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,1
* FARME Institute, Homer, NY 13077
Zinpro Corporation, Eden Prairie, MN 55344
1 Corresponding author: msocha{at}zinpro.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Key Words: trace mineral • dairy cattle • claw lesion
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Amino acid complexes of trace minerals are more bioavailable (Wedekind et al., 1992; Paripatananont and Lovell, 1995) and are better retained by the body (Nockels et al., 1993) than inorganic sources of trace minerals. Clinical responses to improved bioavailability and retention of Zn, Mn, and Cu AA complexes have been demonstrated in numerous studies. For instance, in 12 studies, feeding cattle specific AA complexes of Zn reduced SCC (Kellogg et al., 2004), increased milk production (Kellogg et al., 2004), and improved claw integrity (Moore et al., 1989). Further improvements in milk production or reproduction, or both, have been demonstrated when Co glucoheptonate and AA complexes of Zn, Mn, and Cu (OTM) were added to diets of dairy cattle (Campbell et al., 1999; Kellogg et al., 2003; Griffiths et al., 2007) and when OTM replaced inorganic forms of these trace minerals (Uchida et al., 2001; Ballantine et al., 2002; Kellogg et al., 2003; Ferguson et al., 2004a; Kincaid and Socha, 2004; Kinal et al., 2005; Nocek et al., 2006).
The effect of OTM on claw integrity has been examined to a lesser extent. Although research has shown that adding OTM to diets of dairy cattle improves claw integrity (Nocek et al., 2000; Drendel et al., 2005), improvements in claw integrity when OTM replaced sulfate trace minerals have not been as consistent, with one study showing improvement (Ballantine et al., 2002) and another study showing no improvements (Nocek et al., 2006). Thus, the first objective of this study was to further investigate the effect of replacing sulfate trace minerals with OTM on claw integrity of dairy cattle. The second objective of this study was to determine the effect of replacing sulfate trace minerals with OTM on lactation performance and fertility of dairy cattle.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Primiparous and multiparous cows were commingled in the prefresh and postfresh pens. At 30 to 40 DIM, primiparous cows were separated from multiparous cows and housed in separate pens. All treatment groups were housed in common pens, thus eliminating any potential pen effect. Pens contained free stalls that were covered with a rubber-filled mattress. Approximately 2 cm of sawdust was applied to the top of the mattresses.
Treatments were blended into a pelleted concentrate at a commercial feed mill (Round House Mill, Cortland, NY; Table 1
). The pelleted concentrate was formulated such that 1.5 kg of concentrate provided 360 mg of Zn, 200 mg of Mn, 125 mg of Cu, and 12 mg of Co from either Sulfate or CTM. The Availa-4 added to the CTM pelleted concentrate contained a tracer (Microtracer, MicroTracers Inc., San Francisco, CA), whereas the sulfate source added to the Sulfate pelleted concentrate did not contain a tracer. Samples of each concentrate batch were collected and analyzed for tracer by Micro-Tracers Inc. to ensure that the correct trace mineral source was added to the correct pelleted concentrate.
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Other than the pelleted concentrate delivered to cows via computerized feeders, all other dietary components were identical between treatments with cows receiving a common TMR. The basal diet was formulated to meet the nutrient requirements of cows (i.e., prefresh, post-fresh, primiparous, multiparous), based upon DM intakes, weekly forage analyses, and consumption of the pelleted concentrate (Tables 2 and 3).
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Cows were milked thrice daily, and milk yield was recorded at each milking. Milk was sampled twice monthly, and fat, protein, SCC, and MUN were determined (DairyOne, Ithaca, NY) according to approved procedures of AOAC (2000). Recombinant bovine somatotropin (Posilac, Protiva Inc., St. Louis, MO) was administered to cows beginning at d 63 postpartum with reinjection occurring every 14 d thereafter.
Reproduction
At 24 to 30 d postpartum, all cows were given a 5-cc intramuscular injection of PGF2
(Lutalyse, Pfizer, New York, NY). Fourteen days later, all cows were given a second PGF2 injection. Cow activity was measured electronically and used for estrus detection (ALPRO activity meter, DeLaval). Cows were deemed in estrus if activity increased at least 50% in a 1-h time period. Cows deemed in estrus after 45 DIM were artificially inseminated following the a.m.–p.m. rule.
