Effects of adding a concentrated pomegranate extract to the ration of lactating cows on performance and udder health parameters

Animal Feed Science and Technology 175 (2012) 24–32

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Animal Feed Science and Technology journal homepage: www.elsevier.com/locate/anifeedsci

Effects of adding a concentrated pomegranate extract to the ration of lactating cows on performance and udder health parameters A. Shabtay a , M. Nikbachat a , A. Zenou a , E. Yosef a , O. Arkin b , O. Sneer b , A. Shwimmer c , A. Yaari d , E. Budman d , G. Agmon d , J. Miron a,∗ a b c d

Department of Ruminant Sciences, Institute of Animal Science, Agricultural Research Organization, P.O. Box 6, 50250, Bet-Dagan, Israel Darom dairy farm, Kibbutz Gat, Israel The National Service for Udder Health and Milk Quality, Israel Dairy Board (IDB), Yahud, Israel Gan Shmuel Food Ltd., Kibbutz Gan-Shmuel, Israel

a r t i c l e

i n f o

Article history: Received 7 November 2011 Received in revised form 4 April 2012 Accepted 7 April 2012

Keywords: Pomegranate extract Dairy cow Milk production Udder health

a b s t r a c t Two experiments were conducted to investigate effects of dietary supplementation of concentrated pomegranate extract (CPE) on performance of lactating cows. In Experiment 1, we determined effects of dose of CPE on cows’ voluntary intake and milk performance whereas, in Experiment 2, we measured effects of 40 g CPE addition per kg total mixed ration (TMR) dry matter (DM) fed to lactating cows divided into three subgroups on udder health and milk production. In Experiment 1, effects of 10 g CPE/kg DM and 40 g CPE/kg DM addition resulted in a 3.9% and 4.9% increase in voluntary intake, respectively, compared to control cows. The higher intake and lower clinical mastitis incidences in cows fed 40, 20 and 10 g CPE/kg DM supplements were reflected in a concomitant increase of 8.2%, 2.65% and 5.4%, respectively, in milk production compared to control cows. Milk antioxidant activity in cows fed the CPE supplements increased by 15.0–17.2% relative to control cows. In Experiment 2, 200 cows were divided into pairs to produce three subgroups fed control TMR and three subgroups fed 40 g CPE/kg DM supplement mixed into the TMR. Each of the two low somatic cell count (L-SCC) subgroups (control versus CPE) used 34 cows in mid-lactation, whereas the two high somatic cell count (H-SCC) subgroups (control versus CPE) each used 33 cows in mid-lactation, and two other subgroups (control versus CPE) each used 33 cows in early lactation. The three subgroups fed CPE produced more milk than their respective control subgroups, their milk SCC was lower, and the proportion of H-SCC cows at the end of the experiment was lower. Larger response to CPE addition on milk and milk energy yields seemed to occur in cows suffering from chronic mastitis and in cows in early lactation. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Global production and consumption of pomegranate has greatly increased in recent years, at least partly due to recognition of the health-promoting potential of various components of this fruit to its human consumers (Aviram et al., 2008). Accordingly, 2800 ha of pomegranate trees were planted in Israel through 2010. This has led to development of advanced

Abbreviations: ADFom, acid detergent fiber expressed exclusive of residual ash; aNDFom, neutral detergent fiber assayed with a heat stable amylase and expressed exclusive of residual ash; AOA, antioxidative activity; BW, body weight; CNS, coagulate-negative staphylococci; CON, control cows; CPE, concentrated pomegranate extract; DIM, days in milk; DM, dry matter; H-SCC, high somatic cell count; LDCL, luminol-dependent chemiluminescence; L-SCC, low somatic cell count; TMR, total mixed ration. ∗ Corresponding author. Tel.: +972 3 9683370; fax: +972 3 9604023. E-mail addresses: [email protected], [email protected] (J. Miron). 0377-8401/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.anifeedsci.2012.04.004

