org/10.1016/j.smallrumres.2014.08.005 0921-4488/© 2014 Elsevier B.V. All rights reserved.

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Table 1 Native pasture hay and supplements composition on a dry matter basis fed to sheep. Item

Treatmentsc

Native a

Pasture hay Components (%) Barley grain, ground Wheat middlings Sunflower meal Urea Mineral premixb Nutrient composition (%) Dry matter Crude protein NDF ADF ADL Ash Supplement mineral content (%) P Ca S Zn Supplement intake (g d−1 )

– – – – – 93.3 5.3 77.4 47.4 7.7 9.6 – – – – –

CON

PROT

– – – – 100 96.2 5.7 12.8 4.5 2.0 67.7 2.91 6.19 5.33 0.01 30.0

CZProt

SZProt

28.0 17.9 36.8 4.0 13.3

28.0 16 37.8 4.0 14.2

28.0 17.5 37.4 4.0 13.1

90.6 30.3 28.0 15.2 4.4 17.0

90.7 31.4 27.7 15.2 4.3 18.0

90.6 30.6 27.9 15.2 4.4 18.2

1.18 1.43 1.25 0.05 150.0

1.17 1.44 1.23 0.46 150.0

1.17 1.44 1.25 0.46 150.0

a

Native pasture hay was the basal diet fed ad libitum. Wheat middlings was used as a carrier. Included NaCl, bone ash (to sustain the Ca:P ratio 1.5:1) and NaSO4 (to parallel S intake across treatments). Treatments: CON (control, only mineral mix without a source of Zn); PROT (protein supplement without a source of Zn); CSProt (PROT + ZnCl2 ); and SZProt (PROT + Zn SO4 ). Supplements were 30% CP. b c

1. Introduction Manipulation of rumen environment by the dietary addition of different additives would improve fermentation efficiency and consequently increase animal performance. Adesogan (2009) reviewed several additives such as ionophores, yeasts, fibrolytic enzymes, essential oils, buffers and organic acids; their modes of action and to which extent these additives could influence rumen parameters, as well as animal health and performance. Because of strict regulations on feed additives use, such as that imposed by Regulation 1831/2003/EC (European Union, 2003), researchers focused on finding new additives of natural origin to modulate rumen fermentation. Several novel natural products are evaluated. An example is the experimental use of chitosan to enhance energetic efficiency (Goiri et al., 2010). A recent review discusses progress in decreasing rumen methane through different additives by rumen microbial manipulation (Kobayashi, 2010). Some essential elements could also impact on rumen microbial fermentation as well (Martinez and Church, 1970; Spears and Hatfield, 1979; Rodriguez et al., 1995; Arelovich et al., 2000; Faixova and Faix, 2002; Arelovich et al., 2008; Richter, 2011). Previous research indicated that dietary addition of 250 to 400 mg Zn kg−1 DM to low-quality forage supplemented with urea retarded ammonia accumulation and increased molar proportions of propionate in beef cattle (Arelovich et al., 2000). However, this effect of Zn could be associated diet quality because a significant effect of Zn addition upon rumen parameters was not evident for sheep fed a diet based on barley grain, alfalfa hay and sunflower meal rather than urea (Arelovich et al., 2008). Potential interactions with type of diet, mineral concentration, as well as mineral source are relevant factors for Zn to be considered as

