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Intake, milk production and heat stress of dairy cows fed a citrus extract during summer heat
Accepted Manuscript Title: Intake, milk production and heat stress of dairy cows fed a citrus extract during summer heat Author: J.M. Havlin P.H. Robinson PII: DOI: Reference:
S0377-8401(15)00223-0 http://dx.doi.org/doi:10.1016/j.anifeedsci.2015.06.022 ANIFEE 13318
To appear in:
Animal
Received date: Revised date: Accepted date:
17-4-2015 23-6-2015 24-6-2015
Feed
Science
and
Technology
Please cite this article as: Havlin, J.M., Robinson, P.H.,Intake, milk production and heat stress of dairy cows fed a citrus extract during summer heat, Animal Feed Science and Technology (2015), http://dx.doi.org/10.1016/j.anifeedsci.2015.06.022 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Highlights (for review)
Highlights
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A citrus extract fed to lactating cows had no impact on animal performance Milk SCC count reductions with citrus extract feeding suggested improved mammary health Naturally occurring bioactive compounds can impact animal performance
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*Manuscript (final version with changes or corrections highlighted)
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4 Intake, milk production and heat stress of dairy cows
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fed a citrus extract during summer heat
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J.M. Havlina, P.H. Robinsona,*
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University of California, Davis, CA, 95616, USA
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Department of Animal Science
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*Corresponding author: Tel: 530-754-7565; Fax: 530-752-0175 EM: [email protected]
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Submitted to Animal Feed Science and Technology in April 2015
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Revised and resubmitted in May 2015. 1 Page 2 of 53
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Revised and resubmitted in June 2015.
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Abstract This study determined effects of feeding a citrus extract (CE) to high producing dairy cows
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during summer heat on measures of heat stress, as well as milk production and composition, in a
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replicated 2 x 2 Latin square experiment with two 28 d periods on a dairy farm near Hanford
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(CA, USA). Four „high group‟ pens were used (i.e., cows which had cleared the fresh pen but
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were not yet confirmed pregnant) were used, each with ~310 early lactation multiparity
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cows/pen. The two total mixed rations contained 171 g/kg dry matter (DM) crude protein (CP),
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55 g/kg fat, 335 g/kg neutral detergent fiber (aNDF) and 135 g/kg starch, and were the same
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except for inclusion of the CE at 4 g/cow/d in the treatment diet (CED). Average daily high
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temperatures during the study were 35 to 37oC with lows of 16 to 17oC. In general, cows
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showed mild heat stress, but CE feeding had no effect on respiration rate, panting score or rump
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temperature at any time of the day (i.e., 02:45, 09:15, 17:30 h). However at 02:45 h, a higher
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(P<0.01) proportion of CED cows were lying (versus standing) compared with Control cows
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(68.6 versus 53.7 cows/100 cows), which is an indicator of reduced heat stress. Intake of DM
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(25.3 kg/d) and whole tract digestibility of CP (703 g/kg) and aNDF (510 g/kg) did not differ
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between treatments. Milk production (47.3 kg/d) and its fat and true protein levels (35.4, 28.6
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g/kg) did not differ, and changes in body condition and locomotion scores were also not
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impacted by treatment impacted. However mammary health improved based on lower SCC
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(somatic cell counts; P<0.04) of CED versus Control cows (160,000 versus 196,000 cells/µL),
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and lower linear SCC scores (P<0.01; 2.12 versus 2.30). Feeding this CE to lactating dairy cows
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during summer heat decreased SCC with no impact on other aspects of performance.
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Keywords: limonene, vitamin C, citrus, essential oils
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Abbreviations: AA, amino acids; ADF, acid detergent fiber expressed with residual ash;
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ADFom, ADF expressed without residual ash; aNDF, neutral detergent fiber assayed with a heat
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stable amylase and expressed with residual ash; BCS, body condition score; CE, citrus extract;
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CED, CE diet; CP, crude protein; DM, dry matter; EE, ether extract; EO; essential oils; LS,
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locomotion score; PS, panting score; RR, respiration rate; RT, rump temperature; SCC, somatic
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cell count; THI, temperature/humidity index; TMR, total mixed ration
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54 1. Introduction
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A popular contemporary topic in ruminant nutrition is the study of naturally occurring dietary
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additives capable owhichf modulateing rumen fermentation in order to improve nutrient
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utilization by rumen microorganisms, and/or reduce enteric production of greenhouse gases such
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as methane. Essential oils (EO) encompass a wide range of naturally occurring secondary
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compounds which occur in leaves, flowers, stems and seeds of many plants, and may have
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antimicrobial properties which modify rumen microbial fermentation.
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Reviews of various EO compounds, their possible modes of action and effects on rumen
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fermentation have been described (e.g., Benchaar et al., 2008), and limonene, an EO found in
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citrus products, has been suggested to have beneficial effects on rumen fermentation, and animal
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production, in ruminants (Calsamiglia et al., 2007). While few in vivo studies have examined
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EO mixtures containing limonene, Tassoul and Shaver (2009) did reported increased milk
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protein production, with an overall trend to increased feed efficiency (i.e., kg of fat corrected
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milk/kg of DM intake), when an EO mixture including limonene was fed to dairy cows. In
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contrast, Benchaar et al. (2006, 2007) had earlier reported that the same EO mixture used by
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Tassoul and Shaver (2009) had no effect on milk production, dry matter (DM) intake or feed
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efficiency.
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Vitamin C, found at high levels in citrus products, is not considered an essential nutrient for
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healthy cattle because their liver can synthesize it from glucose at levels believed to be sufficient 4 Page 5 of 53
to meet their needs (Padh, 1990). However cattle under stress, such as those in early lactation at
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high production levels and/or under summer heat, may not synthesize adequate amounts of
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vitamin C to compensate for oxidative stress due to sub-clinical infection or illness (Weiss et al.,
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2004; Ranjan et al., 2005), and feeding supplemental vitamin C can replace depleted reserves
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(Weiss, 2001). As high somatic cell counts (SCC) in milk are the result of degraded mammary
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health, and have a negative impact on animal performance and milk quality, the limonene and
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vitamin C in citrus products may help minimize SCC in dairy cows, thereby improving
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efficiency of feed utilization as a result of improved immune function (Chaiyotwittayakun et al.,
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2002; Castillejos et al., 2006). Indeed Jaramillo et al., (2009) and Giannenas et al. (2011) fed
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citrus pulp to ewes and reduced milk SCC.
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In a review of citrus product feeding to ruminants, Bampidis and Robinson (2006) concluded
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that their feeding did not affect DM digestibility, but decreased crude protein (CP) digestibility,
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while increasing neutral detergent fiber (NDF) and acid detergent fiber (ADF) digestibility.
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Our objective was to evaluate impacts of a CE on feed intake and productive performance of dairy cows during hot weather.
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2. Materials and methods
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2.1. Animals, management, and experimental design
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High group multiparity Holstein cows (i.e., those cows which had cleared the fresh pen but
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were not yet confirmed pregnant) in 4 early lactation pens of ~310 cows each, on a commercial
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dairy farm near Hanford (CA, USA), were used in a study with two 28 d experimental periods in
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a replicated 2 x 2 crossover design during summer. Each pen had 300 head gates and free stalls.
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The only source of cooling was bunk line misters which switched on automatically at 24°C, and
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were on in each pen for 4 mins and then off for 12. Cows were assigned weekly to one of the 4 5 Page 6 of 53
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pens at random from a common fresh cow pen, and moved to a mid pen by ~200 days in milk
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once they were confirmed pregnant. Cows were milked three times daily in a double 40 herringbone parlor. Pen 1 started milking
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at 04:00, 12:00 and 20:00 h. Pens 2, 3, and 4 were milked in sequence after pen 1, at intervals of
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~45 min. The total mixed ration (TMR) was fed twice daily, between 04:00 and 08:00 h, prior to
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cows returning to their pens from milking, and again between 11:30 and 12:30 h. The amount of
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TMR delivered was determined from the previous days intake to create orts equal to ~10 g/kg of
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total TMR delivered (as fed). Orts were removed daily and weighed individually by pen while
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cows were in the parlor during the first milking, just prior to the first TMR feeding.
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Head-locks were set prior to cows returned from the morning milking, to facilitate artificial
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insemination, such that the cows were head locked in the stanchions for 45 to 60 min daily.
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Cows were housed in covered barns with access to free stalls bedded with dried manure, which
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was renewed weekly and groomed bi-weekly. Cows also had free access to an uncovered manure
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pack drylot at all times. Inside alleyways were flushed with water three times daily while the
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cows were being milked. Cows had ad libitum access to clean drinking water at all times.
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At the start of the study, two pens were fed the CE containing diet (CED), while the two
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other pens were fed the Control diet. After the first 28 d period, treatments were reversed. Each
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period contained a 21 d adjustment and 7 d collection period during which all samples were
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collected.
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2.2. Environmental conditions
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Four portable weather data loggers (HOBO U23; Onset, Bourne, MA, USA) were recorded
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ambient temperatures and relative humidity every 30 min throughout the study. One station was
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placed in each experimental pen on a pole at its center ~3 m above the floor and out of direct
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sunlight. The study took place in the 2 mo after the summer solstice to minimize variation of day 6 Page 7 of 53
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length and weather, while maximizing expected temperature/humidity indices (THI) which was
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calculated as:
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THI = tdb – [0.55-(.55*RH/100)]*(tdb – 58.8) where: tdb = dry bulb air temperature (°F) and RH = relative humidity (%). THI ≤ 74 is
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considered “normal”, 75 to 78 is “alert”, 79 to 83 is “danger”, and ≥ 84 is “emergency”
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according to the Livestock Weather Safety Index (LCI, 1970).
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2.3. Diets
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The TMR for the CED and Control groups was formulated identically except for inclusion of
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the CE at 4 g/cow/d in the CED TMR. Rate of inclusion of the CE product (VéO Premium;
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Cristalfeed® Gold Rush; Reference ST 232 P2; Phodé, Terssac, France) in TMR loads was
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calculated weekly based on the number of cows in the pen, and used to create bags of CE which
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the feeding staff added to the CED TMR. The CE contained natural citrus extracts, natural and
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artificial flavoring as well as corn flour, calcium carbonate and silicic acid as flow enhancers.
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2.4. Sample collection
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2.4.1. Feed and total mixed rations
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At the start and end of the collection week of each period (i.e., days 21 and 27) all dietary
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ingredients were sampled. Hays were sampled with a „golf club‟ style hay probe (Sierra Testing
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Services, Acampo, CA, USA), with 12 core samples pooled into a plastic bag. All other
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ingredients, with the exception of liquid whey, were sampled by hand (6 handfuls of each) and
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composited to plastic bags. Silages were sampled from the loose pile which had been knocked
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off the silage face for use that day and wet by-products (i.e., fresh chop alfalfa, tomato pomace)
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were sampled from the storage area. Samples of all other feeds from the start and end of the
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collection weeks were combined to create one sample per ingredient prior for chemical analysis.
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Ten handfuls (~200 g each) of each TMR by pen were collected according to Robinson and
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Meyer (2010) at pre-marked posts with evenly spaced intervals along the bunk-line immediately
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after feeding and before the cows had access to it. These TMR samples were pooled within
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period and pen to create 8 TMR samples for chemical analysis.
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2.4.2. Milk
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At the end of each collection week (i.e., day 28), the entire herd was tested for milk
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production and composition by Dairy Herd Improvement Association (Kings County, CA, USA)
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personnel. Milk weights and representative milk samples collected into tubes containing
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bronopol and natamycin as preservatives were collected from all cows using Tru-test milk meters
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(Tru-Test Ltd., Auckland, New Zealand).
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2.4.3. Body condition and locomotion scores
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Body condition (BCS) was scored at the start of the study (i.e., day 0) and at the end (i.e., day
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28) of each experimental period while the cows were locked in stanchions immediately after
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morning milking. From each pen, 80 cows having the lowest days in milk at the start of the study
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were selected. Cows were scored on the 1 to 5 scale of Edmonson et al. (1989), with intermediate
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values between the ¼ points if a ¼ point score was not clear. One person scored all cows.
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Locomotion was scored (LS) at the end (i.e., day 28) of each experimental period while the
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cows were returning from the parlor from milking starting at 14:00 h. All cows in the pen were
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observed for lameness using the 1 to 5 scoring system of Sprecher et al. (1997). One person
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scored all cows on both occasions.
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2.4.4. Fecal
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Fecal samples were collected from the same 18 cows during the morning lock-up on day 28
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of each period. Feces were manually collected (minimum 250 g) from the rectum of the cow
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into plastic containers, and immediately stored at -18°C until processing for chemical analysis. 8 Page 9 of 53
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2.4.5. Heat stress behavior and skin temperature Respiration rate (RR), panting score (PS), lying versus standing behavior, and rump
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temperature (RT) was measured three times over a 24 h period on days 27 and 28 of each period.
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Skin temperature was recorded on days 26 and 28 during morning lock-up during both periods,
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as they had not yet had access to the dry lot where they would be in direct sunlight at this time. A
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subgroup of 50 cows which had been scored for BCS from each pen was observed for heat stress
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parameters. Identification collars were placed on the cows during morning lock-up to facilitate
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easier identification of cows from a distance during observations. Behavioral observations started
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~24 h after the collars were attached. The behavior observation periods for pens 1 and 2 began at
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08:00, 16:15 and 01:30 h, while observations for pens 3 and 4 began directly following
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observations of pens 1 and 2. Observation periods lasted 75 mins/pen. The 08:00 h observation
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time was chosen to ensure that the cows had been out of head-lock for at least 15 min prior to
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observations. The 16:15 h time was chosen to approximate the hottest time of the day and, during
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the 01:30 h observation period, the sun had been down for ~5.5 h and the temperature was
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approaching the daily low. At this time the vast majority of the cows were in the outside dry lot.
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The RR was determined by observing flank movements of the cows where the time it took a
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cow to take 20 breaths was recorded and converted to breaths/min. A 0 to 5 PS was assigned to
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each cow (Gaughan et al., 2008) based on physical signs including degree of chest movement
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and presence of drool. Cows in two pens were simultaneously observed for heat stress behaviors
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by two individual scorers. Each scorer observed the same treatment and Control pen in each
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period. Scorers trained together prior to the study, and between periods, to eliminate scorer bias.
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To obtain RT, two small spots of ~40 x 60 mm each were shaved on each side of the rear
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flank, distal to where the udder meets the leg. Temperatures from the right and left flank were
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measured using an infrared gun (Fluke-561, Everett, WA, USA). 9 Page 10 of 53
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2.5. Analytical Methods
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2.5.1. Ingredient and total mixed ration chemical analysis All wet ingredients and TMR samples were dried at 55oC for 48 h and air equilibrated for 24
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h. Dried samples, with other ingredients which did not require drying, were analyzed at the UC
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Davis Analytical Laboratory (Davis, CA, USA). All samples were ground to pass a 0.4 mm
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screen on an Intermediate Wiley Mill or a 1 mm screen on a model 4 Wiley Mill (Thomas
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Scientific, Swedesboro, NJ, USA). Moisture was determined by gravimetric loss of free water by
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heating to 105°C in a forced air oven for 3 h, and ash was the gravimetric residue after heating to
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550°C for at least 3 h. Total N and N in ADF were determined with infrared detection and
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thermal conductivity (TruSpec CN Analyzers, St. Joseph, MI, USA) by AOAC (2006; #990.03).
