D. Price 1, M. Hersom 1, J. Yelich 1, M. Irsik 2, O. Rae 2

Transcription

1 Effects of Prenatal and Postnatal Trace Mineral Supplement Source Provided To Gestating Angus and Brangus Cows over Two Production Cycles on Performance and Trace Mineral Status of Cows D. Price 1, M. Hersom 1, J. Yelich 1, M. Irsik 2, O. Rae 2 Synopsis The results of this experiment suggest that assessment of an animal s trace mineral status should preferentially be carried out in liver tissue. The source of trace mineral supplementation provided to gestating and lactating cows had minimal effects on cow performance. Summary A 2 2 factorial arrangement of trace mineral (TM) source and breed utilized Angus (AN) and Brangus (BN) cows supplemented with inorganic (ING, salt sulfate) or organic (ORG, proteinates and Se-yeast) forms of Co, Cu, Mn, Se, and Zn over two production cycles to evaluate liver and serum TM status, and performance of cows. Supplementation initiated 82 ± 2 d prior to calving in yr 1 and was fed as a pellet until breeding with the total TM amount divided equally and fed 3 times/wk at 1.0 lb 1000 lb BW -1 cow - 1 d -1, after which loose mineral was fed through weaning at 4 oz 1000 lb BW -1 cow -1 d -1. Cows remained on same TM sources for yr 2 and received TM as a pellet from post-weaning to breeding and as loose mineral from breeding to weaning. Cows (n = 25 in yr 1 and 24 in yr 2) had serum and liver collected for TM analysis prior to TM initiation (yr 1 only), at pre-calving, breeding, and weaning. Cow BW and BCS were not affected (P>0.05) by TM source, but varied over time (P<0.05). Liver Se was greater (P<0.05) in ORG compared to ING cows in yr 2. Breed differences were observed for cow liver Cu (yr 1) and Mn (both yrs), with greater (P<0.05) concentrations in BN compared to AN, suggesting differences in Cu and Mn absorption and/or utilization may be present. The TM source provided to gestating and lactating cows had minimal effects on cow performance and inconsistent effects on cow reproduction and calf performance. Introduction Trace minerals (TM) are essential components in biochemical, physiological, immunological, and metabolic processes in an animal s body (Suttle, 2010). Sufficient concentrations of circulating and stored TM are necessary for proper growth, reproductive and immune functions in animals, while deficiencies result in decreased growth, innate and adaptive immunity (Spears, 2000), fertility and reproductive functions in males and females (Hidiroglou, 1979). Moreover, as the gestating dam supplies nutrients to the fetus in utero, TM deficiencies of the dam may impact the health and production potential of its future offspring (Ashworth and Antipatis, 2001; Hostetler et al., 2003). An animal s TM status may be influenced by the animal s age, breed or genetics, health status, or amount of TM or TM antagonists consumed, and even the season of the year (Miranda et al., 2006; Suttle, 2010). Additionally, the form of the TM, either as inorganic or organic may impact its bioavailability and ultimately its utilization by the animal (Spears, 2003). Supplementation of the gestating and lactating dam and the effects on offspring TM status warrant investigation. Therefore, the hypothesis of this experiment was that supplementation of pregnant cows with ORG compared to ING TM during the pre-and postnatal periods would improve cow performance, reproduction and maintenance of TM status, and improve neonatal and growing calf TM status and performance through weaning. Materials and Methods Over two production cycles (yr 1 and yr 2), a 2 2 factorial arrangement of TM source (inorganic vs. organic) and breed (Angus vs. Brangus) utilized pregnant cows to investigate the effect of prenatal and 1 Department of Animal Sciences, University of Florida, Gainesville, FL 2 College of Veterinary Medicine, University of Florida, Gainesville, FL