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industrial technologies which provide consumers with “ready to eat” pomegranate grains and fresh fruit juices. These processes have led to production of high quantities of pomegranate byproduct biomass. Pomegranate components have attracted attention for their apparent wound-healing properties (Chidambara et al., 2004), immunomodulatory activity (Gracious et al., 2001; Shabtay et al., 2008), antibacterial activity (Navarro et al., 1996), as well as antiatherosclerotic and antioxidative capacities (Tzulker et al., 2007). Recent studies (Li et al., 2006; Tzulker et al., 2007) demonstrated higher antioxidant capacity of the peels relative to the juice, mainly due to water-soluble polyphenols, anthocyanins and hydrolyzable tannins (Gil et al., 2000; Tzulker et al., 2007). However, fresh pomegranate biomass contains high levels of moisture and soluble sugars (Shabtay et al., 2008), creating problems related to disposal, drying and preservation. Moreover, processing of pomegranate is limited to its short harvest season. These limitations complicate preservation and standardization of the byproduct feed as a continuous and steady ingredient in ruminant rations. Therefore, the Israeli company Gan Shmuel Food Ltd. (Gan-Shmuel, Israel) recently developed an industrial process to produce and standardize a proprietary “Gan Shmuel’s CPE” which contains 5-fold the phenolic compounds compared to the original bulk pomegranate byproduct. Packed CPE reduces storage expenses at −20 ◦ C, and makes CPE available for year round use as a feed supplement for ruminants. Shabtay et al. (2008) demonstrated that dietary supplementation of wet fresh pomegranate peels promotes an increase in feed intake, with a tendency to increased weight gain, in bull calves. They suggested that the antioxidant and immunomodulatory properties of pomegranate peels might improve immune function, which could benefit calf health. In contrast, Oliveira et al. (2010) found that feeding a pomegranate extract to young calves for the first 70 d of life suppressed intake of grain and whole tract digestibility of fat and crude protein, likely because of its high tannin content. Recently, Modarasi et al. (2011) found that feeding pomegranate seed pulp to lactating goats little affected milk and milk solids yield, although milk fat concentration increased. To the best of our knowledge, there is a lack of published information on effects of adding pomegranate components to rations on milking performance and health of high producing lactating cows. Moreover, regarding the discrepancies between Shabtay et al. (2008) and Oliveira et al.’s (2010) and Modarasi et al.’s (2011) findings, it appears that different pomegranate components may have different nutritive effects and influence milk production in different ways. Although polyphenolic compounds may improve animal health, they can also decrease proteolytic activity, thereby compromising protein digestion (Broderick et al., 1991). Therefore, potential benefits of added pomegranate components on cow health and production should be considered relative to the potential decline in nutrient digestion and milk production. This conflict dictates a need to measure optimal dosages of pomegranate compounds, specifically CPE inclusion, in lactating cows’ total mixed ration (TMR). In our study, we assumed that addition of CPE in the low range of 10–40 g/kg TMR dry matter (DM) would not interfere with milk protein synthesis, but would be effective in promoting animal health and milk production. Two consecutive experiments were conducted. The objective of Experiment 1 was to determine the most appropriate dose of CPE by supplementing it at 0, 10, 20 or 40 g/kg TMR DM to four groups of 10 individually fed cows. Dry matter intake, milk yield and composition, udder health and antioxidant capacity of the milk were measured. The objective of Experiment 2 was to measure effects on udder health and milk production of 40 g CPE addition per kg TMR DM of 100 lactating cows divided into three subgroups including: low somatic cell count (L-SCC) cows in mid-lactation; cows with initially high SCC (H-SCC) in mid lactation; and cows in early lactation. The three control subgroups with similar initial milk yields, days in milk (DIM) and milk SCC levels were fed the same TMR without CPE addition. 2. Materials and methods 2.1. Cows, diets, and sampling procedures 2.1.1. Experiment 1 Forty pregnant Israeli Holstein cows in mid-lactation were housed for 7 d of adaptation followed by 42 d of experimental time at the Agricultural Research Organization dairy farm, Bet-Dagan (Israel), in one shaded corral with free access to water in the autumn of 2010. Cows were divided at the onset of the experiment into four groups of 10 cows, which were similar in average lactation number (3.3 ± 0.05), DIM (147 ± 1.2) and milk production (41.0 ± 0.20 kg/cow/d). Ten cows in the control group CON) were individually fed a typical Israeli TMR (Table 1), whereas the three experimental groups were individually fed the same TMR with CPE supplement at levels of 10, 20 or 40 g/kg DM ingested. The CPE was added once daily at 10:00 h on top of the ration and lightly mixed with the TMR to ensure full consumption. The amount of CPE weighed to each cow was determined daily according to her previous days DM intake. The CPE used in Experiments 1 and 2 was 451 g DM/kg containing (g/kg): total phenolics as gallic acid (92), soluble sugars (288), crude protein (20.8), ash (50.2). The phenolics contained punicalagins A + B (26.5) and ellagic acid (2.5). The CPE was preserved by addition of 0.75 g/L sorbate and kept at 4 ◦ C until used when its pH was 2.65. This CPE was prepared by chopping the pomegranate parts remaining after pomegranate juice production, including peels, residual grains and inner parts of the fruit, and subjecting them to water extraction and evaporation which resulted in a 5-folds concentration of the active compounds. The final concentrated extract was standardized to ensure uniform and constant biological activity and a CPE available all year for feeding to ruminants (Fig. 1). The TMR was fed once daily at 10:00 h for ad libitum intake, allowing for 50–100 g/kg TMR of orts, and cows were milked three times daily at 06:00, 14:00 and 22:00 h. Cows were fed individually via a computerized monitoring system designed