additive influencing rumen fermentation. This experiment aims to study the effect of Zn chloride or sulfate added to a protein supplement on intake, digestibility and rumen fermentation parameters in sheep consuming low-quality native pasture hay as a basal diet. 2. Materials and methods 2.1. Animals and treatments Four Corriedale weathers with 31 ±4 kg mean BW were fitted with 40 mm internal diameter ruminal cannulae, and randomly allocated to individual pens. Animal health and welfare was daily monitored according to the standards established by the National Health and Agri-Food Quality Service (SENASA, 2001). All animals were injected with 0.02 mL of Ivermectin/kg of BW immediately before the trial started. A native pasture hay (NPH) chopped in a hammer mill through a 10 mm mesh was used as the basal diet. The NPH, harvested from selected range area, included mainly two native grasses: Piptochaetum napotaense and Stipa tenuis, both in similar proportions. The NPH was offered ad libitum to all animals within four experimental treatments in a 4 × 4 latin square design: (1) control (CON) mineral mix only, (2) protein supplement (PROT), (3) PROT + ZnCl2 (CZProt) and (4) PROT + ZnSO4 (SZProt). All supplements were iso-protein formulated to contain 30% CP and fed at the rate of 150 g animal d−1 . The Zn concentration in the supplements CZProt and SZProt was 460 ppm. Except for Zn content, the mineral mix was equivalent to all treatments. A detailed composition of NPH and supplements is described in Table 1. The daily Zn dose needed to achieve desired rumen Zn concentrations was calculated following procedures described by Arelovich et al. (2000). The Zn levels in this experiment were designed either (1) to meet the dietary Zn requirement (30 ppm in the diet) suggested by NRC (1985), or (2) to assume a concentration of 100 ppm in the total diet (by adding the 30 ppm assumed for the CON and PROT treatments). The dietary tolerance level is set at 300 ppm/animal daily; however, under many circumstances somewhat higher levels have not produced adverse effects in sheep (NRC, 1980). With 100 ppm in the diet about 18 ppm of Zn in the rumen is expected to be reached, estimated to be 10% below the maximum ruminal concentration tolerated by bacteria (Martinez and Church, 1970). As reported by Kennedy et al. (1993) Zn also forms complexes with organic and particulate matter in the rumen. Then, an excess of ruminal Zn concentration was intended to avoid at any time.

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Table 2 Effect of protein supplementation and Zn source on DM intake and digestibility in sheep fed on native pasture hay as a basal diet. Treatments1

Item CON

PROT

CZProt

SZProt

SEM

P-Value

NPH DMI (g d−1 ) Total DMI (g d−1 ) Diet DM digestibility (%) Diet DDMI (g d−1 )

806 836 51.8a 417a

732 882 59.0a,b 496a,b

696 847 59.9a,b 507a,b

738 888 67.7b 571b

48.2 48.2 2.67 26.9

0.4945 0.8389 0.0314 0.0372

Dietary CP intake (g d−1 ) From forage From supplement Total

43 1.7 45

39 45 84

37 47 84

35 46 81

– – –

– – –

1

Treatments: CON (control, only mineral mix without a source of Zn); PROT (protein supplement without a source of Zn); CSProt (PROT + ZnCl2 ); and SZProt (PROT + Zn SO4 ). Supplements were 30% CP. a,b Means with different letters within each row differ (P < 0.05) 2.2. Sampling procedures and determinations Each period of the latin square was divided into an initial adaptation period (d 1 through 7), measurements for NPH consumption and digestibility (d 8 through 12), and rumen sampling on d 13 (at 2, 4, 8, 12, and 24 h after supplement was fed). Fecal grab samples were collected daily on d 10 through 13. Offered and rejected hay samples were immediately dried after collection (48 h at 60 ◦ C), composited within animal and treatment by the end of each period. Fecal samples were refrigerated each day and afterwards composited within animal and treatment by the end of each period and dried (66 h at 50 ◦ C). After drying, samples were ground through a 2-mm screen in a Wiley mill (Standard Model 3, Arthur H. Thomas Co., and Philadelphia, PA) and stored for later analysis. NPH and supplements chemical determinations included dry matter (DM), crude protein (CP = g N 100 g−1 DM × 6.25) by macro-Kjeldahl N analyses, and ash (AOAC, 2000). Neutral detergent fiber (NDF), acid detergent fiber (ADF) and acid detergent lignin (ADL) were determined with an ANKOM Fiber Analyzer (Ankom Technology, Fairport, New York, USA) following the detergent system procedures (Van Soest et al., 1991), including ␣-amylase but without sodium sulfite. The DMI was computed by subtracting dry orts weight from dry weight of NPH. Dry matter digestibility (DMD) was calculated by using ADF-insoluble ash (ADIA, Van Soest et al., 1991) as an inherent marker, and digestible DMI (DDMI) was calculated as the multiple of DMI and DMD. The obtained rumen fluid was filtered through four layers of cheesecloth, pH of the filtrate was measured immediately, and a 100-mL subsample of the filtrate was acidified with 1 mL of a 7.2 N sulfuric acid solution and frozen. Later, ruminal liquor was centrifuged for 20 min at 10,000 × g at 4 ◦ C and the supernatant liquid was analyzed for Zn, ammonia-N (NH3 -N), and volatile fatty acid (VFA) contents. Concentrations of Zn in the supernatant fluid from ruminal contents were determined using inducible plasma emission spectrometry (Shimadzu ICPS1000 III). For measurements of ruminal NH3 -N concentration, 50-mL aliquots were analyzed colorimetrically at 630 nm using a spectrophotometer (Spectronic 20) following the procedure of Broderick and Kang (1980). For VFA a subsample of 8 mL of rumen fluid was mixed with 2 mL of a deproteinizing solution (0.2% (w/v) with HgCl, 2% (w/v) orthophosphoric acid and 0.2% (w/v) methyl valeric acid) and frozen. Previous to VFA determinations these samples were unfrozen at room temperature and centrifuged at 7000 rpm for 20 min at 4 ◦ C. VFA concentrations were measured using a gas chromatograph (Shimadzu GC-14A), equipped with a flame ionization detector with a capillary column Nukol (30 m × 0.25 mm internal diameter), to determine VFA in aqueous media, using helium as a carrier and methyl valeric acid as an internal standard. The temperatures were 220 ◦ C and 250 ◦ C for injection and detection respectively, and final column temperature was 150 ◦ C. The injection volume was 1 ␮L.