2 lactating cow TM supplement source on cow reproductive performance, BW, BCS, and TM status, and calf performance and TM status from birth to weaning. In yr 1 of the experiment, a total of 199 cows were blocked by breed (Angus, AN = 99; Brangus, BN = 100), gestation length, age, BW, and BCS (scale 1 = emaciated, 9 = extremely fat; Wagner et al., 1988) and allocated randomly to receive either 1) inorganic minerals (ING, n =100) with Na selenite and Co, Cu, Mn, and Zn in salt sulfate forms, or 2) organic minerals (ORG, n = 99) with Se-yeast (Sel-plex, Alltech, Nicholasville, KY) and Co, Cu, Mn, and Zn complexed as proteinates (Bioplex, Alltech, Nicholasville, KY). The experimental design resulted in the following four treatment groups: ING-AN (n = 49), ING-BN (n = 51), ORG-AN (n = 50), and ORG-BN (n = 49). Both TM supplements were formulated to meet NRC requirements and were manufactured in either a single pellet or loose mineral batch by the Lakeland Nutrition Group (Lakeland, FL). The TM supplementation was initiated 82 ± 2 d prior to parturition in yr 1 and supplements were fed as a wheat middling based pellet, which was delivered at a rate of 1.0 lb 1000 lb BW -1 cow -1 d -1. The supplement was divided equally and fed 3 times per week (Monday, Wednesday, and Friday) in feed bunks until start of the breeding season. Thereafter, the TM source was offered as a loose mineral supplement on a free choice basis at a rate of 4 oz cow -1 d -1 until weaning. Cows were maintained in 12 pastures (3 per treatment) of approximately ac from the initiation of the trial until the start of the breeding season. Cows had ad libitum access to water and hay throughout the experiment and were fed stored forage (bermudagrass hay, Cynodon dactylon) and supplemented with soybean hulls as needed to maintain a mean cow BCS of 5.0 from initiation of TM supplementation until the start of the breeding season when cows were maintained in four breeding groups (one per treatment) and grazed on bermuda and bahiagrass pastures until calves were weaned. In yr 2 of the experiment, cows (n = 161; ING-AN = 41, ING-BN = 44, ORG-AN = 38, and ORG-BN = 38) remained on the same TM supplement source they were assigned in yr 1 of the experiment and were fed the TM supplement as a wheat middling pellet from post-weaning through breeding and as a loose mineral from breeding to weaning. The TM supplements were fed at an equivalent rate and frequency as in yr 1 of the experiment. Cows were fed stored forage (i.e., bermudagrass hay) and supplemented with soybean hulls as needed to maintain a mean cow BCS of 5.0 from initiation of TM supplementation until the start of the breeding season each year. Cows and calves had ad libitum access to water and hay throughout the experiment and were maintained in 8 bahiagrass pastures (2 pastures per treatment group) from post-weaning in yr 1 until breeding (yr 2) when they were combined into 4 bermuda and bahiagrass pastures (1 pasture per treatment group) until calves were weaned. Daily intake was not measured in yr 1 or yr 2, though each pen consumed all feed that was offered over both years of the trial. Analysis of TM supplements, feed, and forage, and pasture offered to the cows was carried out at a commercial laboratory (Dairy One, Ithaca, NY) and averages from both years are presented in Table 1. To determine cow TM status over yr 1 of the production cycle a subset of multiparous Angus and Brangus cows were selected out of each treatment based on calving date, BW and BCS and had liver biopsy samples and blood collected at 4 time points over the production cycle: prior to mineral supplementation (pre-min, d -20), pre-calving (d 57), breeding (d 155), and at weaning (d 287). All cows on the trial had BW and BCS recorded at pre-min, pre-calving, breeding, and at weaning for determination of cow performance. Along with the cows used for production cycle liver and serum TM status, an additional 17 cows (total of 42) were selected out of the 4 treatments based on expected calving date, BW, and BCS at the initiation of TM delivery, and utilized at calving and at 30 d post-calving to determine calf serum TM concentrations. This resulted in the following subset of calves ING-AN = 10, ING-BN = 11, ORG-AN = 11, and ORG-BN = 11. One ORG-BN cow had twins. In yr 2, a total of 24 cows (6 per treatment) were selected out of each treatment group based on expected calving date, age, BW, and BCS for liver biopsy collection at 3 time points (pre-calving, breeding, and weaning) of the production cycle. There was no pre-min sample in yr 2, since all cows had been on their respective TM treatments from the previous yr of the experiment.