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Table 1 Ingredients (g/kg dry matter) of the basal total mixed ration (TMR) fed as the control diet in Experiments 1 and 2. Experiment

Wheat, silage Oat, hay Wheat, straw Sunflower, straw Corn, silage Clover, hay Soybean, hulls Soybean, meal solvent-extracted (450 g/kg CP) Corn, grain ground Barley, grain ground Wheat, grain ground Cottonseed, whole linted Corn, gluten feed Corn, distillers dried grains with solubles Canola, meal solvent-extracted (360 g/kg CP) Whey, solids NaHCO3 NaCl CaCO3 Ca-LCFAa Soy, molasses Urea Trace mineral + vitamin mixtureb a b

1

2

100.0 80.0 – – 100.0 23.0 78.0 22.0 129.0 87.0 44.0 20.0 96.0 89.0 38.0 36.0 7.4 6.0 9.0 14.0 17.0 4.0 0.6

89.0 80.0 67.0 44.0 0 0 120.0 0 100.0 75.5 60.0 69.0 190.0 0 32.0 50.0 7.4 4.5 9.0 13.0 28.0 1.0 0.6

Calcium salts of long-chain fatty acids, Koppolk Ltd., Petach-Tikva, Israel. Contains (g/kg DM): Zn 24, Fe 24, Cu 12.8, Mn 24, I 1.44, Co 0.32, Se 0.32; 16,000,000 IU Vitamin A; 3,200,000 IU Vitamin D3 ; 48,000 IU Vitamin E.

to electronically identify individual cows and to automatically control and record daily feed intake of each cow (Miron et al., 2003). Voluntary daily DM intake of individual cows was determined based on the DM content of the TMR sampled daily and in individual cows feed refusals. Milk yield of each cow was recorded daily by automatic meters (Afimilk SAE, Afikim, Israel). Milk samples were collected in three sequential milkings on a weekly basis throughout the study. Each set of milk samples for each cow was stored at 4 ◦ C in the presence of 2-bromo-2-nitropropane-1,3-diol, until analyses of fat, true protein, lactose, urea and SCC by infrared analysis (Israeli Cattle Breeders Association laboratory, Caesaria, Israel), using a Milkoscan 4000 (Foss Electric, Hillerod, Denmark). Recumbence time for each cow, an indicator of animal welfare (Drissler et al., 2005), was recorded daily by an automatic meter located within each cow’s pedometer (Afimilk SAE), and body weight (BW) data were recorded by an automatic walk-over scale three times a day while cows were entering the milking parlor. Changes in BW were calculated as the gap between the week before the onset of the experiment and the last week of the experiment. Luminol-enhanced chemiluminescence assay (Ginsburg et al., 2005) was used to measure the reducing antioxidant potential of milk. Briefly, milk samples (20 ␮l) were added to a reaction mixture containing 10 ␮l luminol (10 ␮M), 10 ␮l morpholinosydononimine (SIN-1, 1 mM), 20 ␮l sodium selenite (2 mM) and 10 ␮l Co2+ (SIN-1 cocktail). This radical-generating cocktail simultaneously generates a flux of peroxide and NO. The capacity of milk samples to quench the luminol-enhanced chemiluminescence generated by the cocktail was measured in a LUMAC/3M Biocounter M2010 connected to a linear recorder. The resulting light output was recorded as counts/min. for 6 min. The luminol-dependent chemiluminescence (LDCL) values were expressed as equivalent of gallic acid, using a calibration curve in which gallic acid was plotted against LDCL. The antioxidative potential of the milk was expressed, each minute, relative to the SIN-1 cocktail. Milk energy (MJ/kg milk) was calculated as: 4.183 × {(0.00929 × milk fat g/kg) + (0.00547 × milk true protein g/kg) + (0.00395 × milk lactose g/kg)}, according to NRC (2001). 2.1.2. Experiment 2 Israeli Holstein cows (200) were housed at the “Darom” dairy farm, located in Kibbutz Gat in the southern part of Israel, in one shaded corral during the 2010/2011 winter. Cows were divided into pairs at the onset of the 3 mo experiment to create two groups of 100 cows each housed in the two sides of the open shed. In each side of the corral three pens similar in size were separated by fences to house three sub-groups of 33 or 34 cows each, assigned randomly to the three pens, with free access to water and food. Cows of the control sub-groups were fed a typical Israeli TMR (Table 1), whereas the experimental sub-groups were fed the same TMR with CPE supplement (40 kg CPE mixed in mixing wagon with 1000 kg TMR DM). The three subgroups fed the control or CPE TMR, were: (i) 34 low SCC cows (SCC < 150,000/ml milk in 3 consecutive weekly measures) in mid lactation (L-SCC group); (ii) 33 cows in mid lactation with high SCC > 150,000/ml milk in 3 weekly measures, (H-SCC group); (iii) 33 cows in early lactation entering into the control or experimental groups immediately after calving (early lactation group). The early lactation cows fed the CPE TMR received 40 kg CPE/100 kg TMR DM from 3 wk before calving to 80 DIM, whereas the control early lactation cows did not receive CPE during the dry period or through the first