2.3. Statistical analysis All data were analyzed as a 4 × 4 Latin square, using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC). The DMD, DMI and DDMI data were analyzed according to the model including period, animal, and treatment: Yij =  + ai + pj + εij where Yij represents the value of each individual observation,  is the mean; ai is the random effect associated with the ith animal; pj is the random effect associated with the jth period; εij is

the residual error associated with the experimental unit animal i and period j. For analysis of ruminal parameters (Zn concentration, pH, VFA and NH3 -N), sampling time (2, 4, 8, 12, and 24 h postfeeding) was added to the model including the sampling time × treatment interaction, and studied by the MIXED procedure of SAS for repeated measures (Littell et al., 1998). The statistical analysis of results was subjected to different possible covariance structures. The unstructured, power and simple covariances structures yielded the smallest Schwarz Bayesian criterion for reliable analysis (Wang and Goonewardene, 2004). When applicable, orthogonal contrasts were used and means compared by LSD (P < 0.05), with results reported as least squares means.

3. Results 3.1. Nutrient intake and digestibility Table 2 reports results on diet intake and digestibility. Average treatment CP intake was added to illustrate that CP intake on supplementation treatments resulted as intended. Mean NPH intake and total DMI were not affected by protein supplementation or Zn addition to the diet (Table 2). However, DM digestibility as well as DDMI increased 31 and 37% respectively for SZProt compared to CON (P < 0.05). Although these parameters were also numerically higher for both PROT and CZProt than CON, they did not significantly differ from either CON or SZProt, neither among themselves.

3.2. Rumen measurements Data for rumen variables are presented in Table 3. Differences in Zn content of ruminal fluid indicated that Zn sources influenced Zn availability within the rumen environment, with SZProt exhibiting the highest mean values (1.90 mg dL−1 ; P < 0.05). A significant effect of sampling time was also observed for ruminal concentration of Zn. Sampling hour affected most rumen variables with the exception of butyric and total VFA (P < 0.05). In Fig. 1 a trend line for overall ruminal Zn concentrations illustrates an increasing pattern with a peak between 12 and 14 h after feeding and then a decrease. The pH trend line followed an opposite pattern compared to that of Zn concentrations with lowest values 12 h after feeding (Fig. 2). The highest pH observed values were 6.71, 6. 81 6.76 and 6.73 and the lowest 6.19, 6.29, 6.23 and 6.23 measured at 2 and 12 h after

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Table 3 Effect of Zn source and protein supplementation on ruminal parameters in sheep fed native pasture hay as a basal diet. Treatments1

Rumen variables CON Zn (mg/L) pH NH3 -N (mg/L) VFA Acetic (%) Propionic (%) Isobutyric (%) Butyric (%) Isovaleric (%) Valeric (%) Acetic/propionic ratio Total VFA (mmol L−1 )