3 Blood samples for TM analysis were collected for TM analysis. All TM analysis (Co, Cu, Fe, Mn, Mo, Se, and Zn) for serum and liver (dry matter basis) was carried out by a commercial laboratory (DCPAH, Michigan State University, Lansing, Michigan). Statistical Analysis Data were analyzed in SAS 9.4 (SAS Institute Inc., Cary, NC) with cow or calf as the experimental unit where appropriate. The random statement included cow (or calf) nested within TM source breed. The cow BW and BCS data were analyzed separately for yr 1 and yr 2 by repeated measures PROC MIXED with fixed effects of TM source, breed, time, and their interactions. Cow age was included as a covariate in the yr 1 BW and BCS analysis and considered significant when P Cow serum and liver TM data were analyzed by repeated measures PROC MIXED with fixed effects of TM source, breed, time, and their interactions. Natural logarithm transformations where used on TM concentrations when necessary. The P-values are from the natural logarithm data are presented along with the back calculated LSMEANS. Natural logarithm transformations where used on TM concentrations when necessary. The P- values are from the natural logarithm data are presented along with the back calculated LSMEANS. Pearson correlations between serum and liver TM concentrations used PROC CORR. Data are presented as LSM ± S.E. Results Cow Production Cycle Performance Cow BW and BCS over yr 1 and yr 2 of the production cycle are presented in Table 2. In yr 1, there was no affect (P>0.05) of TM source, breed, TM source breed, nor TM source time on cow BW over the production cycle. However, as expected cow BW varied (P<0.001) across the production cycle, as cows weighed more at pre-mineral and pre-calving than at breeding or at weaning. A breed time, and TM source breed time effect occurred (P = 0.01) for cow BW (yr 1); however, cow BW did not differ at any individual time points, rather the breeds and treatments changed relative rank. Year 2 cow BW varied (P<0.001) over time but was not (P>0.05) affected by TM source, breed or any interactions. Cow BCS was affected (P<0.001) by time during both years of the experiment, where cow BCS was greatest pre-calving. There tended (P = 0.08) to be a TM source time (yr 1) effect; however, cow BCS for all treatments remained within 0.3 BCS at all time points. Year 1 cow BCS was not affected (P>0.05) by TM source, breed or any other interactions. Year 2 BCS was affected (P<0.001) by breed time and tended (P<0.08) to be affected by TM source breed time as BN cows had greater pre-calving and breeding BCS compared to AN cows. Cow BCS did not differ between treatments at breeding or weaning. Similarly, yr 2 BCS varied (P<0.001) over time, but was greater (P = 0.02) in BN compared to AN when pooled across all time points. Cow Trace Mineral Status Year 1 Year 1 cow serum and liver TM concentrations over the production cycle pooled across TM sources and breeds are presented in Table 3 along with the pearson correlations between serum and liver values. With the exception of serum Co (P = 0.13), all serum and liver TM values varied (P 0.01) over time. Liver TM concentrations with the exception of Fe were decreased at breeding and at weaning, relative to their pre-calving values. Positive relationships between serum and liver values were observed (P<0.01) for Co, Mo, and Se when correlations were examined across all liver values and time points, Table 3. When serum and liver correlations were examined at individual sample times, Mo was only correlated (P<0.01) at weaning (r = 0.57), while Co was correlated (P 0.05) at all time points, and Se was correlated at all time points except (P = 0.76) at breeding. Serum Mn concentrations were greater (P = 0.05) in ING (2.18 ± 0.08 ng/ml) compared to ORG (1.96 ± 0.07 ng/ml) cows when pooled across time. Serum Co, Cu, Fe, Mo, Se and Zn were not affected (P>0.10) by TM source. The BN (2.18 ± 0.08 ng/ml) cows tended (P = 0.06) to have greater Mn