Any cow that did not elicit estrus by 74 d postpartum entered a timed breeding program. A 2-cc intramuscular injection of GnRH (Cystorelin, Merial, Duluth, GA) was given at 74 d postpartum. Seven days later, cows received a 5-cc intramuscular injection of PGF2. Forty-eight hours later, cows received a second 2-cc intramuscular injection of GnRH. Cows were artificially inseminated 12 h after the last GnRH injection.
Activity of all cows was monitored. Any cows deemed in estrus before being examined for pregnancy were artificially inseminated following the a.m.–p.m. insemination rule. Cows were checked for pregnancy 34 to 40 d postbreeding via rectal palpation. Cows that were not pregnant and were not observed in estrus reentered the timed breeding program outlined above.
Liver Biopsies
Twenty cows per treatment were selected for collection of liver biopsies before initiation of treatments and again at approximately 14 wk after calving. Liver biopsies were taken between the 10th and 11th right intercostals space using previously defined procedures (Arthington et al., 1995). Samples were placed in Whirl-Pak bags (Nasco, Fort Atkinson, WI) and stored at –20°C until all biopsies were collected. Samples were shipped, frozen, to the Michigan State Diagnostic Laboratory (Lansing) for determination of mineral concentration by flame atomic absorption spectroscopy.
Claw Evaluations
Claws of all cows were examined at dry-off, before treatment administration, and at 16 and 36 wk postpartum by personnel trained in identifying claw lesions. Claws were examined by placing cows in a trimming chute equipped with a hydraulic tilt table. All claws were directly examined for lesions after being functionally trimmed by trained personnel. Lesions were noted in all 8 claws were and scored for severity according to the following protocol: 1) dorsal wall ridge, 0 = none, 1 = ridges and grooves, and 2 = dorsal wall curls up; 2) heel erosion, 0 = smooth heels, 1 = corrugated or skin flaps, 2 = deep cracks >6 mm, and 3 = exposed corium in crack; 3) dorsal wall fissure, 0 = none, 1 = transverse cracks, thimbling, and 2 = vertical cracks; 4) double sole, 0 = none, 1 = separation of <1/3 sole, and 2 = separation of >1/3 sole; 5) white line separation, 0 = no defects, minor manure staining, 1 = red discoloration after trimming, 2 = white line abnormally wide, pancake foot, 3 = crevice still packed with manure after trimming, and 4 = wall separated and broken away from white line; 6) white line abscess, 0 = none, 1 = small abscess, <1/2 cc of pus, 2 = larger abscess undermining wall and some sole, and 3 = abscess breaking out at heel or coronary band; 7) sole hemorrhage, 0 = none, 1 = faint speckles or pink tinge or yellow tinge, 2 = red area larger than 21 mm, and 3 = more than 1/2 of sole colored; 8) sole ulcer, 0 = none, 1 = soft discolored sole at ulcer site, 2 = exposed corium at ulcer site, and 3 = complicated sole ulcer; 9) digital dermatitis, 0 = none and 1 = present; 10) interdigital dermatitis, 0 = none, smooth interdigital skin, 1 = roughened or cratered interdigital skin, and 2 = interdigital fibroma, quittor, corn, and 11) foot rot, 0 = none and 1 = present. For cows exhibiting a noted claw disorder, a claw lesion index was calculated by taking the number of claws affected and multiplying it by the average severity score of the lesions.
Statistical Analysis
Removal of cows from the study occurred only if cows did not adapt to the computer feeders or if health problems (contagious or recurrent mastitis) or injury necessitated pen changes. Data from cows removed from the study before the claw evaluation at 16 wk postpartum (22 cows fed the CTM diet and 14 cows fed the Sulfate diet) were not included in the data analysis. The UNIVARIATE procedure of SAS 9.1 (SAS Institute, Cary, NC) was used to determine outlier cows. An observation that was greater than 2.5 standard deviations from the mean for 3.5% FCM and ECM was considered an outlier. Two cows receiving the Sulfate diet were considered outliers, and their data were excluded from the analyses. Data for milk yield and composition were analyzed using the MIXED procedure of SAS 9.1 according to the following model
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where Yij = dependent variable; µ = overall mean; Ci = fixed effect of the ith treatment, i = 1, 2; cij = random effect of the jth cow within the ith treatment, j = 1,...250; Wk = fixed effect of week of lactation, k = 1,... ,35; CWik = fixed effect of the interaction between the ith treatment and the kth week; Pl = t fixed effect of parity, l = 1, 2; and Eijkl = random residual 
N (0, 
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Data for claw lesions and lesion severity were analyzed using the MIXED procedure of SAS 8.2 (SAS Institute) according to the following model
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where Yij = dependent variable; µ = overall mean; β = the regression (covariate) coefficient; Xij = covariate measurement for the ith cow on the jth treatment; Cj = fixed effect of the jth treatment, j = 1, 2; cij = random effect of the ith cow within the jth treatment, j = 1,...250; Wk = fixed effect of week of lactation, k = 16, 35; CWjk = fixed effect of the interaction between the jth treatment and the kth week; Pl = fixed effect of parity, l = 1, 2; and Eijkl = random residual N (0, ).