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Analysis of aNDF used a heat stable amylase (AOAC, 2006; #2002-04), and aNDF and ADF are
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expressed exclusive of residual ash (i.e., aNDFom, ADFom) or with residual ash (i.e., aNDF).
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ADFom analysis was by boiling in acid detergent for 1 h (AOAC, 1997; #973.18).
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Crude fat, as ether extract (EE), was determined by extraction in ethyl ether (AOAC,
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2006; #2003.05). Free sugars are the sum of glucose, fructose and sucrose as determined by
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HPLC (Johansen et al., 1996). Enzymatic hydrolysis was used to determine the amount of total
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glucose, and free glucose was subtracted from total glucose, and the difference multiplied by 0.9
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to calculate starch (Smith, 1969). Lignin(sa) was determined by the sulfuric acid procedure
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(AOAC, 1997; #973.18). The Ca, Cu, Fe, Mg, Mn, P, K, Na, S and Zn contents were determined
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by microwave nitric acid/hydrogen peroxide digestion/dissolution by inductively coupled plasma
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atomic emission spectrometry (Meyer and Keliher, 1992, Sah and Miller, 1992). The Cl content
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was determined after water extraction and analysis by ion chromatography with conductivity
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detection (Jones, 2001). Total Se was extracted by nitric/perchloric acid digestion/dissolution
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and determined by vapor generation using ICP-AES (Tracy and Mueller, 1990).
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2.5.2. Fecal Fecal samples from the 18 cows/pen which had been sampled in each experimental period
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were divided into 3 subgroups according to ear tag number order. Samples were individually
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dried at 55oC for 48 h and then 150 g of each sample within a subgroup was pooled to create 3
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fecal sample groups/pen/period. The pooled samples were analyzed by the UC Davis Analytical
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Laboratory as described for the feed samples, as appropriate.
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2.5.3. Milk
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Fat, true protein and lactose, as well as SCC, were determined using infrared spectroscopy at the Dairy Herd Improvement Association laboratory in Hanford (CA, USA).
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2.6. Calculations
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2.6.1. Dry matter intake
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Intake of TMR for each pen was calculated during the collection week (i.e., day 22 through
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day 28, inclusive) of each period based on the total weight of TMR delivered to each pen for that
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period corrected for the orts which were removed prior to the first feeding of each day. The
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analyzed 105oC DM content of the TMR was used to determine DM intake. The number of
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cows/pen during the collection period was the average of the number of cows in the pen on the
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electronic record system (Dairy Comp 305, Valley Ag Systems, Tulare, CA, USA) on the first
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and last day of the final week of each period.
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2.6.2. Energy calculations
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Milk energy (MJ/kg) was calculated for individual cows according to Tyrrell and Reid
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(1965) using milk fat, crude protein and lactose, and the energy (MJ/d) in BCS change was
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calculated by cow according to NRC (2001) as 1255.2 MJ/unit BCS. Maintenance energy
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(MJ/d) was calculated as: (BW0.75*0.08*4.184) according to NRC (2001) for all groups assuming
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an average body weight of 650 kg for all cows. Total energy output was calculated as the sum of 11 Page 12 of 53
milk energy (MJ/d), BCS change energy (MJ/d) and maintenance energy (MJ/d). The NEL
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density of the TMR were calculated by pen and period as: Total Energy Output (MJ/d) / DM
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intake (kg/d).
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2.6.3. Somatic cell count and linear conversion
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Because distribution of SCC in milk has a positive skewness with heterogeneous variance
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(Ali and Shook, 1980), log transformation (Schukken et al., 2003) was used to normalize the data
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to create a linear SCC as: LnS = log2(SCC/100)+3, where: SCC is expressed as cells/µL.
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2.6.4. Whole tract digestibility
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Apparent digestibility of dietary components was calculated by the proportion of feed
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components remaining in the feces from the diet, using lignin(sa) as the indigestible marker but
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assuming 0.95 fecal recovery of lignin(sa) (Stensig and Robinson, 1997).
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2.7. Statistical Analysis
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The chemical composition of the TMR fed was statistically analyzed using the GLM
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procedure of SAS (2012) with pen, period and treatment as effects. The two TMR samples
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collected from each pen at the start and end of each collection week (i.e., day 21 and 27 of each
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period) were combined prior to chemical analysis to be representative of the final week of the
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collection period. The DM intake data was analyzed using the GLM option of SAS (2012) with
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pen, period and treatment as fixed effects.
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The eligibility criteria for cows to be included in the statistical analysis was that they had to
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have been in her ≤6 lactation, and have remained in the same pen for the entire study (n=670).
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However, cows with milk or milk component values determined visually to be biological outliers
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were excluded from statistical analysis. Removal selection was completed blind to treatment and
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pen. A total of 16 cows were so removed, leaving 654 cows which met the criteria for inclusion
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and were included in the data set for statistical analysis. Milk yield and components, as well as 12 Page 13 of 53
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LS, were analyzed using the MIXED option of SAS with cow nested within pens as a random
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effect. The statistical model included fixed effects of pen, period and treatment. Data for cows included in the statistical analysis for BCS, which were a subset of the cows
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used for milk production data, had to meet all criteria required for milk analysis, as well as
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having been scored for BCS at the start of the study and at the end of both experimental periods.
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The BCS (n=168) data was analyzed with the MIXED model of SAS with cows, pen, period and
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treatment, as effects, as described above for analysis of milk parameters.
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Statistical inclusion criteria of cows for digestibility of feed components were the same as the
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milk criteria for each cow, and were a subset of these cows. Digestibility was analyzed with the
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MIXED model of SAS with fecal group, pen, period and treatment as effects, as described above
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for milk parameter statistical analysis. Fecal group was a random effect in this model.
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Treatment differences were accepted if P≤0.05, and tendencies to significance were accepted
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3. Results
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3.1. Environmental conditions
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Diurnal patterns of ambient temperature and THI were similar between experimental periods
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(Figure 1). While the daily temperature highs were similar to historical averages, the nighttime
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lows were slightly cooler than normal. During the study period, highs were 36.5°C and 35.6°C
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with lows of 16.9°C and 15.8°C, for period 1 (July) and 2 (August), respectively.
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3.2. Chemical composition of feeds and total mixed ration
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The chemical composition of feeds in the TMR (Table 1) were generally consistent with
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NRC (2001) values, and t. The chemical profile of the TMR (Table 12) met or exceeded
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recommendations of the NRC (2001) for dairy cattle at similar production levels. There were no 13 Page 14 of 53
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differences between the chemical profile of the TMR fed to the Control and CED groups, except
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for a slightly higher level of Ca (P=0.02) of the CED diet.
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3.3. Feed intake, milk production and body condition score DM intake was not affected by feeding CE (Table 23), and milk and milk component yields
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also did not differ between groups. Milk SCC (P=0.04) and linear score of SCC (P<0.01) were
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lower for cows fed CE. The BCS and LS, as well change of BCS, did not differ between groups.
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3.4. Heat stress parameters, body temperatures and locomotion score
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The RR, PS and RT (Table 34) did not differ between treatment groups at any observation
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time. The proportion of cows lying versus standing was higher (P<0.01) for cows fed the CED
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(68.6 versus 53.7 cows/100 cows) at 02:45 h, but did not differ at other times.
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3.5. Whole tract apparent digestibility
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ranges for cows with high DM intake (Colucci et al., 1982; NRC, 2001).
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Feeding the CE did not impact whole tract digestibility (Table 45), with values within normal
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4. Discussion
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4.1. Effect of citrus extract on heat stress, body temperature and locomotion
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Cows in the study were judged to have experienced mild heat stress because, even though
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daytime high THI levels were often in the “alert” or “danger” levels, THI values of <74 (i.e.,
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normal) occurred for ~10 h/d. This judgement is consistent with the measured RR of 58.7 to
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64.0 breaths/min, which contrasts to RR of cattle of 100 breaths/min, or more, under extreme
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heat stress (Turner, 1992). Likewise, the low PS of 0.89 to 1.27 of cows in both treatment groups
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suggests that they were not substantively heat stressed, and rump temperatures were also within
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the range of mildly heat stressed cows (Di Costanzo et al., 1997). This low level of observed
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heat stress is probably because there was consistent night-time cooling, and because the cows 14 Page 15 of 53
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were fully shaded from the sun with access to bunk line misters (West, 2003; Gaughan et al.,
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2008). It is clear that feeding the CE had little or no effect on heat stress of our cows since RR, PS
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and RT were not influenced. That the RR of both groups was lower at 17:30 than at 2:45 h
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differed from expectations due to temperatures at those times of day (Di Costanzo et al., 1997).
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In contrast, the PS pattern for both groups was highest at 17:30 h, the hottest time of the day, and
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the lowest at 09:15 h probably due to cows having recovered during the cooler night. At 2:45 h
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little TMR remained in the feed bunks and very few cows were eating, with the majority in the
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open drylot where more CED cows were lying versus standing. As Hillman et al. (2005) found
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that the body temperature of cows rises when they are lying, and that they tend to stand and seek
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cooling when core body temperature rises above 38.9°C, the higher number of CED cows lying
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at 02:45 h suggests that CE may have reduced core body temperatures and heat stress, at least at
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this time.
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4.2. Production responses to citrus extracts
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Lack of a treatment difference on DM intake was not unexpected because studies with
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limonene fed in concert with other EO have shown no DM intake effect (Benchaar et al., 2006,
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2007), or a slight trend to a reduction (Santos et al., 2010). Vitamin C supplementation also does
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not appear to effect DM intake (Weiss, 2001; Chaiyotwittayakun et al., 2002; Weiss et al., 2004).
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While there are no in vivo studies that have only fed limonene, there is little support from in
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vitro studies that limonene affects microbial fermentation, feed efficiency or nutrient utilization
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to support milk production (Dorman and Deans, 2000). This is consistent with our similar
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dietary NEL density (7.18 versus 7.08 MJ/kg) of the Control and CED TMR (Table 45). In a
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review of citrus by-product feeding, Bampidis and Robinson (2006) reported that adding citrus
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by-products to diets of lactating dairy cows did not change milk yield or composition if the diets
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had a similar nutrient profile, which is also consistent with our finding that there were no
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production differences between the Control and CED groups.
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4.3. Milk somatic cell count (SCC) response to citrus extracts Based on our SCC differences between treatments, the CED diet appears to have reduced the
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incidence and/or extent of mastitis. Indeed Lund et al. (1994) found a high genetic correlation
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between SCC and clinical mastitis (0.97), concluding that SCC is an indicator of clinical
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mastitis. In addition, increased SCC has been shown to be positively correlated with increased
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mastitis risk (Ward and Schultz, 1972; Kirk, 1984; Barkema et al., 1998), and Harmon (1994)
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found that, in cows with higher SCC, lactose tends to leak out of the infected mammary gland
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into the blood, resulting in less milk lactose, which is consistent with the slightly lower lactose
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output of Control cows, which also had higher SCC.
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4.3.1. Effects of feeding citrus pulp or limonene in an EO blend on milk SCC
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When lactating ewes were fed whole citrus pulp at 100, 200 and 300 g/kg DM of their ration,
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Jaramillo et al. (2009) reported lower milk SCC. Giannenas et al. (2011) fed an EO blend, which
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included limonene, to dairy ewes at three levels. At day 50, there was no change in SCC but, at
352
day 100, ewes fed the highest EO treatment had lower SCC and, at day 150, all EO treatments
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had a lower SCC, with ewes fed the highest EO level having a lower SCC than the other ewes.
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This suggests a dose-dependent effect to the EO blend containing limonene which is magnified
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with longer feeding time.
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4.3.2. Effects of feeding vitamin C on milk SCC
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In contrast to EO, there has been substantial amount of research on impacts of dietary
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Vitamin C supplementation on udder health, especially mastitis. When pathogenic
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microorganisms enter the mammary gland through the streak canal an inflammatory response is
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triggered in the infected gland, thereby increasing the number of reactive oxygen species (Ranjan 16 Page 17 of 53
et al, 2005). Since two lines of defense are occurring at this time (i.e., sequestering of
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antioxidants and recruiting of leukocytes (Ranjan et al., 2005; Harmon, 1994)), antioxidants such
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as vitamin C can be utilized in high quantities to inactivate reactive oxygen species to decrease
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their concentrations in plasma and the infected mammary quarter (Harmon, 1994). Early studies
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showed that vitamin C is quickly degraded in vitro and in vivo (Knight et al., 1941; Vavich et al.,
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1945), presumably by rumen microbes. However Hidiroglou (1999) found that cows fed vitamin
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C or ethyl cellulose coated vitamin C (i.e., ruminally protected), or had vitamin C infused
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abomasally, all had elevated plasma amino acid (AA) concentrations, but cows fed uncoated
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vitamin C had the smallest increase. This supports the probability that not all dietary vitamin C
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is degraded in the rumen. Weiss et al. (2004) and Ranjan et al. (2005) showed that infected
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mammary glands increase vitamin C utilization, and Weiss et al. (2004) also showed that
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mammary quarters challenged with E. coli had decreased levels of vitamin C in milk and plasma
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due to an inflammatory response which increased AA oxidation. This suggests that depletion of
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vitamin C in plasma and milk occurs during infection, rather than a low vitamin C status per se,
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which allows a more severe infection to occur.
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Although cattle are thought to synthesize adequate vitamin C to maintain healthy mammary
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gland function, vitamin C synthesis is not up-regulated when cows are under immunological
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stress (Weiss et al., 2004), but vitamin C uptake and oxidation is increased in effected tissues.
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Thus it is possible that in a generally healthy mammary gland, such as was the case of cows in
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our study based upon relatively low overall milk SCC counts, supplemental vitamin C may have
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helped maintain a high antioxidant/pro-oxidant ratio, thereby aiding in prevention of oxidative
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damage.
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5. Conclusions 17 Page 18 of 53
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Supplementing the diet of dairy cows with a commercially available citrus extract decreased
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SCC in generally healthy dairy cows under mild heat stress conditions. However measures of
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heat stress and animal production were not substantively treatment impacted.
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388 Acknowledgements
The authors thank all who volunteered to help: Grace Cun, Stacy Wrinkle, Nadia Swanepoel,
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Blanca Comacho and Emma Robinson. We are grateful to Stacy Wrinkle and Pablo Chilibroste
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for sharing their knowledge on heat stress behaviors, and to William Van Die for allowing us to
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conduct the study on his dairy.