4 compared to AN cows (1.96 ± 0.07 ng/ml). Conversely, AN cows had greater (P = 0.02) serum Se (67.62 ± 2.43 ng/ml) and tended (P = 0.08) to have greater serum Zn (0.79 ± 0.02 µg/ml) than BN cows (59.04 ± 2.50 ng/ml, 0.73 ± 0.02 µg/ml for Se and Zn, respectively) when concentrations were pooled across time. Cow breed did not (P>0.10) affect serum Co, Cu, Fe and Mo values. Molybdenum was affected (P = 0.03) by TM source breed, as ING-BN (1.12 ± 0.19 ng/ml) had lesser serum Mo values than ORG- BN (1.83 ± 0.30 ng/ml), but ING-AN (1.72 ± 0.28 ng/ml) and ORG-AN (1.34 ± 0.20 ng/ml) cows did not differ from each other. There was no TM source breed effect (P>0.10) on serum Co, Cu, Fe, Mn, Se, and Zn concentrations. Greater (P<0.01) overall liver Cu concentrations were observed in ING (255 ± 14.4 µg/g) compared to ORG (190 ± 10.1 µg/g) cows and in BN (266 ± 14.5 µg/g) compared to AN (182 ± 10.0 µg/g) cows when concentrations were pooled across time. Concentrations of liver Mn were greater (P<0.01) in BN (11.25 ± 0.29 µg/g) compared to AN (9.69 ± 0.29 µg/g) cows. Cow TM source did not affect (P>0.05) liver Co, Fe, Mn, Mo, Se, and Zn, nor did breed affect (P>0.05) liver Co, Fe, Mo, Se, and Zn concentrations. Liver Co concentrations exhibited a TM source time effect (P<0.01, Figure 4-1), as ING cows had greater liver Co at weaning compared to ORG cows. When concentrations were pooled across time, liver Se concentrations tended (P = 0.06) to be affected by TM source breed, as Se concentrations differed between ING-AN (1.17 ± 0.08 µg/g) and ING-BN (1.48 ± 0.10 µg/g), but not ORG-AN (1.32 ± 0.08 µg/g), and ORG-BN (1.29 ± 0.08 µg/g). An effect of TM source time (P<0.01) occurred for Se liver concentrations (Figure 4-1) with greater Se concentrations in ORG cows pre-calving and greater concentrations in ING cows at breeding. There was no effect (P>0.05) of TM source breed on liver Co, Cu, Fe, Mn, Mo, and Zn, nor was there an effect (P>0.05) of TM source time for liver Cu, Fe, Mn, Mo, and Zn concentrations. Similar to serum Mo, concentrations of liver Mo were affected (P = 0.02) by a breed time (data not shown) and tended (P = 0.08) to be affected by TM source breed time (Figure 4-2), with the greatest liver Mo concentrations observed in ORG-BN cows at weaning. There was no effect (P>0.05) of breed time or TM source breed time for liver Co, Cu, Fe, Mn, Se, and Zn concentrations. Cow Trace Mineral Status Year 2 In yr 2, there was no effect (P>0.05) of TM source on cow liver Cu, Fe, Mo, and Zn concentrations. Breed did not affect (P>0.05) liver Co, Cu, Fe, Se, and Zn concentrations; although, BN cows had numerically greater Cu at all time points compared to AN cows. When pooled across time, mean liver Co (0.35 ± 0.02 µg/g) and Se (1.21 ± 0.05 µg/g) were greater (P 0.01) and Mn (11.26 ± 0.27 µg/g) tended (P 0.10) to be greater in ORG compared to ING (0.29 ± 0.02, 0.94 ± 0.04, ± 0.26 µg/g, respectively) cows. Additionally, mean Mn was greater (P<0.05) in BN (11.70 ± 0.26 µg/g) compared to AN (10.17 ± 0.27 µg/g) cows. With the exception of Fe (P>0.05), all liver TM concentrations were affected (P 0.05) by time. There was no TM source breed effect (P>0.05) for liver Co, Cu, Fe, Mn, Mo, and Zn concentrations. However, liver Se tended (P<0.10) to be affected by TM source breed, where ORG-AN (1.29 ± 0.07 µg/g) cows had greater (P<0.05) mean liver Se concentrations compared to ING-AN (0.91 ± 0.05 µg/g) and ING-BN (0.98 ± 0.05 µg/g) but did not differ (P>0.05) from ORG-BN (1.13 ± 0.06 µg/g) cows. There was no TM source time effect (P>0.05) on any liver TM concentrations. There was no breed time effect (P>0.05) on liver Co, Cu, Fe, Mn, and Se concentrations. There was a breed time, effect (P<0.05; data not shown) on liver Mo and Zn, where BN cows had greater (P<0.05) Mo at pre-calving (2.55 ± 0.12 µg/g) and weaning (3.09 ± 0.12 µg/g) compared to AN (2.22 and 2.69 ± 0.12 µg/g, respectively). Conversely, AN (142 ± 8.4 µg/g) cows had greater (P<0.05) Zn at weaning compared to BN (102 ± 6.1 µg/g) cows. There was no TM source breed time effect (P>0.05) on liver Co and Fe, Table 4. In contrast, liver Cu tended (P<0.10) to be affected by TM source breed time (Table 4), whereby at breeding, ORG-BN had lesser (P<0.05) Cu compared to ING-BN, but neither treatment differed (P>0.05) from ING-AN and ORG-AN. Additionally, liver Mn, Mo, Se, and Zn were affected (P<0.05) by TM source breed time, Table 4.