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Pomegranate Juice

27

Pomegranate Juice extraction by pressure Pomegranate Fruits residues - Peel, Core, fruit, membrane, seed, juice traces,

Water extraction

Liquification Extraction

Refining Separation

Pasteurization

Residues for feed meal

Evaporation By forced cyrculation To 450g DM/kg

Preservation Standardization Blending

Storage at

Packing

o

-20 C Fig. 1. Concentrated pomegranate extract (CPE) preparation (flow chart); (PCT # 26906-WO-10).

80 d postpartum. The two L-SCC groups (control versus CPE) were similar in lactation number (2.89 ± 0.110), DIM (145 ± 1.3), milk production (40.8 ± 0.23 kg/cow/d) and pregnancy status (67 ± 1.0 cows/100 cows) at the onset of the experiment. The two H-SCC groups (control versus CPE) were also similar in lactation number (2.63 ± 0.122), DIM (160 ± 1.3), milk production (38.7 ± 0.26 kg/cow/d) and pregnancy status (57 ± 1.5 cow/100 cows) at the onset of the experiment. The two early lactation groups (control versus CPE) were also similar in lactation number (3.3 ± 0.2), and total milk production during the previous lactation (12,100 ± 15.6 kg/cow). The control and CPE TMR were fed twice daily at 09:00 and 16:00 h individually to each sub-group for ad libitum intake, allowing for 50 g/kg orts weighed for each subgroup daily. Cows were milked three times daily starting at 06:30, 14:30 and 22:30 h, and each subgroup was milked separately. Milk yield per cow was recorded daily by automatic meter (Afimilk SAE) over the 90 d of the experiment. Milk samples were collected during three sequential milkings every 2 wk, on day 15 and 30 of each month, throughout the 90 d of this study to create a total of six samples per cow. The early lactation cows of the two treatments participated for an average of 80 DIM in the study. Each set of milk samples was analyzed for fat, true protein, lactose, urea and SCC by infrared analysis as described for Experiment 1. In the two sub-groups of H-SCC cows, control or CPE, milk samples from each quarter of each cow’s udder were sampled aseptically twice during the week before the experiment, and twice during the last week of the experiment. These milk samples were immediately cooled to 4 ◦ C and transported to the laboratory of the National Service for Udder Health and Milk Quality (NSUHMQ) according to National Mastitis Council guidelines (ISO 17025), for identification of bacterial species involved in chronic udder infection (Klement et al., 2005).

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Table 2 Chemical composition (g/kg DM; +SE) of the basal total mixed ration (TMR) fed as the control diet in Experiments 1 and 2. TMR Experiment 1 Dry matter (DM, g/kg TMR) Crude protein Ether extract aNDFom Hemicellulose Cellulose Lignin(sa) Forage aNDFoma In vitro DM digestibility (fraction)

633 165 48.7 408 208 145 52.7 180 0.687

± ± ± ± ± ± ± ± ±

18.9 3.3 0.96 7.1 4.1 9.3 7.03 7.2 0.0112

Experiment 2 500 164 56.4 438 207 180 51.0 176 0.759

± ± ± ± ± ± ± ± ±

15.1 3.2 0.98 14.6 6.4 4.8 1.08 5.2 0.0151

a Forage aNDFom is based on analysis of neutral detergent fiber (aNDFom) content of the wheat silage, oat hay, corn silage and clover hay in the TMR of Experiment 1, and of the wheat silage, oat hay, wheat straw and sunflower straw in the TMR of Experiment 2.