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394 References
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*Conflict of Interest Statement
Editors, The authors stipulate that they have no conflicts of interest in preparation or submission of this manuscript.
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P.H. Robinson for the authors
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*Manuscript (final version, no corrections or changes highlighted) Click here to download Manuscript (final version, no corrections or changes highlighted): AFSTcev3unmarked.docx Click here to view linked References
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4 Intake, milk production and heat stress of dairy cows
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fed a citrus extract during summer heat
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J.M. Havlina, P.H. Robinsona,*
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University of California, Davis, CA, 95616, USA
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Department of Animal Science
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*Corresponding author: Tel: 530-754-7565; Fax: 530-752-0175 EM: [email protected]
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Submitted to Animal Feed Science and Technology in April 2015
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Revised and resubmitted in May 2015.
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Revised and resubmitted in June 2015.
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Abstract This study determined effects of feeding a citrus extract (CE) to high producing dairy cows
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during summer heat on measures of heat stress, as well as milk production and composition, in a
27
replicated 2 x 2 Latin square experiment with two 28 d periods on a dairy farm near Hanford
28
(CA, USA). Four „high group‟ pens were used (i.e., cows which had cleared the fresh pen but
29
were not yet confirmed pregnant), each with ~310 early lactation multiparity cows/pen. The two
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total mixed rations contained 171 g/kg dry matter (DM) crude protein (CP), 55 g/kg fat, 335 g/kg
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neutral detergent fiber (aNDF) and 135 g/kg starch, and were the same except for inclusion of
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the CE at 4 g/cow/d in the treatment diet (CED). Average daily high temperatures during the
33
study were 35 to 37oC with lows of 16 to 17oC. In general, cows showed mild heat stress, but
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CE feeding had no effect on respiration rate, panting score or rump temperature at any time of
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the day (i.e., 02:45, 09:15, 17:30 h). However at 02:45 h, a higher (P<0.01) proportion of CED
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cows were lying (versus standing) compared with Control cows (68.6 versus 53.7 cows/100
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cows), which is an indicator of reduced heat stress. Intake of DM (25.3 kg/d) and whole tract
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digestibility of CP (703 g/kg) and aNDF (510 g/kg) did not differ between treatments. Milk
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production (47.3 kg/d) and its fat and true protein levels (35.4, 28.6 g/kg) did not differ, and
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changes in body condition and locomotion scores were also not impacted by treatment. However
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mammary health improved based on lower SCC (somatic cell counts; P<0.04) of CED versus
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Control cows (160,000 versus 196,000 cells/µL), and lower linear SCC scores (P<0.01; 2.12
43
versus 2.30). Feeding this CE to lactating dairy cows during summer heat decreased SCC with
44
no impact on other aspects of performance.
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Keywords: limonene, vitamin C, citrus, essential oils
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Abbreviations: AA, amino acids; ADF, acid detergent fiber expressed with residual ash;
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ADFom, ADF expressed without residual ash; aNDF, neutral detergent fiber assayed with a heat
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stable amylase and expressed with residual ash; BCS, body condition score; CE, citrus extract;
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CED, CE diet; CP, crude protein; DM, dry matter; EE, ether extract; EO; essential oils; LS,
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locomotion score; PS, panting score; RR, respiration rate; RT, rump temperature; SCC, somatic
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cell count; THI, temperature/humidity index; TMR, total mixed ration
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52 1. Introduction
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A popular contemporary topic in ruminant nutrition is the study of naturally occurring dietary
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additives which modulate rumen fermentation in order to improve nutrient utilization by rumen
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microorganisms, and/or reduce enteric production of greenhouse gases such as methane.
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Essential oils (EO) encompass a wide range of naturally occurring secondary compounds which
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occur in leaves, flowers, stems and seeds of many plants, and may have antimicrobial properties
59
which modify rumen microbial fermentation.
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Reviews of various EO compounds, their possible modes of action and effects on rumen
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fermentation have been described (e.g., Benchaar et al., 2008), and limonene, an EO found in
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citrus products, has been suggested to have beneficial effects on rumen fermentation, and animal
63
production, in ruminants (Calsamiglia et al., 2007). While few in vivo studies have examined
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EO mixtures containing limonene, Tassoul and Shaver (2009) reported increased milk protein
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production, with an overall trend to increased feed efficiency (i.e., kg of fat corrected milk/kg of
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DM intake), when an EO mixture including limonene was fed to dairy cows. In contrast,
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Benchaar et al. (2006, 2007) had earlier reported that the same EO mixture used by Tassoul and
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Shaver (2009) had no effect on milk production, dry matter (DM) intake or feed efficiency.
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Vitamin C, found at high levels in citrus products, is not considered an essential nutrient for
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healthy cattle because their liver can synthesize it from glucose at levels believed to be sufficient
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to meet their needs (Padh, 1990). However cattle under stress, such as those in early lactation at 3 Page 28 of 53
high production levels and/or under summer heat, may not synthesize adequate amounts of
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vitamin C to compensate for oxidative stress due to sub-clinical infection or illness (Weiss et al.,
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2004; Ranjan et al., 2005), and feeding supplemental vitamin C can replace depleted reserves
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(Weiss, 2001). As high somatic cell counts (SCC) in milk are the result of degraded mammary
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health, and have a negative impact on animal performance and milk quality, the limonene and
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vitamin C in citrus products may help minimize SCC in dairy cows, thereby improving
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efficiency of feed utilization as a result of improved immune function (Chaiyotwittayakun et al.,
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2002; Castillejos et al., 2006). Indeed Jaramillo et al., (2009) and Giannenas et al. (2011) fed
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citrus pulp to ewes and reduced milk SCC.
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In a review of citrus product feeding to ruminants, Bampidis and Robinson (2006) concluded
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that their feeding did not affect DM digestibility, but decreased crude protein (CP) digestibility,
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while increasing neutral detergent fiber (NDF) and acid detergent fiber (ADF) digestibility.
dairy cows during hot weather.
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Our objective was to evaluate impacts of a CE on feed intake and productive performance of
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2. Materials and methods
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2.1. Animals, management, and experimental design
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High group multiparity Holstein cows (i.e., those cows which had cleared the fresh pen but
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were not yet confirmed pregnant) in 4 early lactation pens of ~310 cows each, on a commercial
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dairy farm near Hanford (CA, USA), were used in a study with two 28 d experimental periods in
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a replicated 2 x 2 crossover design during summer. Each pen had 300 head gates and free stalls.
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The only source of cooling was bunk line misters which switched on automatically at 24°C, and
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were on in each pen for 4 mins and then off for 12. Cows were assigned weekly to one of the 4
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pens at random from a common fresh cow pen, and moved to a mid pen by ~200 days in milk
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once they were confirmed pregnant. Cows were milked three times daily in a double 40 herringbone parlor. Pen 1 started milking
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at 04:00, 12:00 and 20:00 h. Pens 2, 3, and 4 were milked in sequence after pen 1, at intervals of
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~45 min. The total mixed ration (TMR) was fed twice daily, between 04:00 and 08:00 h, prior to
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cows returning to their pens from milking, and again between 11:30 and 12:30 h. The amount of
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TMR delivered was determined from the previous days intake to create orts equal to ~10 g/kg of
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total TMR delivered (as fed). Orts were removed daily and weighed individually by pen while
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cows were in the parlor during the first milking, just prior to the first TMR feeding.
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Head-locks were set prior to cows returned from the morning milking, to facilitate artificial
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insemination, such that the cows were head locked in the stanchions for 45 to 60 min daily.
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Cows were housed in covered barns with access to free stalls bedded with dried manure, which
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was renewed weekly and groomed bi-weekly. Cows also had free access to an uncovered manure
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pack drylot at all times. Inside alleyways were flushed with water three times daily while the
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cows were being milked. Cows had ad libitum access to clean drinking water at all times.
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At the start of the study, two pens were fed the CE containing diet (CED), while the two
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other pens were fed the Control diet. After the first 28 d period, treatments were reversed. Each
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period contained a 21 d adjustment and 7 d collection period during which all samples were
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collected.
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2.2. Environmental conditions
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Four portable weather data loggers (HOBO U23; Onset, Bourne, MA, USA) were recorded
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ambient temperatures and relative humidity every 30 min throughout the study. One station was
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placed in each experimental pen on a pole at its center ~3 m above the floor and out of direct
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sunlight. The study took place in the 2 mo after the summer solstice to minimize variation of day 5 Page 30 of 53
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length and weather, while maximizing expected temperature/humidity indices (THI) which was
120
calculated as:
121
THI = tdb – [0.55-(.55*RH/100)]*(tdb – 58.8) where: tdb = dry bulb air temperature (°F) and RH = relative humidity (%). THI ≤ 74 is
123
considered “normal”, 75 to 78 is “alert”, 79 to 83 is “danger”, and ≥ 84 is “emergency”
124
according to the Livestock Weather Safety Index (LCI, 1970).
125
2.3. Diets
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The TMR for the CED and Control groups was formulated identically except for inclusion of
127
the CE at 4 g/cow/d in the CED TMR. Rate of inclusion of the CE product (VéO Premium;
128
Cristalfeed® Gold Rush; Reference ST 232 P2; Phodé, Terssac, France) in TMR loads was
129
calculated weekly based on the number of cows in the pen, and used to create bags of CE which
130
the feeding staff added to the CED TMR. The CE contained natural citrus extracts, natural and
131
artificial flavoring as well as corn flour, calcium carbonate and silicic acid as flow enhancers.
132
2.4. Sample collection
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2.4.1. Feed and total mixed rations
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At the start and end of the collection week of each period (i.e., days 21 and 27) all dietary
135
ingredients were sampled. Hays were sampled with a „golf club‟ style hay probe (Sierra Testing
136
Services, Acampo, CA, USA), with 12 core samples pooled into a plastic bag. All other
137
ingredients, with the exception of liquid whey, were sampled by hand (6 handfuls of each) and
138
composited to plastic bags. Silages were sampled from the loose pile which had been knocked
139
off the silage face for use that day and wet by-products (i.e., fresh chop alfalfa, tomato pomace)
140
were sampled from the storage area. Samples of all other feeds from the start and end of the
141
collection weeks were combined to create one sample per ingredient prior for chemical analysis.
6 Page 31 of 53
Ten handfuls (~200 g each) of each TMR by pen were collected according to Robinson and
143
Meyer (2010) at pre-marked posts with evenly spaced intervals along the bunk-line immediately
144
after feeding and before the cows had access to it. These TMR samples were pooled within
145
period and pen to create 8 TMR samples for chemical analysis.
146
2.4.2. Milk
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At the end of each collection week (i.e., day 28), the entire herd was tested for milk
148
production and composition by Dairy Herd Improvement Association (Kings County, CA, USA)
149
personnel. Milk weights and representative milk samples collected into tubes containing
150
bronopol and natamycin as preservatives were collected from all cows using Tru-test milk meters
151
(Tru-Test Ltd., Auckland, New Zealand).
152
2.4.3. Body condition and locomotion scores
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Body condition (BCS) was scored at the start of the study (i.e., day 0) and at the end (i.e., day
154
28) of each experimental period while the cows were locked in stanchions immediately after
155
morning milking. From each pen, 80 cows having the lowest days in milk at the start of the study
156
were selected. Cows were scored on the 1 to 5 scale of Edmonson et al. (1989), with intermediate
157
values between the ¼ points if a ¼ point score was not clear. One person scored all cows.
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Locomotion was scored (LS) at the end (i.e., day 28) of each experimental period while the
159
cows were returning from the parlor from milking starting at 14:00 h. All cows in the pen were
160
observed for lameness using the 1 to 5 scoring system of Sprecher et al. (1997). One person
161
scored all cows on both occasions.
162
2.4.4. Fecal
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Fecal samples were collected from the same 18 cows during the morning lock-up on day 28
164
of each period. Feces were manually collected (minimum 250 g) from the rectum of the cow
165
into plastic containers, and immediately stored at -18°C until processing for chemical analysis. 7 Page 32 of 53
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2.4.5. Heat stress behavior and skin temperature Respiration rate (RR), panting score (PS), lying versus standing behavior, and rump
168
temperature (RT) was measured three times over a 24 h period on days 27 and 28 of each period.
169
Skin temperature was recorded on days 26 and 28 during morning lock-up during both periods,
170
as they had not yet had access to the dry lot where they would be in direct sunlight at this time. A
171
subgroup of 50 cows which had been scored for BCS from each pen was observed for heat stress
172
parameters. Identification collars were placed on the cows during morning lock-up to facilitate
173
easier identification of cows from a distance during observations. Behavioral observations started
174
~24 h after the collars were attached. The behavior observation periods for pens 1 and 2 began at
175
08:00, 16:15 and 01:30 h, while observations for pens 3 and 4 began directly following
176
observations of pens 1 and 2. Observation periods lasted 75 mins/pen. The 08:00 h observation
177
time was chosen to ensure that the cows had been out of head-lock for at least 15 min prior to
178
observations. The 16:15 h time was chosen to approximate the hottest time of the day and, during
179
the 01:30 h observation period, the sun had been down for ~5.5 h and the temperature was
180
approaching the daily low. At this time the vast majority of the cows were in the outside dry lot.
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181
The RR was determined by observing flank movements of the cows where the time it took a
182
cow to take 20 breaths was recorded and converted to breaths/min. A 0 to 5 PS was assigned to
183
each cow (Gaughan et al., 2008) based on physical signs including degree of chest movement
184
and presence of drool. Cows in two pens were simultaneously observed for heat stress behaviors
185
by two individual scorers. Each scorer observed the same treatment and Control pen in each
186
period. Scorers trained together prior to the study, and between periods, to eliminate scorer bias.
187
To obtain RT, two small spots of ~40 x 60 mm each were shaved on each side of the rear
188
flank, distal to where the udder meets the leg. Temperatures from the right and left flank were
189
measured using an infrared gun (Fluke-561, Everett, WA, USA). 8 Page 33 of 53
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2.5. Analytical Methods
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2.5.1. Ingredient and total mixed ration chemical analysis All wet ingredients and TMR samples were dried at 55oC for 48 h and air equilibrated for 24
193
h. Dried samples, with other ingredients which did not require drying, were analyzed at the UC
194
Davis Analytical Laboratory (Davis, CA, USA). All samples were ground to pass a 0.4 mm
195
screen on an Intermediate Wiley Mill or a 1 mm screen on a model 4 Wiley Mill (Thomas
196
Scientific, Swedesboro, NJ, USA). Moisture was determined by gravimetric loss of free water by
197
heating to 105°C in a forced air oven for 3 h, and ash was the gravimetric residue after heating to
198
550°C for at least 3 h. Total N and N in ADF were determined with infrared detection and
199
thermal conductivity (TruSpec CN Analyzers, St. Joseph, MI, USA) by AOAC (2006; #990.03).