5 Overall, the results of this experiment suggest that assessment of an animal s TM status should preferentially be carried out in liver tissue. Consistent correlations between serum and liver Co and Se concentrations indicate that determination of Co and Se status can be made based on serum samples if liver tissue is not available. The source of TM supplementation provided to gestating and lactating cows had minimal effects on cow performance. Moreover, breed differences were apparent in cow Cu and Mn concentrations and the mechanisms behind these differences warrant further examination. In conclusion, the TM source provided to gestating and lactating cows had minimal effects on cow performance. Literature Cited Ashworth, C., and C. Antipatis Reproduction. 122: Hidiroglou, M J. Dairy Sci. 62: Hostetler, C. E. et al Vet. J. 166: Miranda, M., et al Anim. Sci. 82: Spears, J. W J. Nutr. 133:1506S 1509S. Suttle, N., ed Mineral nutrition of livestock. 4th ed.

6 Table 1. Trace mineral supplement, feed, and forage component analysis (DM basis) Pelleted mineral supplement 1,2 Free choice supplement 2,4 Composited samples 1 Item Inorganic Organic Inorganic Organic Soybean hulls Pasture 3 Hay DM, % CP 4, % TDN 4, % Ca, % P, % Mg, % K, % Na, % < < 0.01 S, % Co, mg/kg Cu, mg/kg I 4, mg/kg ,000 3, Fe, mg/kg Mn, mg/kg , Mo, mg/kg Se, mg/kg Zn, mg/kg ,230 1, Vitamin A 4, IU/kg 14,337 14,332 53,479 53, Vitamin D 34, IU/kg 1,286 1, Vitamin E 4, IU/kg Analysis of feedstuffs was carried out at Dairy One, (Ithaca, NY). Analysis of trace mineral supplements pellets is averaged across both years of the experiment. 2 Trace mineral supplements were formulated and manufactured as a single batch by Lakeland Nutrition Group (Lakeland, FL) and provided to cows in a pellet at 1.0 lb 100 lb BW -1 cow -1 d -1 and free choice mineral was provided at 4 oz cow -1 d Samples were collected monthly and composited for final analysis. 4 Formulation values for TM supplements.

7 Table 2. Effect of an inorganic (ING) or organic (ORG) trace mineral (TM) supplement source provided to Angus (AN) and Brangus (BN) cows over two production cycles on cow BW and BCS TM Source Breed (B) P-value Item ING-AN ING-BN ORG-AN ORG-BN SEM TM B TM B T 1 TM T B T TM B T Year 1 Cows 2, n BW 3, lb pre-mineral 1142 y 1166 y 1147 y 1158 y < pre-calving 1246 z 1250 z 1255 z 1250 z 17.6 breeding 1114 x 1125 x 1109 w 1133 x 17.6 weaning 1098 w 1129 x 1125 x 1122 x 17.6 BCS 4 pre-mineral < pre-calving breeding weaning Year 2 Cows, n BW, lb pre-calving < breeding weaning BCS pre-calving < < breeding weaning T = Time 2 Cow age (P<0.01) used as a covariate for BW and BCS statistics in year 1. 3 Cow BW did not differ among treatments within any time point, (P>0.05). 4 Cow BCS measured on a scale of 1 = emaciated to 9 = extremely. w-z Means within a treatment differed across time, (P<0.05).