These animal studies were conducted according to the guidelines, and under the supervision of the ARO Animal Care Committee. 2.2. Chemical and in vitro analyses Samples of the TMR fed and orts of each subgroup in experiment 2 were composited weekly. Samples of TMR and individual orts from each cow in experiment 1 were also composited weekly. Sub samples were collected from these composites, dried for 48 h in an aerated 60 ◦ C oven, ground to pass a 1 mm screen and analyzed by methods of AOAC (1990) for DM (#925.40), N (#984.13), ether extract (#920.39) and ash (#923.03). Neutral detergent fiber (aNDFom) was assayed with a heat stable amylase without sodium sulfite and expressed exclusive of residual ash (Van Soest et al., 1991). Acid detergent fiber (ADFom) was determined using the sequential method on aNDF residue and expressed exclusive of residual ash (Van Soest et al., 1991). Hemicellulose was calculated as aNDFom – ADFom. Lignin(sa) was assayed as the residual organic matter remaining after hydrolysis of ADF with 720 g/kg sulfuric acid. Cellulose was calculated as ADFom – lignin(sa). Ankom apparatus (Ankom220 , Macedon, NY, USA) was used to extract and filter aNDF, ADF and lignin(sa). In vitro DM digestibility of the TMR was determined according to the two-stage technique of Tilley and Terry (1963). 2.3. Statistical analyses 2.3.1. Experiment 1 The effect of CPE levels (treatments) on weekly parameters was evaluated using the GLM procedure of SAS (1998), and variances between groups were also analyzed with SAS. Differences between means were determined according to the model: y =  + Ti + eij , where y denotes the dependent variable,  denotes the mean, T is the treatment effect and e is random residual error. All values in Table 3 are means and SEM of the 6 wk measurement period. Linear and quadratic responses were determined with polynomial contrasts (SAS, 1998). Differences among the four groups with respect to changes during the experiment in milk antioxidative activity and BW based on 10 replicates (cows) for each treatment were tested for significance with the GLM option of JMP-5 software (SAS, 1996, Table 3). 2.3.2. Experiment 2 Comparisons between the two subgroups (CON and CPE) of L-SCC (34 cows subgroup), the two subgroups (33 cows each) of H-SCC cows, and the two subgroups (33 cows each) of early lactating cows, with respect to milk yield, milk composition and SCC used the GLM option of SAS (1996) and data are in Tables 4–6 as weekly means for the 80–90 d of the experiment. Tukey’s test was used for means comparison. Differences in Experiment 2 between the CON and CPE subgroups with respect to number of cows with H-SCC levels (Tables 4–6) of each sub-group were tested for significance by Chi-square based on the average SCC of the L-SCC as expected basal reference using JMP-5 software. 3. Results 3.1. Intake, performance and udder health in Experiment1 Ingredients and chemical composition of the basal TMR fed to the cows individually with various levels of added CPE are in Tables 1 and 2. Analyses of the polynomial impacts of CPE level on each of the parameters shows a lack of linear significance (P=0.23–0.53), but significant quadratic impacts (P=0.01–0.05) in most response parameters which suggest higher values at intermediate addition levels of CPE (i.e. milk lactose, AOA on week-4, changes in AOA activity, recumbence time and SCC, Table 3). In

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Table 3 Performance, health and antioxidant activity (AOA)a data of mid-lactation cows individually fed the same total mixed ration (TMR) with different levels of concentrated pomegranate extract (CPE) top dressed for 42 d in Experiment 1. Diet (g/kg CPE) 0 n Dry matter intake (kg/cow/d) Milk yield on healthy daysb (kg/d) Milk yield (kg/d) Milk fat (g/kg) Milk true protein (g/kg) Milk lactose (g/kg) Milk energy (MJ/cow/d) AOA on week 0 (gallic Equiv. ␮M) AOA on week 4 (gallic Equiv. ␮M) Change during 4 wk in AOA activity (fraction) BW change (kg/6 wk) Average SCC (×103 /ml milk, 6 measures/cow) New clinical mastitis incidences Recumbence time (h/d) a b