200
Analysis of aNDF used a heat stable amylase (AOAC, 2006; #2002-04), and aNDF and ADF are
201
expressed exclusive of residual ash (i.e., aNDFom, ADFom) or with residual ash (i.e., aNDF).
202
ADFom analysis was by boiling in acid detergent for 1 h (AOAC, 1997; #973.18).
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Crude fat, as ether extract (EE), was determined by extraction in ethyl ether (AOAC,
204
2006; #2003.05). Free sugars are the sum of glucose, fructose and sucrose as determined by
205
HPLC (Johansen et al., 1996). Enzymatic hydrolysis was used to determine the amount of total
206
glucose, and free glucose was subtracted from total glucose, and the difference multiplied by 0.9
207
to calculate starch (Smith, 1969). Lignin(sa) was determined by the sulfuric acid procedure
208
(AOAC, 1997; #973.18). The Ca, Cu, Fe, Mg, Mn, P, K, Na, S and Zn contents were determined
209
by microwave nitric acid/hydrogen peroxide digestion/dissolution by inductively coupled plasma
210
atomic emission spectrometry (Meyer and Keliher, 1992, Sah and Miller, 1992). The Cl content
211
was determined after water extraction and analysis by ion chromatography with conductivity
212
detection (Jones, 2001). Total Se was extracted by nitric/perchloric acid digestion/dissolution
213
and determined by vapor generation using ICP-AES (Tracy and Mueller, 1990).
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2.5.2. Fecal Fecal samples from the 18 cows/pen which had been sampled in each experimental period
216
were divided into 3 subgroups according to ear tag number order. Samples were individually
217
dried at 55oC for 48 h and then 150 g of each sample within a subgroup was pooled to create 3
218
fecal sample groups/pen/period. The pooled samples were analyzed by the UC Davis Analytical
219
Laboratory as described for the feed samples, as appropriate.
220
2.5.3. Milk
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Fat, true protein and lactose, as well as SCC, were determined using infrared spectroscopy at the Dairy Herd Improvement Association laboratory in Hanford (CA, USA).
223
2.6. Calculations
224
2.6.1. Dry matter intake
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Intake of TMR for each pen was calculated during the collection week (i.e., day 22 through
226
day 28, inclusive) of each period based on the total weight of TMR delivered to each pen for that
227
period corrected for the orts which were removed prior to the first feeding of each day. The
228
analyzed 105oC DM content of the TMR was used to determine DM intake. The number of
229
cows/pen during the collection period was the average of the number of cows in the pen on the
230
electronic record system (Dairy Comp 305, Valley Ag Systems, Tulare, CA, USA) on the first
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and last day of the final week of each period.
232
2.6.2. Energy calculations
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Milk energy (MJ/kg) was calculated for individual cows according to Tyrrell and Reid
234
(1965) using milk fat, crude protein and lactose, and the energy (MJ/d) in BCS change was
235
calculated by cow according to NRC (2001) as 1255.2 MJ/unit BCS. Maintenance energy
236
(MJ/d) was calculated as: (BW0.75*0.08*4.184) according to NRC (2001) for all groups assuming
237
an average body weight of 650 kg for all cows. Total energy output was calculated as the sum of 10 Page 35 of 53
milk energy (MJ/d), BCS change energy (MJ/d) and maintenance energy (MJ/d). The NEL
239
density of the TMR were calculated by pen and period as: Total Energy Output (MJ/d) / DM
240
intake (kg/d).
241
2.6.3. Somatic cell count and linear conversion
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Because distribution of SCC in milk has a positive skewness with heterogeneous variance
243
(Ali and Shook, 1980), log transformation (Schukken et al., 2003) was used to normalize the data
244
to create a linear SCC as: LnS = log2(SCC/100)+3, where: SCC is expressed as cells/µL.
245
2.6.4. Whole tract digestibility
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Apparent digestibility of dietary components was calculated by the proportion of feed
247
components remaining in the feces from the diet, using lignin(sa) as the indigestible marker but
248
assuming 0.95 fecal recovery of lignin(sa) (Stensig and Robinson, 1997).
249
2.7. Statistical Analysis
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The chemical composition of the TMR fed was statistically analyzed using the GLM
251
procedure of SAS (2012) with pen, period and treatment as effects. The two TMR samples
252
collected from each pen at the start and end of each collection week (i.e., day 21 and 27 of each
253
period) were combined prior to chemical analysis to be representative of the final week of the
254
collection period. The DM intake data was analyzed using the GLM option of SAS (2012) with
255
pen, period and treatment as fixed effects.
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256
The eligibility criteria for cows to be included in the statistical analysis was that they had to
257
have been in her ≤6 lactation, and have remained in the same pen for the entire study (n=670).
258
However, cows with milk or milk component values determined visually to be biological outliers
259
were excluded from statistical analysis. Removal selection was completed blind to treatment and
260
pen. A total of 16 cows were so removed, leaving 654 cows which met the criteria for inclusion
261
and were included in the data set for statistical analysis. Milk yield and components, as well as 11 Page 36 of 53
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LS, were analyzed using the MIXED option of SAS with cow nested within pens as a random
263
effect. The statistical model included fixed effects of pen, period and treatment. Data for cows included in the statistical analysis for BCS, which were a subset of the cows
265
used for milk production data, had to meet all criteria required for milk analysis, as well as
266
having been scored for BCS at the start of the study and at the end of both experimental periods.
267
The BCS (n=168) data was analyzed with the MIXED model of SAS with cows, pen, period and
268
treatment, as effects, as described above for analysis of milk parameters.
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Statistical inclusion criteria of cows for digestibility of feed components were the same as the
270
milk criteria for each cow, and were a subset of these cows. Digestibility was analyzed with the
271
MIXED model of SAS with fecal group, pen, period and treatment as effects, as described above
272
for milk parameter statistical analysis. Fecal group was a random effect in this model.
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Treatment differences were accepted if P≤0.05, and tendencies to significance were accepted
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3. Results
277
3.1. Environmental conditions
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Diurnal patterns of ambient temperature and THI were similar between experimental periods
279
(Figure 1). While the daily temperature highs were similar to historical averages, the nighttime
280
lows were slightly cooler than normal. During the study period, highs were 36.5°C and 35.6°C
281
with lows of 16.9°C and 15.8°C, for period 1 (July) and 2 (August), respectively.
282
3.2. Chemical composition of feeds and total mixed ration
283
The chemical composition of feeds in the TMR were generally consistent with NRC (2001)
284
values, and the chemical profile of the TMR (Table 1) met or exceeded recommendations of the
285
NRC (2001) for dairy cattle at similar production levels. There were no differences between the 12 Page 37 of 53
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chemical profile of the TMR fed to the Control and CED groups, except for a slightly higher
287
level of Ca (P=0.02) of the CED diet.
288
3.3. Feed intake, milk production and body condition score DM intake was not affected by feeding CE (Table 2), and milk and milk component yields
290
also did not differ between groups. Milk SCC (P=0.04) and linear score of SCC (P<0.01) were
291
lower for cows fed CE. The BCS and LS, as well change of BCS, did not differ between groups.
292
3.4. Heat stress parameters, body temperatures and locomotion score
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The RR, PS and RT (Table 3) did not differ between treatment groups at any observation
294
time. The proportion of cows lying versus standing was higher (P<0.01) for cows fed the CED
295
(68.6 versus 53.7 cows/100 cows) at 02:45 h, but did not differ at other times.
296
3.5. Whole tract apparent digestibility
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ranges for cows with high DM intake (Colucci et al., 1982; NRC, 2001).
d
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Feeding the CE did not impact whole tract digestibility (Table 4), with values within normal
299
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297
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4. Discussion
301
4.1. Effect of citrus extract on heat stress, body temperature and locomotion
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302
Cows in the study were judged to have experienced mild heat stress because, even though
303
daytime high THI levels were often in the “alert” or “danger” levels, THI values of <74 (i.e.,
304
normal) occurred for ~10 h/d. This judgement is consistent with the measured RR of 58.7 to
305
64.0 breaths/min, which contrasts to RR of cattle of 100 breaths/min, or more, under extreme
306
heat stress (Turner, 1992). Likewise, the low PS of 0.89 to 1.27 of cows in both treatment groups
307
suggests that they were not substantively heat stressed, and rump temperatures were also within
308
the range of mildly heat stressed cows (Di Costanzo et al., 1997). This low level of observed
309
heat stress is probably because there was consistent night-time cooling, and because the cows 13 Page 38 of 53
310
were fully shaded from the sun with access to bunk line misters (West, 2003; Gaughan et al.,
311
2008). It is clear that feeding the CE had little or no effect on heat stress of our cows since RR, PS
313
and RT were not influenced. That the RR of both groups was lower at 17:30 than at 2:45 h
314
differed from expectations due to temperatures at those times of day (Di Costanzo et al., 1997).
315
In contrast, the PS pattern for both groups was highest at 17:30 h, the hottest time of the day, and
316
the lowest at 09:15 h probably due to cows having recovered during the cooler night. At 2:45 h
317
little TMR remained in the feed bunks and very few cows were eating, with the majority in the
318
open drylot where more CED cows were lying versus standing. As Hillman et al. (2005) found
319
that the body temperature of cows rises when they are lying, and that they tend to stand and seek
320
cooling when core body temperature rises above 38.9°C, the higher number of CED cows lying
321
at 02:45 h suggests that CE may have reduced core body temperatures and heat stress, at least at
322
this time.
323
4.2. Production responses to citrus extracts
te
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312
Lack of a treatment difference on DM intake was not unexpected because studies with
325
limonene fed in concert with other EO have shown no DM intake effect (Benchaar et al., 2006,
326
2007), or a slight trend to a reduction (Santos et al., 2010). Vitamin C supplementation also does
327
not appear to effect DM intake (Weiss, 2001; Chaiyotwittayakun et al., 2002; Weiss et al., 2004).
328
While there are no in vivo studies that have only fed limonene, there is little support from in
329
vitro studies that limonene affects microbial fermentation, feed efficiency or nutrient utilization
330
to support milk production (Dorman and Deans, 2000). This is consistent with our similar
331
dietary NEL density (7.18 versus 7.08 MJ/kg) of the Control and CED TMR (Table 4). In a
332
review of citrus by-product feeding, Bampidis and Robinson (2006) reported that adding citrus
333
by-products to diets of lactating dairy cows did not change milk yield or composition if the diets
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14 Page 39 of 53
334
had a similar nutrient profile, which is also consistent with our finding that there were no
335
production differences between the Control and CED groups.
336
4.3. Milk somatic cell count (SCC) response to citrus extracts Based on our SCC differences between treatments, the CED diet appears to have reduced the
338
incidence and/or extent of mastitis. Indeed Lund et al. (1994) found a high genetic correlation
339
between SCC and clinical mastitis (0.97), concluding that SCC is an indicator of clinical
340
mastitis. In addition, increased SCC has been shown to be positively correlated with increased
341
mastitis risk (Ward and Schultz, 1972; Kirk, 1984; Barkema et al., 1998), and Harmon (1994)
342
found that, in cows with higher SCC, lactose tends to leak out of the infected mammary gland
343
into the blood, resulting in less milk lactose, which is consistent with the slightly lower lactose
344
output of Control cows, which also had higher SCC.
345
4.3.1. Effects of feeding citrus pulp or limonene in an EO blend on milk SCC
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When lactating ewes were fed whole citrus pulp at 100, 200 and 300 g/kg DM of their ration,
347
Jaramillo et al. (2009) reported lower milk SCC. Giannenas et al. (2011) fed an EO blend, which
348
included limonene, to dairy ewes at three levels. At day 50, there was no change in SCC but, at
349
day 100, ewes fed the highest EO treatment had lower SCC and, at day 150, all EO treatments
350
had a lower SCC, with ewes fed the highest EO level having a lower SCC than the other ewes.
351
This suggests a dose-dependent effect to the EO blend containing limonene which is magnified
352
with longer feeding time.
353
4.3.2. Effects of feeding vitamin C on milk SCC
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354
In contrast to EO, there has been substantial amount of research on impacts of dietary
355
Vitamin C supplementation on udder health, especially mastitis. When pathogenic
356
microorganisms enter the mammary gland through the streak canal an inflammatory response is
357
triggered in the infected gland, thereby increasing the number of reactive oxygen species (Ranjan 15 Page 40 of 53
et al, 2005). Since two lines of defense are occurring at this time (i.e., sequestering of
359
antioxidants and recruiting of leukocytes (Ranjan et al., 2005; Harmon, 1994)), antioxidants such
360
as vitamin C can be utilized in high quantities to inactivate reactive oxygen species to decrease
361
their concentrations in plasma and the infected mammary quarter (Harmon, 1994). Early studies
362
showed that vitamin C is quickly degraded in vitro and in vivo (Knight et al., 1941; Vavich et al.,
363
1945), presumably by rumen microbes. However Hidiroglou (1999) found that cows fed vitamin
364
C or ethyl cellulose coated vitamin C (i.e., ruminally protected), or had vitamin C infused
365
abomasally, all had elevated plasma amino acid (AA) concentrations, but cows fed uncoated
366
vitamin C had the smallest increase. This supports the probability that not all dietary vitamin C
367
is degraded in the rumen. Weiss et al. (2004) and Ranjan et al. (2005) showed that infected
368
mammary glands increase vitamin C utilization, and Weiss et al. (2004) also showed that
369
mammary quarters challenged with E. coli had decreased levels of vitamin C in milk and plasma
370
due to an inflammatory response which increased AA oxidation. This suggests that depletion of
371
vitamin C in plasma and milk occurs during infection, rather than a low vitamin C status per se,
372
which allows a more severe infection to occur.
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373
Although cattle are thought to synthesize adequate vitamin C to maintain healthy mammary
374
gland function, vitamin C synthesis is not up-regulated when cows are under immunological
375
stress (Weiss et al., 2004), but vitamin C uptake and oxidation is increased in effected tissues.
376
Thus it is possible that in a generally healthy mammary gland, such as was the case of cows in
377
our study based upon relatively low overall milk SCC counts, supplemental vitamin C may have
378
helped maintain a high antioxidant/pro-oxidant ratio, thereby aiding in prevention of oxidative
379
damage.
380 381
5. Conclusions 16 Page 41 of 53
382
Supplementing the diet of dairy cows with a commercially available citrus extract decreased
383
SCC in generally healthy dairy cows under mild heat stress conditions. However measures of
384
heat stress and animal production were not substantively treatment impacted.
386
ip t
385 Acknowledgements
The authors thank all who volunteered to help: Grace Cun, Stacy Wrinkle, Nadia Swanepoel,
388
Blanca Comacho and Emma Robinson. We are grateful to Stacy Wrinkle and Pablo Chilibroste
389
for sharing their knowledge on heat stress behaviors, and to William Van Die for allowing us to
390
conduct the study on his dairy.