0.82a 6.5 7.6 63.8 20.7 1.3 12.0a 1.2a 0.9 3.2 27.1

SEM

P-Values

PROT

CZProt

SZProt

Treatment

Hour

TxH

0.52a 6.6 23.1

1.27a 6.49 23.6

1.90b 6.53 25.6

0.310 0.091 1.581

0.0235 0.8114 0.0001

0.0122 0.0002 0.0001

0.8596 0.4446 0.0048

59.5 21.8 1.4 14.2b 1.9c 1.2 2.8 26.6

60.9 21.0 1.3 14.0b 1.6b 1.2 2.9 29.9

61.1 21.0 1.4 13.9b 1.5b 1.1 2.9 31.5

1.026 1.273 0.213 0.341 0.055 0.121 0.204 1.748

0.0877 0.9404 0.9310 0.0004 0.0001 0.5329 0.6042 0.1691

0.0177 0.0002 0.0003 0.0767 0.0001 0.0003 0.0001 0.3009

0.5046 0.1336 0.4195 0.9203 0.1204 0.1090 0.2919 0.5682

1

Treatments: CON (control, only mineral mix without a source of Zn); PROT (protein supplement without a source of Zn); CSProt (PROT + ZnCl2 ); and SZProt (PROT + Zn SO4 ). Supplements were 30% CP. a,b Means with different letters within each row differ (P < 0.05) Table 4 Effect of Zn source and protein supplementation on ruminal N-ammonia by treatment at different time periods in sheep fed native pasture hay as a basal diet. Treatmenta

CON PROT CZProt SZProt SEM

Time (h) *

2

4

8**

12**

24***

9.87a 43.84b 37.10b 36.52b 4.715

8.76a 30.92b 29.25b 39.76b 3.328

6.04a 15.51b 21.34b 21.20b 2.180

4.31a 10.55b 15.99b 15.04b 1.892

8.80 14.49 14.22 15.31 1.412

Orthogonal contrast CON vs. all. Means with different letters (a, b) within each column differ (P < 0.05). a CON (control, only mineral mix without a source of Zn); PROT (protein supplement without a source of Zn); CSProt (PROT + ZnCl2 ); and SZProt (PROT + Zn SO4 ). Supplements were 30% CP. CZProt and SZProt provided 460 ppm of Zn. * Orthogonal contrast: CZProt vs. SZProt non-significant trend 4 h after feeding (P = 0.0897). ** Orthogonal contrast: PROT vs. CZProt + SZProt non-significant trend 8 and 12 h after feeding (P = 0.0627 and 0.0570 respectively). *** Non-significant effect of treatment (P = 0.1337).

feeding for CON, PROT, CZProt and SZProt. The pH across treatments and time averaged 6.53. A treatment by sampling hour interaction (TxH, P < 0.05) was only found for ruminal NH3 -N concentration (Table 3). Then the effect of treatments was evaluated separately for each sampling interval by means of orthogonal contrasts (Table 4). At 2, 4, 8 and 12 h, NH3 -N was much lower in

Fig. 1. Trend line for mean ruminal Zn concentration by time after morning feeding. Points represent means for each sampling hour across dietary treatments represented by the equation Zn = − 0.2286t2 + 0.14634t − 0.748; R2 = 0.6735, n = 80; where Zn = Zn concentration; t = time.

CON than in protein supplemental treatments. Although, numerically lower, CON (8.80 mg dL−1 ) did not differ from the others 24 h after feeding (P > 0.05). All supplemental treatments have similar concentrations 24 h after feeding with values of 14.8, 14.2 and 15.3 mg dL−1 for PROT, CZProt and SZProt respectively. The protein supplements with added Zn followed a similar pattern with initial values for NH3 -N of 37.1 and 36.1 mg dL−1 for CZProt and SZProt

Fig. 2. Trend line for mean ruminal pH by time after morning feeding. Points represent means for each sampling hour across dietary treatments represented by the equation pH = 0.0039t2 − 0.11053t + 6.9574; R2 = 0.3458, n = 80; where t = time.

H.M. Arelovich et al. / Small Ruminant Research 121 (2014) 175–182

Fig. 3. Trend lines for mean ruminal NH3 -N concentrations by time and diets. Treatments at each time period are described by the following equations: (control), only mineral mix added, NH3 CON N = 1.0514t2 − 12.211t + 48.646; R2 = 0.9963, n = 20; PROT (protein supplement); NH3 -N = 3.2064t2 − 27,074t + 69,084; R2 = 0.9785, n = 20; CSProt (PROT + ZnCl2 ), NH3 -N = 0.4607t2 − 9.4793t + 48.938; R2 = 0.8152, n = 20; SZProt (PROT + Zn SO4 ), NH3 -N = 0.8707t2 − 5.8833t + 15.628; R2 = 0.7060, n = 20; where NH3 -N = ammonia-N concentration; t = time.