10 28.3 44.2 41.5 32.5 29.5 48.3 113.5 20.1 20.0 −0.005 +8.1 139 4 9.11

P

10

20

40

10 29.4 44.5 43.8 29.0 29.5 48.4 114.0 20.8 23.8 +0.144 +11.0 155 1 9.96

10 28.6 45.0 42.6 31.9 30.4 49.1 117.0 20.4 23.8 +0.167 +6.1 172 1 9.40

10 29.7 44.9 44.9 29.8 30.3 48.5 119.1 20.0 23.0 +0.150 +6.7 119 0 9.59

SEM

Linear

Quadratic

0.11 0.19 0.22 0.20 10. 0 0.10 0.52 0.83 0.61 0.0150 3.44 14.3

0.53 0.25 0.23 0.35 0.42 0.27 0.23 0.35 0.25 0.34 0.44 0.38

0.05 0.04 0.05 0.05 0.04 0.05 0.03 0.22 0.03 0.01 0.32 0.04

0.06

0.26

0.04

Antioxidant activity in milk defined as gallic equivalents. Average milk yield excluding days in which cows were identified as suffering from clinical mastitis and treated with antibiotics until recovered.

Table 4 Lactational performance of low somatic cell count (SCC < 150,000/ml milk) mid-lactation cows in Experiment 2 fed for 90 d a total mixed ration (TMR) containing 40 g/kg dry matter concentrated pomegranate extract (CPE) versus control TMR. CPE n Dry matter intakea (kg/cow/d) Milk yield (kg/cow/d) Milk fat (g/kg) Milk true protein (g/kg) Milk lactose (g/kg) Milk energy (MJ/cow/d) Average SCC (×103 /ml milk, 6 measures) Cows with H-SCC > 200,000/ml (fraction) New clinical mastitis incidence

34 27.6 43.4 35.0 32.3 47.0 124.8 119 0.059 0

Control 34 28.1 42.6 34.0 32.4 47.6 121.4 172 0.171 3

SEM

P

0.60 0.09 0.31 0.13 0.2 2 0.80 13.5 0.0150

0.007 0.057 0.75 0.16 0.046 0.042 0.047

a Dry matter intake was calculated on the basis of daily group feeding, therefore only SEM values are provided (repeated measures) but measurement of significance is not possible.

contrast, the response parameters DM intake, milk yield, milk true protein as well as milk energy showed evidence of higher response at the highest CPE addition. In contrast, milk fat proportion was lowest at intermediate addition level of CPE. The CON cows tended to be more sensitive to clinical mastitis during the experimental period than the CPE-fed cows, as four of the CON cows developed clinical mastitis versus only one in the 10 g/kg CPE group, one in the 20 g/kg CPE group and none in the 40 g/kg CPE group. During active clinical mastitis, milk yield was sharply reduced, and this resulted in a high gap between milk yield on healthy days and average milk yield within all groups except of the 40 g/kg CPE group (Table 3). Antioxidant activity of the milk tended to decrease slightly with increasing DIM, as reflected by 0.5% reduction in antioxidant activity in CON cows in the 4th wk of the experiment compared to their activity at the onset of the experiment. 3.2. Performance and udder health in Experiment 2 The L-SCC cows fed 40 g/kg CPE produced, over the 90 d experiment, 1.9% more milk (P<0.01) and 2.8% more milk energy (P<0.05) than their control counterparts (Table 4). The effect of CPE was expressed in a 31% lower SCC (P<0.04) and 66% lower proportion (P<0.05) of H-SCC cows. The CPE cows tended to have lower clinical mastitis events (0 versus 3) than control cows. The H-SCC cows in mid-lactation fed 40 g/kg CPE produced, over the 90 d experiment, 9.4% more milk (P<0.01) and 8.8% more milk energy (P<0.01) than their control counterparts (Table 5). The effect of CPE was also expressed in a 22.7% lower SCC (P<0.05) than their control counterparts. Addition of CPE reduced the proportion of H-SCC cows (i.e. those with SCC > 200,000) by 22.8% (P<0.05) compared to their control counterparts. In the H-SCC cows (data not shown) the coagulate-negative staphylococci (CNS) caused most of the chronic mastitis events in the CPE cows (98% of udder quarters infected by CNS and 4% by Bacillus at the onset and end of the experiment) and most chronic mastitis events in the control cows (88% of udder quarters infected by CNS, 3% by Bacillus, 7% by Corina and 2% by Pseodomonas at the onset and end of the experiment). All clinical mastitis events were caused by Escherichia coli