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391 References
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Ali, A.K.A., Shook, G.E., 1980. An optimum transformation for somatic cell concentration in
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AOAC, 1997. Official Methods of Analysis of AOAC International, 16th ed. Association of
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AOAC, 2006. Official Methods of Analysis of AOAC International, 18th ed. Association of Official Analytical Chemists, Arlington, VA, USA. Bampidis, V.A., Robinson P.H., 2006. Citrus by-products as ruminant feeds: A review. Anim. Feed Sci. Technol. 128, 175-217.
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Barkema, H.W., Schukken, Y.H., Lam, T.J.G.M., Beiber, M.L., Wilmink, H., Benedictus G.,
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production, and milk composition in dairy cows. J. Dairy Sci. 89, 4352–4364. Benchaar, C., Petit H.V., Berthiaume, R., Ouellet, D.R., Chiquette, J., Chouinard, P.Y., 2007.
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Castillejos, L., Calsamiglia, S., Ferret, A., 2006. Effect of essential oil active compounds on
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Hillman, P.E., Lee, C.N., Willard, S.T., 2005. Thermoregulatory responses associated with lying and standing in heat-stressed dairy cows. American Soc. Agric. Eng. 48, 795-801.
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Jaramillo, D.P., Garcia, T., Buffa, M., Rodrigues, M., Guamis, B., Trujillo, A., 2009. Effect of
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the inclusion of whole citrus in the ration of lactating ewes on the properties of milk and
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cheese characteristics during ripening. J. Dairy Sci. 92, 469-476.
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Johansen, H.N., Glitso, V., Knudsen, K.E.B., 1996. Influence of Extraction Solvent and
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Temperature on the Quantitative Determination of Oligosaccharides from Plant Materials by
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High-Performance Liquid Chromatography. J. Agric. Food Chem. 44, 1470-1474.
447 448 449 450
Jones, J.B., 2001. Laboratory Guide for Conducting Soil Tests and Plant Analysis, CRC Press LLC, Boca Raton, FL, USA. pp. 227-228. Kirk, J.H., 1984. Programmable calculator program for linear somatic cell scores to estimate mastitis yield losses. J. Dairy Sci. 67, 441-443.
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Knight, C.A., Dutcher, R.A., Guerrant, N.B., Bechdel, S.I., 1941. Excretion of ascorbic acid by the dairy cow. J. Dairy Sci. 4, 567-577. LCI, 1970. Patterns of transit losses. Livestock Conservation, Inc. Omaha, NE, USA.
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Lund, T., Miglior, F., Dekkers, J.C.M., Burnside, E.B., 1994. Genetic relationships between
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clinical mastitis, somatic cell count, and udder conformation in Danish Holsteins. Livest.
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Prod. Sci. 39, 243-251.
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Meyer, G.A., Keliher, P.N., 1992. An overview of analysis by inductively coupled plasma-
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atomic emission spectrometry. In: Montaser, A., Golightly, D.W. (Eds.), Inductively Coupled
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Plasmas in Analytical Atomic Spectrometry. VCH. Publishers Inc., New York, NY, USA.
460
pp. 473-505.
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National Research Council, 2001. Nutrient Requirements of Dairy Cattle. 7th rev. ed. National
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Academy of Sciences, Washington, DC, USA.
Padh, H., 1990. Cellular functions of ascorbic acid. Biochem. Cell Biol. 68, 1166-1173.
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Ranjan, R., Swarup, D., Naresh, R., Patra, R.C., 2005. Enhanced erythrocytic lipid peroxides and
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reduced plasma ascorbic acid, and alteration of blood trace elements level in dairy cows with
466
mastitis. Vet. Res. Comm. 29, 27-34.
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Robinson, P.H., Meyer, D., 2010. Total mixed ration (TMR) sampling protocol. ANR Publication 8413, ANR Publications, University of California, Berkeley, CA, USA. Roth, J.A., Kaeberle, M.L., 1985. In vivo effect of ascorbic acid on neutrophil function in healthy and dexamethasone-treated cattle. Journal of Dairy Science 46, 2434-2436. Sah R.N., Miller, R.O., 1992. Spontaneous reaction for acid dissolution of biological tissues in closed vessels. Anal. Chem. 64, 230-233.
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Santos, M.B., Robinson, P.H., Williams, P., Losa, R., 2010. Effects of addition of an essential oil
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complex to the diet of lactating dairy cows on whole tract digestion of nutrients and
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productive performance. Anim. Feed Sci. Technol. 157, 64-71. SAS® User‟s Guide, Statistics, 2012. SAS Institute Inc., Cary, NC, USA.
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Schukken, Y.H., Wilson, D.J., Welcome, F., Garrison-Tikofsky, L., Gonzalez, R.N., 2003.
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Monitoring udder health and milk quality using somatic cell counts. Vet. Res. 34, 579-596.
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Smith, D., 1969. Removing and Analyzing Total Nonstructural Carbohydrates from Plant Tissue.
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Wisconsin Agric. Exp. Sta. Res. Report 41, University of Wisconsin, Madison, WI, USA.
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Sprecher, D.J., Hostetler, D.E., Kaneene J.B., 1997. A lameness scoring system that uses posture
482
and gait to predict dairy cattle reproductive performance. Theriogenology 47, 1179-1187.
483
Stensig, T., Robinson, P.H., 1997: Digestion and passage kinetics of forage fiber in dairy cows as
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affected by fiber-free concentrate in the diet. J. Dairy Sci. 80, 1339-1352. Tassoul, M.D., Shaver, R.D., 2009. Effect of a mixture of supplemental dietary plant essential
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oils on performance of periparturient and early lactation dairy cows. J. Dairy Sci, 92, 1734-
487
1740.
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Tracy, M.L., Mueller, G., 1990. Continuous flow vapor generation for inductively coupled argon
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plasma spectrometric analysis. Part 1. Selenium. J. Assoc. Off. Anal. Chem. 73, 404–410.
490
Turner, L.W., Chastain, J.P., Hemken, R.W., Gates, R.S., Crist, W.L., 1992. Reducing heat stress
491 492 493 494 495
in dairy cows through sprinkler and fan cooling. Appl. Eng. in Agric. 8, 251-256. Tyrrell, H.F., Reid, J.T., 1965. Prediction of the energy value of cow's milk. J. Dairy Sci. 48, 1215-1223. Vavich, M.G., Dutcher, R.A., Guerrant, N.B., 1945. Utilization and excretion of ingested ascorbic acid by the dairy cow. J. Dairy Sci. 28, 759-770.
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497 498 499
Ward, G.E., Schultz, L.H., 1972. Relationship of somatic cells in quarter milk to type of bacteria and production. J. Dairy Sci. 55, 1428-1431. Weiss, W.P., 2001. Effect of dietary vitamin C on concentrations of ascorbic acid in plasma and milk. J. Dairy Sci. 84, 2302-2307.
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Weiss, W.P., Hogan, J.S., Smith, K.L., 2004. Changes in vitamin C concentrations in plasma
501
and milk from dairy cows after an intramammary infusion of escherichia coli. J Dairy Sci.87,
502
32-37.
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2144.
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West, J.W., 2003. Effects of heat-stress on production in dairy cattle. J. Dairy Sci. 86, 2131-
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Table
Table 1 Ingredient and chemical composition of Control and orange extract (CED) total mixed rations. Control
CED
167 72 60 159 144 33 19 5 64 71 11 23 36 22 33 38 14 29
164 72 60 160 145 34 19 5 64 71 11 23 36 21 33 38 0.16 14 29
SEM
P
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Alfalfa, hay Cottonseed, whole uplandb Cottonseed, cracked pimab Almond, hullsb Canola, meal - solventb Wheat, strawb Mineral, premixb Limestone, crushedb DDGSb Corn gluten, pelletsb Fat, rumen inert b,c Molasses, liquidb Alfalfa, fresh chop Wheat, whole crop silage Corn, flaked grain Corn, whole crop silage “Cristalfeed® Gold Rush” d Tomato, pomace - wet Whey, liquid
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Ingredient composition (g/kg DM) a
574
578
1.7
0.36
Crude protein ADICP (g/kg CP)e aNDF aNDFom ADFom Lignin(sa) Starch Sugarsf Crude Fat
172 64 335 316 230 5.0 125 37 54
170 66 334 317 231 5.2 144 37 55
1.5 1.5 2.3 2.0 3.3 0.4 7.3 0.4 0.4
0.37 0.50 0.88 0.69 0.82 0.09 0.25 0.51 0.31
Ash Calcium Phosphorus Magnesium Potassium Sulfur Sodium Chloride
94 9.3 5.4 3.0 19.3 2.9 2.8 6.5
93 9.8 5.5 3.1 19.2 2.9 2.8 6.6
0.2 0.05 0.06 0.02 0.15 0.04 0.04 0.11
0.07 0.02 0.63 0.27 0.66 0.77 0.92 0.75
Chemical composition (g/kg DM) Dry matter (g/kg)
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71.5 39.4 212 13.6 0.41 0.42
0.311 0.336 17.1 0.08 0.018 0.005
a
TMR ingredients listed in the order in which they entered the mixer. Ingredients combined to create a premix which was added to create the TMR. c EngerG II, Virtus Nutrition, Corcoran, CA, USA. b
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Acid detergent insoluble CP. Free glucose, sucrose and fructose.
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VéO Premium (Cristalfeed® Gold Rush; Reference ST 232 P2; Phodé, Terssac, France).
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0.86 0.63 0.35 0.73 0.70 0.31
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4 5 6 7 8 9 10
71.6 39.7 245 13.6 0.43 0.43
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Zinc (mg/kg DM) Manganese (mg/kg DM) Iron (mg/kg DM) Copper (mg/kg DM) Cobalt (mg/kg DM) Selenium (mg/kg DM)
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Table 2 Effects of feeding citrus extract (CE) on DM intake, milk yield, milk components and body condition score. SEM
DM intake (kg/d; n=8)
25.39
25.18
0.390
Yield (kg/d; n=654) Milk Fat True Protein Lactose Energy (MJ/d)
47.04 1.66 1.34 2.25 135.3
47.46 1.67 1.35 2.27 136.6
0.270 0.013 0.007 0.013 0.826
Components (g/kg milk) Fat True protein Lactose SCC (cells/µL) SCC (linear score)
35.4 28.6 47.8 196 2.30
0.13 0.46 0.08 0.06 0.19
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0.20 0.08 0.06 16.5 0.795
0.79 0.49 0.14 0.04 <0.01
2.59 0.027
0.016 0.0133
0.53 0.93
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Body Condition Score (n=168) Average (units) 2.59 Change (units/28 d) 0.028
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CED
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CED
SEM
Respiration Rate (breaths/min) 2:45 h 9:15 h 17:30 h
63.2 62.9 59.8
61.6 63.9 58.7
1.18 1.34 1.14
Panting Score (unit)b 2:45 h 9:15 h 17:30 h
1.09 0.89 1.27
1.12 0.92 1.22
0.037 0.038 0.034
0.55 0.46 0.28
68.6 39.7 27.3
0.038 0.038 0.034
< 0.01 0.79 0.18
34.1
34.1
0.076
0.50
1.15
1.15
0.015
0.88
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Rump temperature (°C)
a
0.36 0.59 0.50
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Locomotion score (unit; n=654)
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Lying versus standing (cow/100 cows)c 2:45 h 53.7 9:15 h 41.1 17:30 h 33.7
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Control
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Table 3 Effects of feeding citrus extract (CE) on respiration rates, panting scores, lying versus standing behavior at three times of the daya, as well as skin temperature and locomotion scores in early lactation cows (n=168).
Midpoint of the period starting 75 min prior and ending 75 min after the listed time. A measurement on a scale of 0 to 5 of the extent that a cow was is panting (Figure 2). c The proportion of cows scored as lying versus standing based on the position in which they were first found. b
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Table 4 Effects of feeding citrus extract (CE) on whole tract digestibility (g/kg) of some dietary nutrients (n=24), and partial energy balance (MJ/d). Control
CED
SEM
P
724
715
4.7
0.18
513
508
6.3
0.62
aNeutral detergent fiberb
501
492
Crude Protein
711
695
135.3
136.6
0.83
0.19
1.3
1.2
0.60
0.93
179.7
180.9
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-
7.18
-
-
Partial energy balance
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Milk Change in BCS
M
NELc NEL densitye (MJ/kg DM)
7.08
a
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aNeutral detergent
fiberoma
7.5
0.41
7.3
0.14
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Organic matter
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Whole tract digestibility
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aNDFom expressed exclusive of residual ash. aNDF expressed inclusive of residual ash. c Net energy of lactation; calculated by summing maintenance, milk and change in BCS energy, where maintenance energy is 41.8 MJ/d assuming cow average body weight was 650 kg. d Net energy of lactation divided by measured DM intake. e Reported as listed values with arithmetic means of treatment means. b
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Figure
Figure 1. The 24 h pattern of ambient temperature and temperature humidity index (THI) during the 3 day period of behavioral observations in periods 1 and 2. Weather data was
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recorded every 30 min. Graphs show the averages of each weather data point.
1 Page 53 of 53
S0377-8401(15)00223-0 http://dx.doi.org/doi:10.1016/j.anifeedsci.2015.06.022 ANIFEE 13318
To appear in:
Animal
Received date: Revised date: Accepted date:
17-4-2015 23-6-2015 24-6-2015
Feed
Science
and
Technology
Please cite this article as: Havlin, J.M., Robinson, P.H.,Intake, milk production and heat stress of dairy cows fed a citrus extract during summer heat, Animal Feed Science and Technology (2015), http://dx.doi.org/10.1016/j.anifeedsci.2015.06.022 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Highlights (for review)
Highlights
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A citrus extract fed to lactating cows had no impact on animal performance Milk SCC count reductions with citrus extract feeding suggested improved mammary health Naturally occurring bioactive compounds can impact animal performance
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*Manuscript (final version with changes or corrections highlighted)
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4 Intake, milk production and heat stress of dairy cows
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fed a citrus extract during summer heat
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J.M. Havlina, P.H. Robinsona,*
15 16 17 18 19 20 21
University of California, Davis, CA, 95616, USA
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Department of Animal Science
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*Corresponding author: Tel: 530-754-7565; Fax: 530-752-0175 EM: [email protected]
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Submitted to Animal Feed Science and Technology in April 2015
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Revised and resubmitted in May 2015. 1 Page 2 of 53
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Revised and resubmitted in June 2015.