respectively 2 h after feeding. A trend was observed for increased NH3 -N concentration in SZProt (39.76 mg dL−1 ) vs. CZProt (29.25 mg dL−1 ) at 4 h (P = 0.0897); and for the average of Zn treatments contrasted with PROT at 8 and 12 h after feeding (P = 0.0627 and 0.0570 respectively). In general, the largest single NH3 -N concentration was 43.8 mg dL−1 for PROT at 2 h, followed by a fast drop on time after feeding, as illustrated by the trend lines drawn for each experimental treatment at the different sampling times in Fig. 3. A less sharp fall of NH3 -N concentration in time was observed for CZProt and SZProt compared with PROT. The NH3 -N concentration showed the lowest daily variation for CON with the lowest concentration of 4.31 mg dL−1 12 h after feeding. For VFA a treatment effect was just observed for the molar proportions of butyric and valeric acids, which increased with protein supplementation (P < 0.05). Besides, molar proportion of isovaleric acid (1.9%; P < 0.05) was highest for PROT, but all three supplemental treatments resulted higher than CON (1.2%; P < 0.05). The molar proportion of acetic acid followed a non-significant decreasing trend (P = 0.0877) more evident for PROT (59.5%) than for CSProt (60.9%) and SZProt (61.1%) when compared to CON (63.8%). Although an effect of treatment was not detected for total VFA (P = 0.1691) the mean concentration for SZProt was 31.5 mmol L−1 being numerically larger (16%) than 27.1 mmol L−1 for CON. 4. Discussion 4.1. Diet intake and digestibility In contrast with other studies protein supplementation, with or without Zn, has not increased either NPH or total DMI compared with CON treatment, a substitution effect was observed. Numerous authors reported a positive effect of CP supplementation on voluntary intake of low quality forages (CP content below 7%; Paterson et al., 1994), and

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with different N sources and feeding strategies (Arelovich et al., 1992; Hennessy, 1996; Bandyk et al., 2001; Lardy and Endecott, 2010). For NDF levels up to 35% a linear increase in DMI could be expected (Arelovich et al., 2008). However, Mertens (1985, 1994) indicated that DMI could be maximized when NDF intake is approximately 12.5 g kg BW−1 . Then a large NDF intake could partially explain the overall lack of response of DMI to CP supplementation, since the NDF content for NPH was 77.4% (Table 1) and the calculated NDF intakes for the forage alone were 20, 18, 17 and 18 g kg−1 BW for CON, PROT, CZProt and SZProt. In previous trials, a concentration of 250 ppm of Zn improved rumen fermentation efficiency without affecting intake and digestibility in beef cattle (Arelovich et al., 2000). However higher concentrations of dietary Zn reduced feed consumption by sheep (Puls, 1990) and DDMI tended to be linearly decreased in beef cattle (Arelovich et al., 2000). If high dietary levels of Zn would allow manipulation of rumen fermentation, then the wide gap existing between animal requirements of Zn and tolerance level could be considered advantageous. For sheep Zn requirements between 10 and 33 ppm were suggested for normal physiological functions, growth and wool production (Underwood and Suttle, 1999). Tolerance levels range between 750 and 1000 ppm (NRC, 1980; Underwood and Suttle 1999). In this study the Zn levels supplied by CZProt and SZProt (460 ppm in the supplement or approximately 70 ppm of total diet) were far below suggested maximum tolerance level for either sheep or ruminants in general. Both, Zn requirements as well as tolerance levels could be influenced by type of diet, Zn rumen bioavailability (source of Zn) and Zn interaction with other minerals. Martinez and Church (1970) found that additions of 20 ppm of Zn washed suspensions of ruminal microorganisms decreased cellulose digestibility by 31%. This effect on fiber digestion was also detected in vitro by Arelovich et al. (2000), but alleviated when Mn was added to the incubation tubes. However, in vivo, the addition of more than 200 ppm of Zn as ZnCl (total diet) to low quality hay fed to steers and supplemented with urea did not show a negative impact on DM digestibility (Arelovich et al., 2000). Little information can be found because of interaction among mineral supply, rumen microorganisms and enzyme production or activity in the rumen. An in vitro study from Eryavuz and Dehority (2009) reported that 50 ␮g Zn mL−1 of rumen fluid reduced cellulose digestion after 24 h but not at 48 h incubation, and did not affect cellulolytic and total bacterial counts. The authors also suggested an inhibitory effect on the cellulolytic enzymes produced by the bacteria. The rumen fluid concentration of Zn in our study was highest for SZProt as well with largest values across treatments between 10 and 16 h after morning meal (Table 3 and Fig. 1 respectively). However, Zn never reached concentrations that appear to limit fiber or DM digestibility. Conversely, rumen Zn availability from SZProt (ZnSO4 ) increased by 30% DM digestibility compared to CON, which in turn induced a higher greater DDMI. The sources of Zn and CP as well as the whole diet quality appeared to be involved in the magnitude of response to Zn if any. The significant increase in DMD with Zn SO4 (SZProt) observed in this study was not found in previous