In the models used to analyze the lactation and claw lesion data, the random effect of cows within treatment subclasses was used as the error term for the effect of trace mineral source. The residual errors, which are errors within cows across time and represent errors from repeated measurements in the experimental units (cows), were modeled using a first-order autoregressive covariance structure. Degrees of freedom were calculated using the Kenward-Roger option of the MIXED procedure of SAS. Least squares means were determined for the main effects of trace mineral source and trace mineral source x week interactions.
Data for reproduction were analyzed using the MIXED procedure of SAS according to the following model
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where Yij = dependent variable; µ = overall mean; Ci = fixed effect of the ith treatment, i = 1, 2; cij = random effect of the jth cow within the ith treatment, j = 1,...250; Pk = fixed effect of parity, k = 1, 2; and Eijk = random residual N (0, ).
Data for liver trace mineral concentrations were analyzed using the MIXED procedure of SAS according to the following model
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where Yij = dependent variable; µ = overall mean; β = regression (covariate) coefficient; Xi = covariate measurement; Cj = fixed effect of the jth treatment, j = 1, 2; cij = random effect of the ith cow within the jth treatment, j = 1,... 250; Pk = fixed effect of parity, k = 1, 2; and Eijk = random residual N (0, ).
In the models used to analyze the reproduction and liver data, the random effect of cows within treatment subclasses was used as the error term for the effect of trace mineral source. Degrees of freedom were calculated using the Kenward-Roger option of the MIXED procedure of SAS.
The LIFETEST procedure of SAS was used to perform the survival-curve analyses for days from calving to culling and days from calving to pregnancy. Culled cows were treated as censored on the day the cow left the herd; nonpregnant cows were censored on the last day of the 35-wk lactation period. Significant treatment effects were noted at P 
0.05, and trends for treatment effects were noted at 0.05 < P 0.10.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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). Yields of milk and ECM increased 2.9 and 3.2%, respectively, when Sulfate minerals were replaced with minerals supplied by CTM. Cows fed CTM also had increased yields of (P 0.05) milk protein and solids (fat + protein) as compared with cows fed the Sulfate diet. Content of fat, protein, solids, and somatic cells in milk were not affected (P > 0.10) by treatment.
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Level of trace mineral fortification in diets fed in the forementioned studies was not indicative of potential lactation performance in response to replacing inorganic trace minerals with OTM. According to NRC (2001), a second-lactation, 635-kg cow (38 mo of age, mature BW 680 kg, 14 DIM, producing 36.3 kg of milk containing 3.74% fat and 3.08% true protein and consuming 16.6 kg of DM) has Zn, Mn, Cu, and Co requirements of 70, 19, 15, and 0.11 mg/kg of DM, respectively, when using absorption coefficients assigned to feed-stuffs. In general (on a mg/kg of DM basis), trace mineral requirements are highest in early lactation and lowest in midlactation. Lactating diets in the Ballantine et al. (2002) study were fortified well in excess of NRC (2001) requirements, containing 155 mg/kg of Zn, 119 mg/kg of Mn, 23 mg/kg of Cu, and 1.5 mg/kg of Co. Similarly, lactating diets fed in the Uchida et al. (2001) study were fortified well in excess of NRC (2001) requirements, containing, on average, 134 mg/kg of Zn, 77 mg/kg of Mn, 26 mg/kg of Cu, and 1.2 mg/kg of Co. In contrast, only dietary Mn, Cu, and Co concentrations in the Kincaid and Socha (2004) and Ferguson et al. (2004a) studies were fortified well in excess of NRC (2001) trace mineral requirements. Lactating diets in the Kincaid and Socha (2004) study contained 75 mg/kg of Zn, 67 mg/kg of Mn, 20 mg/kg of Cu, and 1.7 mg/kg of Co, whereas lactating diets in the Ferguson et al. (2004a) study contained, on average, 80 mg/kg of Zn, 74 mg/kg of Mn, 23 mg/kg of Cu, and 1.4 mg/kg of Co.