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Abstract This study determined effects of feeding a citrus extract (CE) to high producing dairy cows
28
during summer heat on measures of heat stress, as well as milk production and composition, in a
29
replicated 2 x 2 Latin square experiment with two 28 d periods on a dairy farm near Hanford
30
(CA, USA). Four „high group‟ pens were used (i.e., cows which had cleared the fresh pen but
31
were not yet confirmed pregnant) were used, each with ~310 early lactation multiparity
32
cows/pen. The two total mixed rations contained 171 g/kg dry matter (DM) crude protein (CP),
33
55 g/kg fat, 335 g/kg neutral detergent fiber (aNDF) and 135 g/kg starch, and were the same
34
except for inclusion of the CE at 4 g/cow/d in the treatment diet (CED). Average daily high
35
temperatures during the study were 35 to 37oC with lows of 16 to 17oC. In general, cows
36
showed mild heat stress, but CE feeding had no effect on respiration rate, panting score or rump
37
temperature at any time of the day (i.e., 02:45, 09:15, 17:30 h). However at 02:45 h, a higher
38
(P<0.01) proportion of CED cows were lying (versus standing) compared with Control cows
39
(68.6 versus 53.7 cows/100 cows), which is an indicator of reduced heat stress. Intake of DM
40
(25.3 kg/d) and whole tract digestibility of CP (703 g/kg) and aNDF (510 g/kg) did not differ
41
between treatments. Milk production (47.3 kg/d) and its fat and true protein levels (35.4, 28.6
42
g/kg) did not differ, and changes in body condition and locomotion scores were also not
43
impacted by treatment impacted. However mammary health improved based on lower SCC
44
(somatic cell counts; P<0.04) of CED versus Control cows (160,000 versus 196,000 cells/µL),
45
and lower linear SCC scores (P<0.01; 2.12 versus 2.30). Feeding this CE to lactating dairy cows
46
during summer heat decreased SCC with no impact on other aspects of performance.
47
Keywords: limonene, vitamin C, citrus, essential oils
48
Abbreviations: AA, amino acids; ADF, acid detergent fiber expressed with residual ash;
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ADFom, ADF expressed without residual ash; aNDF, neutral detergent fiber assayed with a heat
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stable amylase and expressed with residual ash; BCS, body condition score; CE, citrus extract;
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CED, CE diet; CP, crude protein; DM, dry matter; EE, ether extract; EO; essential oils; LS,
52
locomotion score; PS, panting score; RR, respiration rate; RT, rump temperature; SCC, somatic
53
cell count; THI, temperature/humidity index; TMR, total mixed ration
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54 1. Introduction
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A popular contemporary topic in ruminant nutrition is the study of naturally occurring dietary
57
additives capable owhichf modulateing rumen fermentation in order to improve nutrient
58
utilization by rumen microorganisms, and/or reduce enteric production of greenhouse gases such
59
as methane. Essential oils (EO) encompass a wide range of naturally occurring secondary
60
compounds which occur in leaves, flowers, stems and seeds of many plants, and may have
61
antimicrobial properties which modify rumen microbial fermentation.
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Reviews of various EO compounds, their possible modes of action and effects on rumen
63
fermentation have been described (e.g., Benchaar et al., 2008), and limonene, an EO found in
64
citrus products, has been suggested to have beneficial effects on rumen fermentation, and animal
65
production, in ruminants (Calsamiglia et al., 2007). While few in vivo studies have examined
66
EO mixtures containing limonene, Tassoul and Shaver (2009) did reported increased milk
67
protein production, with an overall trend to increased feed efficiency (i.e., kg of fat corrected
68
milk/kg of DM intake), when an EO mixture including limonene was fed to dairy cows. In
69
contrast, Benchaar et al. (2006, 2007) had earlier reported that the same EO mixture used by
70
Tassoul and Shaver (2009) had no effect on milk production, dry matter (DM) intake or feed
71
efficiency.
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Vitamin C, found at high levels in citrus products, is not considered an essential nutrient for
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healthy cattle because their liver can synthesize it from glucose at levels believed to be sufficient 4 Page 5 of 53
to meet their needs (Padh, 1990). However cattle under stress, such as those in early lactation at
75
high production levels and/or under summer heat, may not synthesize adequate amounts of
76
vitamin C to compensate for oxidative stress due to sub-clinical infection or illness (Weiss et al.,
77
2004; Ranjan et al., 2005), and feeding supplemental vitamin C can replace depleted reserves
78
(Weiss, 2001). As high somatic cell counts (SCC) in milk are the result of degraded mammary
79
health, and have a negative impact on animal performance and milk quality, the limonene and
80
vitamin C in citrus products may help minimize SCC in dairy cows, thereby improving
81
efficiency of feed utilization as a result of improved immune function (Chaiyotwittayakun et al.,
82
2002; Castillejos et al., 2006). Indeed Jaramillo et al., (2009) and Giannenas et al. (2011) fed
83
citrus pulp to ewes and reduced milk SCC.
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In a review of citrus product feeding to ruminants, Bampidis and Robinson (2006) concluded
85
that their feeding did not affect DM digestibility, but decreased crude protein (CP) digestibility,
86
while increasing neutral detergent fiber (NDF) and acid detergent fiber (ADF) digestibility.
89
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Our objective was to evaluate impacts of a CE on feed intake and productive performance of dairy cows during hot weather.
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2. Materials and methods
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2.1. Animals, management, and experimental design
92
High group multiparity Holstein cows (i.e., those cows which had cleared the fresh pen but
93
were not yet confirmed pregnant) in 4 early lactation pens of ~310 cows each, on a commercial
94
dairy farm near Hanford (CA, USA), were used in a study with two 28 d experimental periods in
95
a replicated 2 x 2 crossover design during summer. Each pen had 300 head gates and free stalls.
96
The only source of cooling was bunk line misters which switched on automatically at 24°C, and
97
were on in each pen for 4 mins and then off for 12. Cows were assigned weekly to one of the 4 5 Page 6 of 53
98
pens at random from a common fresh cow pen, and moved to a mid pen by ~200 days in milk
99
once they were confirmed pregnant. Cows were milked three times daily in a double 40 herringbone parlor. Pen 1 started milking
101
at 04:00, 12:00 and 20:00 h. Pens 2, 3, and 4 were milked in sequence after pen 1, at intervals of
102
~45 min. The total mixed ration (TMR) was fed twice daily, between 04:00 and 08:00 h, prior to
103
cows returning to their pens from milking, and again between 11:30 and 12:30 h. The amount of
104
TMR delivered was determined from the previous days intake to create orts equal to ~10 g/kg of
105
total TMR delivered (as fed). Orts were removed daily and weighed individually by pen while
106
cows were in the parlor during the first milking, just prior to the first TMR feeding.
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Head-locks were set prior to cows returned from the morning milking, to facilitate artificial
108
insemination, such that the cows were head locked in the stanchions for 45 to 60 min daily.
109
Cows were housed in covered barns with access to free stalls bedded with dried manure, which
110
was renewed weekly and groomed bi-weekly. Cows also had free access to an uncovered manure
111
pack drylot at all times. Inside alleyways were flushed with water three times daily while the
112
cows were being milked. Cows had ad libitum access to clean drinking water at all times.
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At the start of the study, two pens were fed the CE containing diet (CED), while the two
114
other pens were fed the Control diet. After the first 28 d period, treatments were reversed. Each
115
period contained a 21 d adjustment and 7 d collection period during which all samples were
116
collected.
117
2.2. Environmental conditions
118
Four portable weather data loggers (HOBO U23; Onset, Bourne, MA, USA) were recorded
119
ambient temperatures and relative humidity every 30 min throughout the study. One station was
120
placed in each experimental pen on a pole at its center ~3 m above the floor and out of direct
121
sunlight. The study took place in the 2 mo after the summer solstice to minimize variation of day 6 Page 7 of 53
122
length and weather, while maximizing expected temperature/humidity indices (THI) which was
123
calculated as:
124
THI = tdb – [0.55-(.55*RH/100)]*(tdb – 58.8) where: tdb = dry bulb air temperature (°F) and RH = relative humidity (%). THI ≤ 74 is
126
considered “normal”, 75 to 78 is “alert”, 79 to 83 is “danger”, and ≥ 84 is “emergency”
127
according to the Livestock Weather Safety Index (LCI, 1970).
128
2.3. Diets
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The TMR for the CED and Control groups was formulated identically except for inclusion of
130
the CE at 4 g/cow/d in the CED TMR. Rate of inclusion of the CE product (VéO Premium;
131
Cristalfeed® Gold Rush; Reference ST 232 P2; Phodé, Terssac, France) in TMR loads was
132
calculated weekly based on the number of cows in the pen, and used to create bags of CE which
133
the feeding staff added to the CED TMR. The CE contained natural citrus extracts, natural and
134
artificial flavoring as well as corn flour, calcium carbonate and silicic acid as flow enhancers.
135
2.4. Sample collection
136
2.4.1. Feed and total mixed rations
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At the start and end of the collection week of each period (i.e., days 21 and 27) all dietary
138
ingredients were sampled. Hays were sampled with a „golf club‟ style hay probe (Sierra Testing
139
Services, Acampo, CA, USA), with 12 core samples pooled into a plastic bag. All other
140
ingredients, with the exception of liquid whey, were sampled by hand (6 handfuls of each) and
141
composited to plastic bags. Silages were sampled from the loose pile which had been knocked
142
off the silage face for use that day and wet by-products (i.e., fresh chop alfalfa, tomato pomace)
143
were sampled from the storage area. Samples of all other feeds from the start and end of the
144
collection weeks were combined to create one sample per ingredient prior for chemical analysis.
7 Page 8 of 53
Ten handfuls (~200 g each) of each TMR by pen were collected according to Robinson and
146
Meyer (2010) at pre-marked posts with evenly spaced intervals along the bunk-line immediately
147
after feeding and before the cows had access to it. These TMR samples were pooled within
148
period and pen to create 8 TMR samples for chemical analysis.
149
2.4.2. Milk
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At the end of each collection week (i.e., day 28), the entire herd was tested for milk
151
production and composition by Dairy Herd Improvement Association (Kings County, CA, USA)
152
personnel. Milk weights and representative milk samples collected into tubes containing
153
bronopol and natamycin as preservatives were collected from all cows using Tru-test milk meters
154
(Tru-Test Ltd., Auckland, New Zealand).
155
2.4.3. Body condition and locomotion scores
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Body condition (BCS) was scored at the start of the study (i.e., day 0) and at the end (i.e., day
157
28) of each experimental period while the cows were locked in stanchions immediately after
158
morning milking. From each pen, 80 cows having the lowest days in milk at the start of the study
159
were selected. Cows were scored on the 1 to 5 scale of Edmonson et al. (1989), with intermediate
160
values between the ¼ points if a ¼ point score was not clear. One person scored all cows.
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161
Locomotion was scored (LS) at the end (i.e., day 28) of each experimental period while the
162
cows were returning from the parlor from milking starting at 14:00 h. All cows in the pen were
163
observed for lameness using the 1 to 5 scoring system of Sprecher et al. (1997). One person
164
scored all cows on both occasions.
165
2.4.4. Fecal
166
Fecal samples were collected from the same 18 cows during the morning lock-up on day 28
167
of each period. Feces were manually collected (minimum 250 g) from the rectum of the cow
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into plastic containers, and immediately stored at -18°C until processing for chemical analysis. 8 Page 9 of 53
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2.4.5. Heat stress behavior and skin temperature Respiration rate (RR), panting score (PS), lying versus standing behavior, and rump
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temperature (RT) was measured three times over a 24 h period on days 27 and 28 of each period.
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Skin temperature was recorded on days 26 and 28 during morning lock-up during both periods,
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as they had not yet had access to the dry lot where they would be in direct sunlight at this time. A
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subgroup of 50 cows which had been scored for BCS from each pen was observed for heat stress
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parameters. Identification collars were placed on the cows during morning lock-up to facilitate
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easier identification of cows from a distance during observations. Behavioral observations started
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~24 h after the collars were attached. The behavior observation periods for pens 1 and 2 began at
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08:00, 16:15 and 01:30 h, while observations for pens 3 and 4 began directly following
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observations of pens 1 and 2. Observation periods lasted 75 mins/pen. The 08:00 h observation
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time was chosen to ensure that the cows had been out of head-lock for at least 15 min prior to
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observations. The 16:15 h time was chosen to approximate the hottest time of the day and, during
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the 01:30 h observation period, the sun had been down for ~5.5 h and the temperature was
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approaching the daily low. At this time the vast majority of the cows were in the outside dry lot.
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The RR was determined by observing flank movements of the cows where the time it took a
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cow to take 20 breaths was recorded and converted to breaths/min. A 0 to 5 PS was assigned to
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each cow (Gaughan et al., 2008) based on physical signs including degree of chest movement
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and presence of drool. Cows in two pens were simultaneously observed for heat stress behaviors
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by two individual scorers. Each scorer observed the same treatment and Control pen in each
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period. Scorers trained together prior to the study, and between periods, to eliminate scorer bias.
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To obtain RT, two small spots of ~40 x 60 mm each were shaved on each side of the rear
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flank, distal to where the udder meets the leg. Temperatures from the right and left flank were
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measured using an infrared gun (Fluke-561, Everett, WA, USA). 9 Page 10 of 53
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2.5. Analytical Methods
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2.5.1. Ingredient and total mixed ration chemical analysis All wet ingredients and TMR samples were dried at 55oC for 48 h and air equilibrated for 24
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h. Dried samples, with other ingredients which did not require drying, were analyzed at the UC
197
Davis Analytical Laboratory (Davis, CA, USA). All samples were ground to pass a 0.4 mm
198
screen on an Intermediate Wiley Mill or a 1 mm screen on a model 4 Wiley Mill (Thomas
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Scientific, Swedesboro, NJ, USA). Moisture was determined by gravimetric loss of free water by
200
heating to 105°C in a forced air oven for 3 h, and ash was the gravimetric residue after heating to
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550°C for at least 3 h. Total N and N in ADF were determined with infrared detection and
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thermal conductivity (TruSpec CN Analyzers, St. Joseph, MI, USA) by AOAC (2006; #990.03).
203
Analysis of aNDF used a heat stable amylase (AOAC, 2006; #2002-04), and aNDF and ADF are
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expressed exclusive of residual ash (i.e., aNDFom, ADFom) or with residual ash (i.e., aNDF).
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ADFom analysis was by boiling in acid detergent for 1 h (AOAC, 1997; #973.18).
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Crude fat, as ether extract (EE), was determined by extraction in ethyl ether (AOAC,
207
2006; #2003.05). Free sugars are the sum of glucose, fructose and sucrose as determined by
208
HPLC (Johansen et al., 1996). Enzymatic hydrolysis was used to determine the amount of total
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glucose, and free glucose was subtracted from total glucose, and the difference multiplied by 0.9
210
to calculate starch (Smith, 1969). Lignin(sa) was determined by the sulfuric acid procedure
211
(AOAC, 1997; #973.18). The Ca, Cu, Fe, Mg, Mn, P, K, Na, S and Zn contents were determined
212
by microwave nitric acid/hydrogen peroxide digestion/dissolution by inductively coupled plasma
213
atomic emission spectrometry (Meyer and Keliher, 1992, Sah and Miller, 1992). The Cl content
214
was determined after water extraction and analysis by ion chromatography with conductivity
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detection (Jones, 2001). Total Se was extracted by nitric/perchloric acid digestion/dissolution
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and determined by vapor generation using ICP-AES (Tracy and Mueller, 1990).