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trials with ZnCl as a source of Zn, added to low quality hay supplemented with urea (Arelovich et al., 2000), or to a high quality mixed ration (Arelovich et al., 2008). Moreover, the numeric increase in DMD observed with CZProt (ZnCl) compared to CON or PROT treatments, was not statistically significant. Then the addition of Zn from ZnSO4 rather than other source could be of relevance when the basal diet is fibrous low quality hay supplemented with true protein. This is a usual production situation in feeding practice. Zn activates several enzymatic systems and plays a role in different metalloenzymes in animal metabolism (NRC, 1980). Similarly, in rumen environment increased Zn concentrations can activate fibrolytic enzymes but also impact on survival of specific rumen microorganisms. In a recent study Zn was found to enhance ␤-glucosidase activity of Bacillus licheniformis, a facultative bacterium from the rumen of a native Korean goat (Seo et al., 2013). Additionally, it was shown that 35 and 70 ppm of Zn in rumen fluid (supplemented as Zn SO4 ) benefit rumen fermentation, but also decreased protozoa counts in crossbred cattle (Mandal et al., 2007). 4.2. Zinc effects on rumen parameters The addition of Zn to the diet increased significantly ruminal concentration of Zn for SZProt. Bioavailability is different among sources of Zn as suggested by Baker and Ammerman (1995). Although the effects of Zn source on ruminal concentrations may not be proportional to bioavailability, it could be also related to alterations in fermentation kinetics. Rumen pH values below 5.0–5.5 would inhibit fibrolytic microorganisms (Hoover, 1986). The lowest values found 12 h after feeding were approximately 6.2, almost identical for all treatments. In the present study, the imposed diets did not decrease pH below critical values for effective activity of fibrolytic bacteria and fiber degradation. The rumen NH3 is the most important source of N for microbial protein synthesis. A reference range for rumen NH3 -N concentration is 5–8 mg dL−1 , suggested and widely cited as the optimum for maximizing microbial growth yield (Satter and Slyter, 1974). However, other studies showed that higher NH3 concentrations may have a positive effect on intake and digestibility as reviewed by Leng (1990) and Kertz (2010). In this study only marginal levels of NH3 -N were found for CON at 12 h; however, at 24 h the level increased again which could be attributed to a slower intake rate of unsupplemented NPH plus the physiological N recycling. A decreased urea hydrolysis was found when high Zn concentrations were supplied (Rodriguez et al., 1995; Arelovich et al., 2000). However, when a source of plant protein is used, independently of the degradation protein rate it will always be slower than urea. The addition of Zn further decreased the degradation rate of plant protein as shown by the treatment of soybean meal with Zn salts (Cecava et al., 1993), which may have affected activity of proteolytic microbes. These results can turn into improved performance; thus, Zn treatment of soybean meal enhanced efficiency of N utilization in growing calves (Britton and Klopfenstein, 1986). Additionally, when true