Although improvements in yield of milk and milk components have been observed previously, in general, milk composition does not appear to be affected by level or source of trace mineral supplementation (Uchida et al., 2001; Ballantine et al., 2002; Kellogg et al., 2003). However, Ferguson et al. (2004a) and Kincaid and Socha (2004) reported greater milk protein content for cows supplemented with OTM.
Claw Parameters
White line separation and sole hemorrhages were the most prominent claw lesions, with more than 85% of cows being afflicted with these lesions at 16 wk postpartum (Table 5). Incidence rates of sole hemorrhages were similar to previous observations (Bergsten, 1994; Nocek et al., 2000), but incidences of white line separation were considerably greater (Nocek et al., 2000; Ballantine et al., 2002). Sole hemorrhages and white line separation are clinical manifestations of disturbances in the microcirculation of the corium of the claw with subsequent degeneration at the dermal-epidermal junction (Lischer and Ossent, 2002) and can be attributed to subclinical acidosis and less-than-desired cow comfort. Factors contributing to poor cow comfort include excess time away from the pen, flooring that is rough and abrasive, and free stalls that are too narrow, too short, or do not provide adequate cushioning (Cook and Nordlund, 2007).
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0.05) incidence of sole ulcers at wk 36 postpartum. In addition, cows fed CTM tended to have less (P = 0.09) incidence of interdigital dermatitis than cows fed the Sulfate diet at wk 16 and 36 postpartum (Table 5
). Severity of heel erosion tended to be less (P = 0.07) for cows fed CTM than cows fed Sulfate minerals (Table 6).
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Decreased incidence of claw lesions with increased mineral availability is a reflection of the role of trace minerals in maintaining claw integrity. The biological basis for Zn in maintaining claw integrity is related to the Zn-dependent enzymes, RNA nucleotide transferases, RNA polymerase, alkaline phosphatase, carbonic anhydrase, and carboxypeptidase, which are integral in differentiation of keratinocytes (Cousins, 1996; NRC, 2001). The Zn-finger proteins (Cousins, 1996) have an integral role in the formation of keratin filaments in the keratinocytes. In addition, Zn affects Ca metabolism through the regulation of calmodulin and inositol phosphate (NRC, 2001). Calcium is needed for activation of epidermal transglutaminase, which is active in cross-linkage of the cell envelope keratin fibers and is involved in the initiation and regulation of the terminal differentiation of the epidermal cells (Tomlinson et al., 2004). Zinc supplementation has been shown to decrease incidence of claw lesions in dairy cattle (Demertizis, 1973; Moore et al., 1989).
Copper’s role in the production of a healthy claw horn is related to the Cu enzyme, thiol oxidase, which strengthens claw horn through the formation of disulfide bonds between Cys residues of adjoining keratin filaments (O’Dell, 1990). The connective tissue that suspends the distal phalanx within the claw capsule is strengthened by the Cu-dependent enzyme lysyl oxidase, which forms the cross-linkages between collagen fibers (Smart and Cymbaluk, 1997). Overloading the suspensory connective tissue of the distal phalanx compresses the corium, resulting in the development of claw lesions such as sole hemorrhages, sole ulcers, and white line separation.
Although Co and Mn appear to have a lesser role in maintaining claw integrity, vitamin B12 deficiency has been shown to increase the risk of lameness (Smart and Cymbaluk, 1997). The Mn-dependent enzymes, galacto-transferase and glycosyl transferase, are required for the formation of proteoglycans (Miller et al., 1988), which are components of synovial fluid, cartilage, and loose connective tissues (Murray et al., 1993). The fibro-cartilaginous insertions in the claw wall and sole and insertion areas of the ligaments and tendons are currently being investigated to determine if failure of these components is a contributing factor to breakdown of the suspensory apparatus of the distal phalanx (Westerfield et al., 2004).