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2.5.2. Fecal Fecal samples from the 18 cows/pen which had been sampled in each experimental period
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were divided into 3 subgroups according to ear tag number order. Samples were individually
220
dried at 55oC for 48 h and then 150 g of each sample within a subgroup was pooled to create 3
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fecal sample groups/pen/period. The pooled samples were analyzed by the UC Davis Analytical
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Laboratory as described for the feed samples, as appropriate.
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2.5.3. Milk
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Fat, true protein and lactose, as well as SCC, were determined using infrared spectroscopy at the Dairy Herd Improvement Association laboratory in Hanford (CA, USA).
226
2.6. Calculations
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2.6.1. Dry matter intake
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Intake of TMR for each pen was calculated during the collection week (i.e., day 22 through
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day 28, inclusive) of each period based on the total weight of TMR delivered to each pen for that
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period corrected for the orts which were removed prior to the first feeding of each day. The
231
analyzed 105oC DM content of the TMR was used to determine DM intake. The number of
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cows/pen during the collection period was the average of the number of cows in the pen on the
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electronic record system (Dairy Comp 305, Valley Ag Systems, Tulare, CA, USA) on the first
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and last day of the final week of each period.
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2.6.2. Energy calculations
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Milk energy (MJ/kg) was calculated for individual cows according to Tyrrell and Reid
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(1965) using milk fat, crude protein and lactose, and the energy (MJ/d) in BCS change was
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calculated by cow according to NRC (2001) as 1255.2 MJ/unit BCS. Maintenance energy
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(MJ/d) was calculated as: (BW0.75*0.08*4.184) according to NRC (2001) for all groups assuming
240
an average body weight of 650 kg for all cows. Total energy output was calculated as the sum of 11 Page 12 of 53
milk energy (MJ/d), BCS change energy (MJ/d) and maintenance energy (MJ/d). The NEL
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density of the TMR were calculated by pen and period as: Total Energy Output (MJ/d) / DM
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intake (kg/d).
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2.6.3. Somatic cell count and linear conversion
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Because distribution of SCC in milk has a positive skewness with heterogeneous variance
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(Ali and Shook, 1980), log transformation (Schukken et al., 2003) was used to normalize the data
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to create a linear SCC as: LnS = log2(SCC/100)+3, where: SCC is expressed as cells/µL.
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2.6.4. Whole tract digestibility
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Apparent digestibility of dietary components was calculated by the proportion of feed
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components remaining in the feces from the diet, using lignin(sa) as the indigestible marker but
251
assuming 0.95 fecal recovery of lignin(sa) (Stensig and Robinson, 1997).
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2.7. Statistical Analysis
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The chemical composition of the TMR fed was statistically analyzed using the GLM
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procedure of SAS (2012) with pen, period and treatment as effects. The two TMR samples
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collected from each pen at the start and end of each collection week (i.e., day 21 and 27 of each
256
period) were combined prior to chemical analysis to be representative of the final week of the
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collection period. The DM intake data was analyzed using the GLM option of SAS (2012) with
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pen, period and treatment as fixed effects.
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The eligibility criteria for cows to be included in the statistical analysis was that they had to
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have been in her ≤6 lactation, and have remained in the same pen for the entire study (n=670).
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However, cows with milk or milk component values determined visually to be biological outliers
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were excluded from statistical analysis. Removal selection was completed blind to treatment and
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pen. A total of 16 cows were so removed, leaving 654 cows which met the criteria for inclusion
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and were included in the data set for statistical analysis. Milk yield and components, as well as 12 Page 13 of 53
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LS, were analyzed using the MIXED option of SAS with cow nested within pens as a random
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effect. The statistical model included fixed effects of pen, period and treatment. Data for cows included in the statistical analysis for BCS, which were a subset of the cows
268
used for milk production data, had to meet all criteria required for milk analysis, as well as
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having been scored for BCS at the start of the study and at the end of both experimental periods.
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The BCS (n=168) data was analyzed with the MIXED model of SAS with cows, pen, period and
271
treatment, as effects, as described above for analysis of milk parameters.
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Statistical inclusion criteria of cows for digestibility of feed components were the same as the
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milk criteria for each cow, and were a subset of these cows. Digestibility was analyzed with the
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MIXED model of SAS with fecal group, pen, period and treatment as effects, as described above
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for milk parameter statistical analysis. Fecal group was a random effect in this model.
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Treatment differences were accepted if P≤0.05, and tendencies to significance were accepted
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3. Results
280
3.1. Environmental conditions
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Diurnal patterns of ambient temperature and THI were similar between experimental periods
282
(Figure 1). While the daily temperature highs were similar to historical averages, the nighttime
283
lows were slightly cooler than normal. During the study period, highs were 36.5°C and 35.6°C
284
with lows of 16.9°C and 15.8°C, for period 1 (July) and 2 (August), respectively.
285
3.2. Chemical composition of feeds and total mixed ration
286
The chemical composition of feeds in the TMR (Table 1) were generally consistent with
287
NRC (2001) values, and t. The chemical profile of the TMR (Table 12) met or exceeded
288
recommendations of the NRC (2001) for dairy cattle at similar production levels. There were no 13 Page 14 of 53
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differences between the chemical profile of the TMR fed to the Control and CED groups, except
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for a slightly higher level of Ca (P=0.02) of the CED diet.
291
3.3. Feed intake, milk production and body condition score DM intake was not affected by feeding CE (Table 23), and milk and milk component yields
293
also did not differ between groups. Milk SCC (P=0.04) and linear score of SCC (P<0.01) were
294
lower for cows fed CE. The BCS and LS, as well change of BCS, did not differ between groups.
295
3.4. Heat stress parameters, body temperatures and locomotion score
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The RR, PS and RT (Table 34) did not differ between treatment groups at any observation
297
time. The proportion of cows lying versus standing was higher (P<0.01) for cows fed the CED
298
(68.6 versus 53.7 cows/100 cows) at 02:45 h, but did not differ at other times.
299
3.5. Whole tract apparent digestibility
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ranges for cows with high DM intake (Colucci et al., 1982; NRC, 2001).
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Feeding the CE did not impact whole tract digestibility (Table 45), with values within normal
302
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4. Discussion
304
4.1. Effect of citrus extract on heat stress, body temperature and locomotion
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Cows in the study were judged to have experienced mild heat stress because, even though
306
daytime high THI levels were often in the “alert” or “danger” levels, THI values of <74 (i.e.,
307
normal) occurred for ~10 h/d. This judgement is consistent with the measured RR of 58.7 to
308
64.0 breaths/min, which contrasts to RR of cattle of 100 breaths/min, or more, under extreme
309
heat stress (Turner, 1992). Likewise, the low PS of 0.89 to 1.27 of cows in both treatment groups
310
suggests that they were not substantively heat stressed, and rump temperatures were also within
311
the range of mildly heat stressed cows (Di Costanzo et al., 1997). This low level of observed
312
heat stress is probably because there was consistent night-time cooling, and because the cows 14 Page 15 of 53
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were fully shaded from the sun with access to bunk line misters (West, 2003; Gaughan et al.,
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2008). It is clear that feeding the CE had little or no effect on heat stress of our cows since RR, PS
316
and RT were not influenced. That the RR of both groups was lower at 17:30 than at 2:45 h
317
differed from expectations due to temperatures at those times of day (Di Costanzo et al., 1997).
318
In contrast, the PS pattern for both groups was highest at 17:30 h, the hottest time of the day, and
319
the lowest at 09:15 h probably due to cows having recovered during the cooler night. At 2:45 h
320
little TMR remained in the feed bunks and very few cows were eating, with the majority in the
321
open drylot where more CED cows were lying versus standing. As Hillman et al. (2005) found
322
that the body temperature of cows rises when they are lying, and that they tend to stand and seek
323
cooling when core body temperature rises above 38.9°C, the higher number of CED cows lying
324
at 02:45 h suggests that CE may have reduced core body temperatures and heat stress, at least at
325
this time.
326
4.2. Production responses to citrus extracts
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Lack of a treatment difference on DM intake was not unexpected because studies with
328
limonene fed in concert with other EO have shown no DM intake effect (Benchaar et al., 2006,
329
2007), or a slight trend to a reduction (Santos et al., 2010). Vitamin C supplementation also does
330
not appear to effect DM intake (Weiss, 2001; Chaiyotwittayakun et al., 2002; Weiss et al., 2004).
331
While there are no in vivo studies that have only fed limonene, there is little support from in
332
vitro studies that limonene affects microbial fermentation, feed efficiency or nutrient utilization
333
to support milk production (Dorman and Deans, 2000). This is consistent with our similar
334
dietary NEL density (7.18 versus 7.08 MJ/kg) of the Control and CED TMR (Table 45). In a
335
review of citrus by-product feeding, Bampidis and Robinson (2006) reported that adding citrus
336
by-products to diets of lactating dairy cows did not change milk yield or composition if the diets
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had a similar nutrient profile, which is also consistent with our finding that there were no
338
production differences between the Control and CED groups.
339
4.3. Milk somatic cell count (SCC) response to citrus extracts Based on our SCC differences between treatments, the CED diet appears to have reduced the
341
incidence and/or extent of mastitis. Indeed Lund et al. (1994) found a high genetic correlation
342
between SCC and clinical mastitis (0.97), concluding that SCC is an indicator of clinical
343
mastitis. In addition, increased SCC has been shown to be positively correlated with increased
344
mastitis risk (Ward and Schultz, 1972; Kirk, 1984; Barkema et al., 1998), and Harmon (1994)
345
found that, in cows with higher SCC, lactose tends to leak out of the infected mammary gland
346
into the blood, resulting in less milk lactose, which is consistent with the slightly lower lactose
347
output of Control cows, which also had higher SCC.
348
4.3.1. Effects of feeding citrus pulp or limonene in an EO blend on milk SCC
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340
When lactating ewes were fed whole citrus pulp at 100, 200 and 300 g/kg DM of their ration,
350
Jaramillo et al. (2009) reported lower milk SCC. Giannenas et al. (2011) fed an EO blend, which
351
included limonene, to dairy ewes at three levels. At day 50, there was no change in SCC but, at
352
day 100, ewes fed the highest EO treatment had lower SCC and, at day 150, all EO treatments
353
had a lower SCC, with ewes fed the highest EO level having a lower SCC than the other ewes.
354
This suggests a dose-dependent effect to the EO blend containing limonene which is magnified
355
with longer feeding time.
356
4.3.2. Effects of feeding vitamin C on milk SCC
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357
In contrast to EO, there has been substantial amount of research on impacts of dietary
358
Vitamin C supplementation on udder health, especially mastitis. When pathogenic
359
microorganisms enter the mammary gland through the streak canal an inflammatory response is
360
triggered in the infected gland, thereby increasing the number of reactive oxygen species (Ranjan 16 Page 17 of 53
et al, 2005). Since two lines of defense are occurring at this time (i.e., sequestering of
362
antioxidants and recruiting of leukocytes (Ranjan et al., 2005; Harmon, 1994)), antioxidants such
363
as vitamin C can be utilized in high quantities to inactivate reactive oxygen species to decrease
364
their concentrations in plasma and the infected mammary quarter (Harmon, 1994). Early studies
365
showed that vitamin C is quickly degraded in vitro and in vivo (Knight et al., 1941; Vavich et al.,
366
1945), presumably by rumen microbes. However Hidiroglou (1999) found that cows fed vitamin
367
C or ethyl cellulose coated vitamin C (i.e., ruminally protected), or had vitamin C infused
368
abomasally, all had elevated plasma amino acid (AA) concentrations, but cows fed uncoated
369
vitamin C had the smallest increase. This supports the probability that not all dietary vitamin C
370
is degraded in the rumen. Weiss et al. (2004) and Ranjan et al. (2005) showed that infected
371
mammary glands increase vitamin C utilization, and Weiss et al. (2004) also showed that
372
mammary quarters challenged with E. coli had decreased levels of vitamin C in milk and plasma
373
due to an inflammatory response which increased AA oxidation. This suggests that depletion of
374
vitamin C in plasma and milk occurs during infection, rather than a low vitamin C status per se,
375
which allows a more severe infection to occur.
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376
Although cattle are thought to synthesize adequate vitamin C to maintain healthy mammary
377
gland function, vitamin C synthesis is not up-regulated when cows are under immunological
378
stress (Weiss et al., 2004), but vitamin C uptake and oxidation is increased in effected tissues.
379
Thus it is possible that in a generally healthy mammary gland, such as was the case of cows in
380
our study based upon relatively low overall milk SCC counts, supplemental vitamin C may have
381
helped maintain a high antioxidant/pro-oxidant ratio, thereby aiding in prevention of oxidative
382
damage.
383 384
5. Conclusions 17 Page 18 of 53
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Supplementing the diet of dairy cows with a commercially available citrus extract decreased
386
SCC in generally healthy dairy cows under mild heat stress conditions. However measures of
387
heat stress and animal production were not substantively treatment impacted.
389
ip t
388 Acknowledgements
The authors thank all who volunteered to help: Grace Cun, Stacy Wrinkle, Nadia Swanepoel,
391
Blanca Comacho and Emma Robinson. We are grateful to Stacy Wrinkle and Pablo Chilibroste
392
for sharing their knowledge on heat stress behaviors, and to William Van Die for allowing us to
393
conduct the study on his dairy.
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394 References
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AOAC, 2006. Official Methods of Analysis of AOAC International, 18th ed. Association of
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AOAC, 1997. Official Methods of Analysis of AOAC International, 16th ed. Association of Official Analytical Chemists, Arlington, VA, USA. AOAC, 2006. Official Methods of Analysis of AOAC International, 18th ed. Association of Official Analytical Chemists, Arlington, VA, USA. Bampidis, V.A., Robinson P.H., 2006. Citrus by-products as ruminant feeds: A review. Anim. Feed Sci. Technol. 128, 175-217.
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Barkema, H.W., Schukken, Y.H., Lam, T.J.G.M., Beiber, M.L., Wilmink, H., Benedictus G.,
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Robinson, P.H., Meyer, D., 2010. Total mixed ration (TMR) sampling protocol. ANR
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Santos, M.B., Robinson, P.H., Williams, P., Losa, R., 2010. Effects of addition of an essential oil
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complex to the diet of lactating dairy cows on whole tract digestion of nutrients and
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productive performance. Anim. Feed Sci. Technol. 157, 64-71.
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SAS® User‟s Guide, Statistics, 2012. SAS Institute Inc., Cary, NC, USA.
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Schukken, Y.H., Wilson, D.J., Welcome, F., Garrison-Tikofsky, L., Gonzalez, R.N., 2003.
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Monitoring udder health and milk quality using somatic cell counts. Vet. Res. 34, 579-596.