protein is fed it would be more effective to sustain an adequate rumen NH3 concentration along the day rather than reach high levels followed by a fast drop, with considerable amounts of NH3 -N lost by absorption through rumen wall. Reduce ammonia loss associated to high peaks would help to maintain microbial activity and extent of microbial protein synthesis in the rumen (Johnson, 1976; Mizwicki et al., 1980). This would provide a long lasting available N source for protein synthesis of rumen microbes, particularly with a slow release energy substrates such as NPH are fed. The impact of dietary addition of Zn on rumen levels of NH3 has shown different results in previous experiments. When urea was the source of N with low quality hay as basal diet (Rodriguez et al., 1995; Arelovich et al., 2000), Zn maintained NH3 -N concentration upon a critical level for a longer period of time. If it was due to a diminished rate of urea hydrolysis or decreased use of urea by the microorganisms it was not known. Thus previous results supported the concept that NH3 utilization can be modified by addition of Zn to ruminal fluid. However, when a high quality ration including a plant source of protein was supplied, the rumen NH3 -N concentrations were not substantially affected (Arelovich et al., 2008). It could be hypothesized that readily available carbohydrates trigger a faster use of NH3 , then; the potential effects of Zn on NH3 levels would not be noticed in these diets. In our experiment with NPH as well as in previous (Arelovich et al., 2000) all protein supplements had greater NH3 -N concentrations than CON at each time period studied. The addition of Zn showed a non-significant but remarkable trend to sustain higher NH3 levels at 4, 8 and 12 h after feeding. Moreover, at 4 h SZProt was higher than CZProt suggesting a potential effect of Zn source which could be associated to the greater DM digestibility found for this treatment. The addition of Zn in the present trial has not affected molar proportions of propionic acid. The non-significant trend (P = 0.0877) for reduced acetic acid levels with protein supplementation were compensated by higher molar proportions of butyric rather than propionic. Although available information is scarce, Zn supply under certain conditions seemed to improve energy utilization efficiency by favoring propionate production. Froetschel et al. (1990) noted that Zn supplementation of a 50% concentrate diet increased the molar proportion of propionic acid and decreased the acetate: propionate ratio of rumen fluid. On a diet based on low quality prairie hay the molar proportion of propionate was increased by added Zn, with 15.5, and 16.9% of VFA being propionate with 250 and 470 ppm supplemental dietary Zn in beef cattle (Arelovich et al., 2000). Several strains of rumen bacteria require branched chain volatile fatty acids for normal growth (Allison et al., 1962). In this study, isovaleric was increased by protein supplementation but levels were lowered with Zn addition, but still larger than CON. These necessary fermentation products can decrease with the addition of a source of low degradability-protein (Broderick, 2004). The slower rate of protein degradation observed when Zn was added could also resemble the effects of supplying a low degradable protein and can explain the differences found between PROT and the Zn-supplements. The larger total VFA production trend, where probably animal variability prevented to

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reach the threshold of significance, indicates that Zn sulfate could become a differential source from Zn chloride to impact on energy efficiency when low quality roughage is fed. Although the findings of this experiment do not show a clear effect of Zn, it proved to alter the populations of specific ruminal microbes and thereby change rates of production of different VFA. Karr et al. (1991) indicated that Zn at concentrations of 10 to 80 ppm inhibited growth of selected strains of ruminal bacteria. Alterations in VFA patterns also might explain why feedlot consultants empirically often formulate cattle feedlot diets with 300 ppm or more of Zn (Galyean, 1996). Sheep receiving a low-quality diet plus supplementary Zn and following similar feeding procedures exhibited different effects on their fermentation patterns (Arelovich et al., 2003). High intakes of Zn can alter absorption or retention of other minerals (Puls, 1990; NRC, 1980), so care in balancing other minerals, particularly Cu, should be exercised. Additionally Suttle (2010) reported that Zn, Cd and Cu can be involved in a triple interaction, since both Cd and Zn separately can affect Cu metabolism in sheep. The net effect of this interaction would depend on which element became limiting first. However, high levels of Zn in lambs could have protecting effect from Cu depletion originated in high levels of Cd in the diet. Also Zn not retained in the animal and excreted could be a potential hazard in areas that are already contaminated by heavy metals (Faixova and Faix, 2002). Any way in this study the levels of Zn supplied by treatments were much lower than previously studied when proposed as an additive to manipulate rumen fermentation. 5. Conclusions Protein supplementation, with or without Zn added has not increased basal diet intake as expected, which could be attributed to its high NDF content. However, protein supplementation plus Zn, from ZnSO4 rather than other source, increased substantially DM digestibility (30%) of the basal low quality roughage compared to CON treatment. The trend to maintain also highest NH3 levels in the first 12 h after feeding by Zn addition, particularly ZnSO4 (at 4 h), is coincident with previous findings. This would improve N availability to the rumen fermentation. Although effects of Zn on VFA were not observed in this study, a trend for larger total VFA production, indicates that ZnSO4 could become a differential source from ZnCl2 to impact on energy efficiency when low quality roughage is fed. The levels of Zn supplied in this study were lower than previously suggested when proposed as an additive to manipulate rumen fermentation. Although not evaluated high Zn intake can affect absorption or retention of other minerals, then caution in balancing other minerals particularly Cu should be exercised. Zinc from sulfate rather than chloride appear to exhibit the best characteristics as an additive for low quality forage diet supplemented with protein. Besides the source of Zn, the type of diet, feed processing, and feeding frequency of the supplement could differentially affect animal response to Zn supply. In the view of these results more research emphasizing these topics would be necessary to