Improvements in claw integrity observed in this study have been observed previously. Ballantine et al. (2002) reported that replacing sulfate minerals with OTM, beginning 21 d prepartum, decreased incidence and severity of white line disease at both 75 and 250 d postpartum, severity of sole ulcers at 250 d postpartum, and severity of heel erosion at 75 d postpartum. Ferguson et al. (2004b) observed a reduction in sole lesions including sole ulcers at 30 d postpartum when OTM replaced inorganic minerals, beginning 60 d prepartum. In contrast, Nocek et al. (2006) only observed a reduction in severity of white line lesions when sulfate minerals were replaced with OTM. Limited effects of trace mineral source on claw integrity may be attributed to a lower incidence of claw lesions in the Nocek et al. (2006) study as compared with incidence of claw lesions observed in this study as well as in the Nocek et al. (2000) and Ballantine et al. (2002) studies.
Fertility Parameters
Despite first service conception tending to be greater (P = 0.07) for cows fed the Sulfate diet, there was no effect of treatment (P > 0.10) on days to first service, services per conception, and days open (Table 7). There was no effect of treatment (P > 0.10) on days to conception (Figure 1), but there was a trend (P = 0.10) for a greater percentage of cows fed the Sulfate diet to be culled from the herd (Figure 2).
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Variability in response does not appear to be dependent on whether OTM was added to the diet (Kellogg et al., 2003; Nocek et al., 2006) or substituted for inorganic minerals (Uchida et al., 2001; Ballantine et al., 2002; Ferguson et al., 2004b). In addition, response does not appear to be dependent on whether cows were fed diets fortified with moderate (Ferguson et al., 2004b) or high levels of supplemental minerals (Uchida et al., 2001; Ballantine et al., 2002) as noted earlier.
However, improvements in fertility in response to increased availability of Zn, Mn, and Cu do have a biological basis. Manganese is necessary for cholesterol synthesis, which, in turn, is required for synthesis of the steroids, estrogen, progesterone, and testosterone (Keen and Zidenberg-Cherr, 1990). In addition, the corpus luteum has a high Mn content and may be affected by level of Mn supplementation (Brown and Casillas, 1986). Symptoms of a Cu deficiency include early embryonic death, resorption of the embryo, increased retained placentas, and necrosis of the placenta (Miller et al., 1988; Puls, 1994). Weak and silent heats have been reported. Kappel et al. (1984) reported that dairy cows with greater serum Cu levels had significantly less days to first service, fewer services per conception, and fewer days open. Inadequate Zn levels have been associated with decreased fertility, abnormal estrus, abortion, and altered myometrial contractibility with prolonged labor (Duffy et al., 1977; Maas, 1987). Campbell and Miller (1998) found that feeding dry cows an additional 800 mg of Zn for the last 6 wk of gestation decreased days to first estrus and days to service in the subsequent lactation despite consuming a basal diet containing 102 mg/kg of Zn.
Liver Parameters
There was no effect of treatment (P > 0.10) on trace mineral content of the liver (Table 8). Average liver concentrations indicate that all groups of cows had adequate Cu, Mn, Zn, and Fe status, and Mo concentrations in the liver were normal (Puls, 1994). Despite cows having adequate Zn, Mn, and Cu status, as indicated by trace mineral content of liver, lactation performance and claw integrity of cows were affected by trace mineral source. These results suggest that either Zn, Mn, and Cu content of liver is a poor indicator of trace mineral status or that trace mineral content of liver is not an accurate predictor of whether cows will respond to different sources of trace minerals. Potential explanations include trace minerals in the liver being in a form that is of limited availability for metabolism or stores of Zn, Mn, and Cu being mobilized at an insufficient rate to compensate for periods when animals are consuming inadequate amounts of these trace minerals to meet metabolic requirements. Similar results have been observed in other studies (Ballantine et al., 2002; Ferguson et al., 2004a,b; Nocek et al., 2006), in which cows had adequate Zn, Mn, Cu, and Co status, and despite minimal or no effect of treatment on liver trace mineral content, lactation performance, fertility, or claw integrity were improved when OTM were included in the diet.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Received for publication October 15, 2007. Accepted for publication January 22, 2008.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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) or Availa-4 (CTM, 
; log-rank test, 
2 = 0.68, Sulfates SEM = 7.0, CTM SEM = 7.1, P = 0.41).