8 Table 3. Cow serum and liver (µg/g) trace mineral (TM) concentrations pooled across TM sources and breeds in year 1 of the production cycle (on a DM basis )1 Premin (d -20) Time (d) relative to TM initiation Pre-Calving Breeding (d 57) (d 155) Weaning (d 287) SEM P-Value Pearson Correlation 2 Item Co Serum, ng/ml 1.29 ab 1.05 a 1.11 ab 1.31 b Liver 0.34 b 0.36 b 0.36 b 0.29 a 0.02 < 0.01 < 0.01 Cu Serum, µg/ml 0.71 c 0.54 a 0.66 b 0.63 b 0.02 < Liver 229 a 242 a 182 b 232 a 12.5 < Fe Serum, µg/dl. 131 a 140 ab 154 b 5.9 < Liver 458 a 544 b 468 a 619 b 23.7 < Mn Serum, ng/ml 2.20 b 1.87 a 2.35 b 1.89 a 0.09 < Liver 9.61 a b b a 0.33 < Mo Serum, ng/ml 1.88 c 1.18 b 0.90 a 2.36 c 0.17 < Liver 2.97 bc 3.08 c 2.64 a 2.85 ab 0.09 < 0.01 < 0.01 Se Serum, ng/ml 60.2 b 70.5 c 71.3 c 51.4 a 2.03 < Liver 1.20 b 1.68 d 1.47 c 0.98 a 0.06 < 0.01 < 0.01 Zn Serum, µg/ml 0.77 b 0.71 a 0.75 ab 0.79 b Liver 118 a 148 c 113 a 134 b 3.5 < Time affected (P 0.01) all serum and liver TM concentrations except serum Co (P = 0.13). 2 Pearson correlations are on first line and P-values are on second line. a-d Means within a row with difference superscripts differed (P 0.05).

9 Serum Co, ng/ml A Serum Se, ng/ml B ING Serum ORG Serum ING Liver ORG Liver b y b x a a Pre-Min Pre-Calve Breed Time Wean y y x x ING Serum ORG Serum ING Liver ORG Liver Pre-Min Pre-Calve Breed Wean Time Liver Co, µg/g Liver Se, µg/g Figure 1. Serum and liver A) Co and B) Se in Angus and Brangus cows that were supplemented with inorganic (ING) or organic (ORG) trace minerals over the production cycle. a-b Serum means with different superscripts differed within that time point, (P 0.05). x-y Liver means with different superscripts differed within that time point, (P 0.05).

10 Serum Mo, ng/ml A ING-AN ING-BN ORG-AN ORG-BN b c a b b a, b a a a a a Pre-Min Pre-Calve Breed Wean Time ING-AN ORG-AN ING-BN ORG-BN b Liver Mo, µg/g a a a 2.0 Pre-Min Pre-Calve Rebreed Wean B Time Figure 2. Concentrations of Mo in A) serum and B) liver of Angus (AN) and Brangus (BN) cows that were supplemented with inorganic (ING) and organic (ORG) trace mineral sources over the production cycle. a-c Means with different superscripts differed within that time point, (P 0.05).

11 Table 4. Liver trace mineral (TM) concentrations (on a DM basis) in Angus (AN) and Brangus (BN) cows which received inorganic (ING) or organic (ORG) sources of trace minerals (TM) in year 2 of the production cycle Time (T) P-value Item 1 Pre-calving Breeding Weaning SEM TM B 2 T TM T B T TM B T Co, µg/g ING-AN < ING-BN ORG-AN ORG-BN Cu, µg/g ING-AN ING-BN ORG-AN ORG-BN Fe, µg/g ING-AN ING-BN ORG-AN ORG-BN Mn, µg/g ING-AN 7.95 ax az 9.84 aby < < ING-BN bxy aby ax ORG-AN bcy ay 9.22 bx ORG-BN 9.83 cx bz cy Mo, µg/g ING-AN 1.94 ax 2.79 y 2.76 aby < ING-BN 2.70 b ab ORG-AN 2.50 bx 3.19 y 2.62 ax ORG-BN 2.40 abx 2.74 x 3.15 by

12 Table 4. Continued Time (T) P-value Item 1 Pre-calving Breeding Weaning SEM TM B 2 T TM T B T TM B T Se 3, µg/g ING-AN 0.93 ax 1.00 ay 0.81 ax 0.08 < < ING-BN 1.10 ay 1.14 acy 0.74 ax ORG-AN 1.35 by 1.58 by 1.02 bx ORG-BN 1.16 by 1.24 cy 1.02 bx Zn 3, µg/g ING-AN 102 x 102 ax 167 ay ING-BN ab 95 b ORG-AN ab 121 c ORG-BN 105 x 130 by 110 bcxy 1 TM source breed, (P>0.05) for all TM concentrations. 2 B= breed. 3 Data were log-transformed before statistical analysis; back transformed means are presented. a-c Means within a column with different superscripts differed within, (P 0.05). x-z Means within a row with different superscripts differed within, (P 0.05).