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elucidate dietary and physiological conditions under which Zn would play a role in manipulating rumen fermentation. Conflict of interest None declared. Acknowledgments We wish to extend our sincere gratitude to our institution, Departamento de Agronomía de la Universidad Nacional del Sur for providing the experimental animals, facilities and funding. We also want to acknowledge the assistance of Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC) for additional funding and miscellaneous support. References Adesogan, A.T., 2009. Using dietary additives to manipulate rumen fermentation and improve nutrient utilization and animal performance. In: 20th Annual Florida Ruminant Nutrition Symposium, February 10, pp. 1–26, http://dairy.ifas.ufl.edu/rns/2009/Adesogan.pdf (accessed 19.11.13). Allison, M.J., Bryant, M.P., Katz, I., Keeney, M., 1962. Metabolic function of branched-chain volatile fatty acids, growth factors for ruminococci. II. Biosynthesis of higher branched-chain fatty acids and aldehydes. J. Bacteriol. 83, 1084–1093. AOAC, 2000. Official Methods of Analysis, 17th ed. Association of Official Analytical Chemists, Washington, DC, USA. Arelovich, H.M., Laborde, H.E., Villalba, J.J., Amela, M.I., Torrea, M.B., 1992. Effects of nitrogen and energy supplementation on the utilization of low quality weeping lovegrass by calves. Agric. Mediterr. 122, 123–129. Arelovich, H.M., Owens, F.N., Horn, G.W., Vizcarra, J.A., 2000. Effects of supplemental zinc and manganese on ruminal fermentation, forage intake and digestion by cattle fed prairie hay and urea. J. Anim. Sci. 78, 2972–2979. Arelovich, H.M., Amela, M.I., Martinez, M.F., Torrea, M.B., Laborde, H.E., 2003. Diferentes fuentes de zinc en el suplemento proteico de ovinos alimentados con un forraje de baja calidad. 2. Parámetros ruminales. Rev. Arg. Prod. Anim. 23 (Suppl. 1), 88 (Abstr.). Arelovich, H.M., Laborde, H.E., Amela, M.I., Torrea, M.B., Martínez, M.F., 2008. Effects of dietary addition of zinc and (or) monensin on performance, rumen fermentation and digesta kinetics in beef cattle. Span. J. Agric. Res. 6, 362–372. Baker, D.H., Ammerman, C.B., 1995. Zinc bioavailability. In: Ammerman, C.B., Baker, D.H., Lewis, A.J. (Eds.), Bioavailability of Nutrients for Animals: Amino Acids, Minerals, and Vitamins. Academic Press, New York, pp. 367–399. Bandyk, C.A., Cochran, R.C., Wicersham, T.A., Titgemeyer, E.C., Farmer, C.G., Higgins, J.J., 2001. Effect of ruminal vs postruminal administration of degradable protein on utilization of low-quality forage by beef steers. J. Anim. Sci. 79, 225–231. Britton, R.A., Klopfenstein, T.J., 1986. Zinc-treated soybean meal: A method to increase bypass., pp. 45–46, Nebraska 1986 Beef Cattle Rep. MP-50. Lincoln. Broderick, G.A., Kang, J.H., 1980. Automated simultaneous determination of ammonia and total amino acids in ruminal fluid and in vitro media. J. Dairy Sci. 63, 64–75. Broderick, G.A., 2004. Effect of low level monensin supplementation on the production of dairy cows fed alfalfa silage. J. Dairy Sci. 87, 359–368. Cecava, M.J., Hancock, D.L., Parker, J.E., 1993. Effects of zinc-treated soybean meal on ruminal fermentation and intestinal amino acid flows in steers fed corn silage-based diets. J. Anim. Sci. 71, 3423–3431. Eryavuz, A., Dehority, B.A., 2009. Effects of supplemental zinc concentration on cellulose digestion and cellulolytic and total bacterial numbers in vitro. Anim. Feed Sci. Technol. 151, 175–183. European Union, 2003. Regulation (EC) No. 1831-2003 of the European Parliament and of the Council of 22 September 2003 on additives for use in animal nutrition. Off. J. Eur. Union L268, 229–243.

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