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Table 5 Lactational performance and udder health of high somatic cell count (SCC > 150,000/ml milk) mid-lactation cows in Experiment 2 fed for 90 d a total mixed ration (TMR) containing 40 g/kg DM concentrated pomegranate extract (CPE) versus the control TMR.

n Dry matter intakea (kg/cow/d) Milk yield (kg/cow/d) Milk fat (g/kg) Milk true protein (g/kg) Milk lactose (g/kg) Milk energy (MJ/cow/d) BW change (kg/cow/90d) Average SCC at the onset of the experiment (×103 /ml milk, 3 measures) Average SCC during the experiment (×103 /ml milk, 6 measures) Cows with H-SCC (> 200,000/ml milk) during the experiment (fraction of group) Infected udder quarters at the onset of the experiment at the end of the experiment New clinical mastitis incidences

CPE

Control

SEM

P

33 25.1 38.3 36.0 34.0 47.0 113.1 +54.0 712 368 0.61

33 24.1 35.0 37.0 34.0 45.8 104.0 +64.1 683 476 0.79

0.58 0.14 0.32 2. 03 22. 0 0.79 5.09 121.0 28.0 0.033

0.006 0.073 0.28 0.008 0.009 0.45 0.92 0.049 0.046

48.0 45.1 1

69.0 37.1 1

5.60 3.00

0.12 0.22

a Dry matter intake was calculated on the basis of daily group feeding, therefore only SEM values are provided (repeated measures) but measurement of significance is not possible.

Table 6 Lactational performance and udder health of early-lactation cows fed for 80 d post partum in Experiment 2 a total mixed ration (TMR) containing 40 g/kg dry matter concentrated pomegranate extract (CPE) versus control TMR.

n Dry matter intakea (kg/cow/d) Milk yield (kg/cow/d) Milk fat (g/kg) Milk protein (g/kg) Milk lactose (g/kg) Milk energy (MJ/cow/d) BW change (kg) Average SCC (×103 /ml milk 4–6 measures) Cows with average H-SCC (>200,000/ml milk) during the experiment (fraction) New clinical mastitis incidence

CPE

Control

SEM

P

3 31.7 50.2 35.1 30.6 47.5 143.0 +18.0 175 0.212 2

3 30.9 47.2 34.7 30.3 47.5 133.4 +13.2 280 0.333 3

0.47 0.19 11. 0 12. 0 0.11 0.43 6.49 39.1 0.0250

0.019 0.40 0.99 0.48 0.001 0.69 0.17 0.046

a Dry matter intake was calculated on the basis of daily group feeding, therefore only SEM values are provided (repeated measures) but measurement of significance is not possible.

in H-SCC cows. However, CPE addition did not have antibacterial effects against CNS since the incidence of CNS-infected udder quarters of the CPE cows at the end of the experiment were similar to that at its onset (Table 5). The cows in early lactation fed 40 g/kg CPE additive produced over 80 DIM, 6.4% more milk (P<0.02) and 7.2% more milk energy (P<0.01) than their control counterparts (Table 6). Effects of CPE feeding was expressed in this group in 36.3% lower proportion of H-SCC cows (P<0.05), compared to control. 4. Discussion 4.1. Immunomodulatory effect of CPE addition Four of the control cows developed clinical mastitis during experiment 1, whereas only one in the cows fed 10 and 20 g/kg CPE and none in the 40 g CPE/kg group. However, within 3 wks of termination of CPE addition, the incidence of clinical mastitis was similar in the four ex-experimental groups at 3–4/10 cows. Similarly, cows fed 40 g/kg CPE responded with lower SCC and reduced proportion of H-SCC cows in each of Experiment 2 sub-groups. These results may be associated with the immunomodulatory activity of pomegranate peels as discussed by Gracious et al. (2001) and Shabtay et al. (2008). The reduction in milk SCC accompanied by a reduction in the proportion of H-SCC cows and increased milk production suggest that CPE stimulated the cows’ immune systems, although it probably reduced the level of immune cells (i.e., SCC) in milk. Consistent with our findings, Oliveira et al. (2010) demonstrated that supplementing polyphenols from pomegranate extracts to pre-weaned dairy calves improved lymphocyte function, which may have improved humoral and cell-mediated immunity as the calves underwent active immunization. 4.2. Antioxidative effect of CPE addition The antioxidant activity of CPE, by an increase in the antioxidative activity of milk from cows fed CPE, may have human health implications, as results from our laboratory (A. Shabtay, ARO Israel, unpublished results), indicate that pasteurization

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does not affect milk antioxidant capacity. This should encourage use of milk enriched with antioxidants in human diets to promote human health and prevent diseases associated with oxidative stress, including cancers (Serrano et al., 1998). 4.3. Effects of CPE tannins on intake and performance Pomegranate peel is rich in tannins (Shabtay et al., 2008), which have been shown to have both adverse and beneficial effects in ruminants (Makkar, 2003). Moderate concentrations of condensed tannins (i.e., 20–40 g/kg of DM) in the diet have improved production efficiency of ruminants, without increasing DM intake, as manifested by increases in wool growth, BW gain, milk yield and ovulation rate (Aerts et al., 1999). Hydrolyzable tannins have been shown to correlate positively with antioxidant activity and polyphenol content in pomegranate peel and juice (Gil et al., 2000; Tzulker et al., 2007). In pomegranate, hydrolyzable tannins include punicalin, ellagic acid, gallagic acid and punicalagin, which account for about half of the antioxidant capacity of pomegranate juice (Gil et al., 2000). Punicalagin was the most abundant tannic component in our CPE at 26.5 g/kg DM. Among tannins, punicalagin has the highest lipid peroxidation-inhibitory and radical-scavenging activities (Kulkarni et al., 2004), and its health promoting attributes may be of relevance to the health of the animal which consume it (Adams et al., 2006). High concentrations of hydrolyzable tannins may reduce DM intake, digestibility of proteins and carbohydrates as well as animal performance, through negative effects on palatability and/or digestion (Broderick et al., 1991; Reed, 1995). Indeed, that 10–40 g/kg CPE addition increased DM intake and milk production, suggests the absence of negative effects of tannins on palatability or energy utilization of the diet in Experiment 1. Similar positive effects of pomegranate peel addition on palatability and DM intake occurred in calves at 11 mo of age fed fresh pomegranate peels in addition to a mixed ration, and peel intake increased linearly during feeding without adverse effects on BW gain (Shabtay et al., 2008). In contrast, addition of 60 or 120 g/kg pomegranate seed pulp to lactating goats little affected DM intake, milk yield and milk solids yield (Modarasi et al., 2011). Our findings suggest that at levels up to 40 g/kg CPE, the hydrolyzable tannins originating from CPE did not interact strongly enough with dietary proteins (Broderick et al., 1991), or inhibit protease activity (Van Leeuwen et al., 1995) sufficient to negatively impair animal performance. The discrepancy between studies with respect to pomegranate effects on DM intake and performance might be associated with differences in type and features of the pomegranate additive, such as CPE in our study, seed pulp in the goats’ study (Modarasi et al., 2011), and fresh wet peels in the calf study (Shabtay et al., 2008). 5. Conclusions Mid lactation Holstein cows fed up to 40 g/kg CPE may be more resistant to clinical mastitis, as only supplementation of CPE at 40 g/kg, reduced SCC level and proportion of cows with high SCC (i.e., >200,000/ml milk). In addition, 40 g/kg CPE supplementation increased milk and milk energy yield, while elevating levels of antioxidants in milk. Larger response to CPE addition on milk and milk energy yields seemed to occur in cows suffering from chronic mastitis and in cows in early lactation. Acknowledgements We express our appreciation to the ARO dairy farm staff for their help with Experiment 1, the staff of Darom dairy farm for their help in Experiment 2, and to the Massuot Itzhak Feeding Center. This study was supported by the Israel Dairy Board Foundation Project #362-0296. References Adams, L.S., Seeram, N.P., Aggarwal, B.B., Takada, Y., Sand, D., Heber, D., 2006. Pomegranate juice, total pomegranate ellagitannins, and punicalagin suppress inflammatory cell signaling in colon cancer cells. J. Agric. Food Chem. 54, 980–985. Aerts, R.J., Barry, T.N., McNabb, W.C., 1999. Polyphenols and agriculture: beneficial effects of proanthocyanidins in forages. Agric. Ecosyst. Environ. 75, 1–12. AOAC, 1990. Official Methods of Analysis, vol. I., 15th ed. Association of Official Analytical Chemists, Arlington, VA, USA. Aviram, M., Volkova, N., Coleman, R., Dreher, M., Reddy, M.K., Ferreira, D., Rosenblat, M., 2008. 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