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Smith, D., 1969. Removing and Analyzing Total Nonstructural Carbohydrates from Plant Tissue.
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Wisconsin Agric. Exp. Sta. Res. Report 41, University of Wisconsin, Madison, WI, USA.
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Sprecher, D.J., Hostetler, D.E., Kaneene J.B., 1997. A lameness scoring system that uses posture
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and gait to predict dairy cattle reproductive performance. Theriogenology 47, 1179-1187.
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Stensig, T., Robinson, P.H., 1997: Digestion and passage kinetics of forage fiber in dairy cows as
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Tassoul, M.D., Shaver, R.D., 2009. Effect of a mixture of supplemental dietary plant essential
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oils on performance of periparturient and early lactation dairy cows. J. Dairy Sci, 92, 1734-
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1740.
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Tracy, M.L., Mueller, G., 1990. Continuous flow vapor generation for inductively coupled argon
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plasma spectrometric analysis. Part 1. Selenium. J. Assoc. Off. Anal. Chem. 73, 404–410.
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Tyrrell, H.F., Reid, J.T., 1965. Prediction of the energy value of cow's milk. J. Dairy Sci. 48,
Vavich, M.G., Dutcher, R.A., Guerrant, N.B., 1945. Utilization and excretion of ingested
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ascorbic acid by the dairy cow. J. Dairy Sci. 28, 759-770.
Ward, G.E., Schultz, L.H., 1972. Relationship of somatic cells in quarter milk to type of bacteria and production. J. Dairy Sci. 55, 1428-1431.
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Turner, L.W., Chastain, J.P., Hemken, R.W., Gates, R.S., Crist, W.L., 1992. Reducing heat stress
Weiss, W.P., 2001. Effect of dietary vitamin C on concentrations of ascorbic acid in plasma and milk. J. Dairy Sci. 84, 2302-2307.
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Weiss, W.P., Hogan, J.S., Smith, K.L., 2004. Changes in vitamin C concentrations in plasma
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*Conflict of Interest Statement
Editors, The authors stipulate that they have no conflicts of interest in preparation or submission of this manuscript.
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P.H. Robinson for the authors
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*Manuscript (final version, no corrections or changes highlighted) Click here to download Manuscript (final version, no corrections or changes highlighted): AFSTcev3unmarked.docx Click here to view linked References
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4 Intake, milk production and heat stress of dairy cows
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fed a citrus extract during summer heat
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J.M. Havlina, P.H. Robinsona,*
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University of California, Davis, CA, 95616, USA
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Department of Animal Science
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*Corresponding author: Tel: 530-754-7565; Fax: 530-752-0175 EM: [email protected]
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Submitted to Animal Feed Science and Technology in April 2015
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Revised and resubmitted in May 2015.
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Revised and resubmitted in June 2015.
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Abstract This study determined effects of feeding a citrus extract (CE) to high producing dairy cows
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during summer heat on measures of heat stress, as well as milk production and composition, in a
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replicated 2 x 2 Latin square experiment with two 28 d periods on a dairy farm near Hanford
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(CA, USA). Four „high group‟ pens were used (i.e., cows which had cleared the fresh pen but
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were not yet confirmed pregnant), each with ~310 early lactation multiparity cows/pen. The two
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total mixed rations contained 171 g/kg dry matter (DM) crude protein (CP), 55 g/kg fat, 335 g/kg
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neutral detergent fiber (aNDF) and 135 g/kg starch, and were the same except for inclusion of
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the CE at 4 g/cow/d in the treatment diet (CED). Average daily high temperatures during the
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study were 35 to 37oC with lows of 16 to 17oC. In general, cows showed mild heat stress, but
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CE feeding had no effect on respiration rate, panting score or rump temperature at any time of
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the day (i.e., 02:45, 09:15, 17:30 h). However at 02:45 h, a higher (P<0.01) proportion of CED
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cows were lying (versus standing) compared with Control cows (68.6 versus 53.7 cows/100
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cows), which is an indicator of reduced heat stress. Intake of DM (25.3 kg/d) and whole tract
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digestibility of CP (703 g/kg) and aNDF (510 g/kg) did not differ between treatments. Milk
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production (47.3 kg/d) and its fat and true protein levels (35.4, 28.6 g/kg) did not differ, and
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changes in body condition and locomotion scores were also not impacted by treatment. However
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mammary health improved based on lower SCC (somatic cell counts; P<0.04) of CED versus
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Control cows (160,000 versus 196,000 cells/µL), and lower linear SCC scores (P<0.01; 2.12
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versus 2.30). Feeding this CE to lactating dairy cows during summer heat decreased SCC with
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no impact on other aspects of performance.
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Keywords: limonene, vitamin C, citrus, essential oils
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Abbreviations: AA, amino acids; ADF, acid detergent fiber expressed with residual ash;
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ADFom, ADF expressed without residual ash; aNDF, neutral detergent fiber assayed with a heat
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stable amylase and expressed with residual ash; BCS, body condition score; CE, citrus extract;
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CED, CE diet; CP, crude protein; DM, dry matter; EE, ether extract; EO; essential oils; LS,
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locomotion score; PS, panting score; RR, respiration rate; RT, rump temperature; SCC, somatic
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cell count; THI, temperature/humidity index; TMR, total mixed ration
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52 1. Introduction
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A popular contemporary topic in ruminant nutrition is the study of naturally occurring dietary
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additives which modulate rumen fermentation in order to improve nutrient utilization by rumen
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microorganisms, and/or reduce enteric production of greenhouse gases such as methane.
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Essential oils (EO) encompass a wide range of naturally occurring secondary compounds which
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occur in leaves, flowers, stems and seeds of many plants, and may have antimicrobial properties
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which modify rumen microbial fermentation.
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Reviews of various EO compounds, their possible modes of action and effects on rumen
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fermentation have been described (e.g., Benchaar et al., 2008), and limonene, an EO found in
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citrus products, has been suggested to have beneficial effects on rumen fermentation, and animal
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production, in ruminants (Calsamiglia et al., 2007). While few in vivo studies have examined
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EO mixtures containing limonene, Tassoul and Shaver (2009) reported increased milk protein
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production, with an overall trend to increased feed efficiency (i.e., kg of fat corrected milk/kg of
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DM intake), when an EO mixture including limonene was fed to dairy cows. In contrast,
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Benchaar et al. (2006, 2007) had earlier reported that the same EO mixture used by Tassoul and
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Shaver (2009) had no effect on milk production, dry matter (DM) intake or feed efficiency.
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Vitamin C, found at high levels in citrus products, is not considered an essential nutrient for
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healthy cattle because their liver can synthesize it from glucose at levels believed to be sufficient
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to meet their needs (Padh, 1990). However cattle under stress, such as those in early lactation at 3 Page 28 of 53
high production levels and/or under summer heat, may not synthesize adequate amounts of
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vitamin C to compensate for oxidative stress due to sub-clinical infection or illness (Weiss et al.,
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2004; Ranjan et al., 2005), and feeding supplemental vitamin C can replace depleted reserves
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(Weiss, 2001). As high somatic cell counts (SCC) in milk are the result of degraded mammary
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health, and have a negative impact on animal performance and milk quality, the limonene and
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vitamin C in citrus products may help minimize SCC in dairy cows, thereby improving
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efficiency of feed utilization as a result of improved immune function (Chaiyotwittayakun et al.,
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2002; Castillejos et al., 2006). Indeed Jaramillo et al., (2009) and Giannenas et al. (2011) fed
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citrus pulp to ewes and reduced milk SCC.
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In a review of citrus product feeding to ruminants, Bampidis and Robinson (2006) concluded
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that their feeding did not affect DM digestibility, but decreased crude protein (CP) digestibility,
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while increasing neutral detergent fiber (NDF) and acid detergent fiber (ADF) digestibility.
dairy cows during hot weather.
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Our objective was to evaluate impacts of a CE on feed intake and productive performance of
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2. Materials and methods
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2.1. Animals, management, and experimental design
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High group multiparity Holstein cows (i.e., those cows which had cleared the fresh pen but
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were not yet confirmed pregnant) in 4 early lactation pens of ~310 cows each, on a commercial
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dairy farm near Hanford (CA, USA), were used in a study with two 28 d experimental periods in
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a replicated 2 x 2 crossover design during summer. Each pen had 300 head gates and free stalls.
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The only source of cooling was bunk line misters which switched on automatically at 24°C, and
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were on in each pen for 4 mins and then off for 12. Cows were assigned weekly to one of the 4
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pens at random from a common fresh cow pen, and moved to a mid pen by ~200 days in milk
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once they were confirmed pregnant. Cows were milked three times daily in a double 40 herringbone parlor. Pen 1 started milking
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at 04:00, 12:00 and 20:00 h. Pens 2, 3, and 4 were milked in sequence after pen 1, at intervals of
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~45 min. The total mixed ration (TMR) was fed twice daily, between 04:00 and 08:00 h, prior to
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cows returning to their pens from milking, and again between 11:30 and 12:30 h. The amount of
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TMR delivered was determined from the previous days intake to create orts equal to ~10 g/kg of
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total TMR delivered (as fed). Orts were removed daily and weighed individually by pen while
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cows were in the parlor during the first milking, just prior to the first TMR feeding.
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Head-locks were set prior to cows returned from the morning milking, to facilitate artificial
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insemination, such that the cows were head locked in the stanchions for 45 to 60 min daily.
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Cows were housed in covered barns with access to free stalls bedded with dried manure, which
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was renewed weekly and groomed bi-weekly. Cows also had free access to an uncovered manure
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pack drylot at all times. Inside alleyways were flushed with water three times daily while the
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cows were being milked. Cows had ad libitum access to clean drinking water at all times.
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At the start of the study, two pens were fed the CE containing diet (CED), while the two
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other pens were fed the Control diet. After the first 28 d period, treatments were reversed. Each
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period contained a 21 d adjustment and 7 d collection period during which all samples were
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collected.
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2.2. Environmental conditions
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Four portable weather data loggers (HOBO U23; Onset, Bourne, MA, USA) were recorded
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ambient temperatures and relative humidity every 30 min throughout the study. One station was
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placed in each experimental pen on a pole at its center ~3 m above the floor and out of direct
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sunlight. The study took place in the 2 mo after the summer solstice to minimize variation of day 5 Page 30 of 53
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length and weather, while maximizing expected temperature/humidity indices (THI) which was
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calculated as:
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THI = tdb – [0.55-(.55*RH/100)]*(tdb – 58.8) where: tdb = dry bulb air temperature (°F) and RH = relative humidity (%). THI ≤ 74 is
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considered “normal”, 75 to 78 is “alert”, 79 to 83 is “danger”, and ≥ 84 is “emergency”
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according to the Livestock Weather Safety Index (LCI, 1970).
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2.3. Diets
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The TMR for the CED and Control groups was formulated identically except for inclusion of
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the CE at 4 g/cow/d in the CED TMR. Rate of inclusion of the CE product (VéO Premium;
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Cristalfeed® Gold Rush; Reference ST 232 P2; Phodé, Terssac, France) in TMR loads was
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calculated weekly based on the number of cows in the pen, and used to create bags of CE which
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the feeding staff added to the CED TMR. The CE contained natural citrus extracts, natural and
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artificial flavoring as well as corn flour, calcium carbonate and silicic acid as flow enhancers.
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2.4. Sample collection
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2.4.1. Feed and total mixed rations
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At the start and end of the collection week of each period (i.e., days 21 and 27) all dietary
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ingredients were sampled. Hays were sampled with a „golf club‟ style hay probe (Sierra Testing
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Services, Acampo, CA, USA), with 12 core samples pooled into a plastic bag. All other
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ingredients, with the exception of liquid whey, were sampled by hand (6 handfuls of each) and
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composited to plastic bags. Silages were sampled from the loose pile which had been knocked
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off the silage face for use that day and wet by-products (i.e., fresh chop alfalfa, tomato pomace)
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were sampled from the storage area. Samples of all other feeds from the start and end of the
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collection weeks were combined to create one sample per ingredient prior for chemical analysis.
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Ten handfuls (~200 g each) of each TMR by pen were collected according to Robinson and
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Meyer (2010) at pre-marked posts with evenly spaced intervals along the bunk-line immediately
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after feeding and before the cows had access to it. These TMR samples were pooled within
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period and pen to create 8 TMR samples for chemical analysis.
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2.4.2. Milk
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At the end of each collection week (i.e., day 28), the entire herd was tested for milk
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production and composition by Dairy Herd Improvement Association (Kings County, CA, USA)
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personnel. Milk weights and representative milk samples collected into tubes containing
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bronopol and natamycin as preservatives were collected from all cows using Tru-test milk meters
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(Tru-Test Ltd., Auckland, New Zealand).
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2.4.3. Body condition and locomotion scores
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Body condition (BCS) was scored at the start of the study (i.e., day 0) and at the end (i.e., day
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28) of each experimental period while the cows were locked in stanchions immediately after
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morning milking. From each pen, 80 cows having the lowest days in milk at the start of the study
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were selected. Cows were scored on the 1 to 5 scale of Edmonson et al. (1989), with intermediate
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values between the ¼ points if a ¼ point score was not clear. One person scored all cows.
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Locomotion was scored (LS) at the end (i.e., day 28) of each experimental period while the
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cows were returning from the parlor from milking starting at 14:00 h. All cows in the pen were
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observed for lameness using the 1 to 5 scoring system of Sprecher et al. (1997). One person
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scored all cows on both occasions.
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2.4.4. Fecal
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Fecal samples were collected from the same 18 cows during the morning lock-up on day 28
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of each period. Feces were manually collected (minimum 250 g) from the rectum of the cow
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into plastic containers, and immediately stored at -18°C until processing for chemical analysis. 7 Page 32 of 53
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2.4.5. Heat stress behavior and skin temperature Respiration rate (RR), panting score (PS), lying versus standing behavior, and rump
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temperature (RT) was measured three times over a 24 h period on days 27 and 28 of each period.
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Skin temperature was recorded on days 26 and 28 during morning lock-up during both periods,
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as they had not yet had access to the dry lot where they would be in direct sunlight at this time. A
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subgroup of 50 cows which had been scored for BCS from each pen was observed for heat stress
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parameters. Identification collars were placed on the cows during morning lock-up to facilitate
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easier identification of cows from a distance during observations. Behavioral observations started
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~24 h after the collars were attached. The behavior observation periods for pens 1 and 2 began at
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08:00, 16:15 and 01:30 h, while observations for pens 3 and 4 began directly following
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observations of pens 1 and 2. Observation periods lasted 75 mins/pen. The 08:00 h observation
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time was chosen to ensure that the cows had been out of head-lock for at least 15 min prior to
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observations. The 16:15 h time was chosen to approximate the hottest time of the day and, during
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the 01:30 h observation period, the sun had been down for ~5.5 h and the temperature was
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approaching the daily low. At this time the vast majority of the cows were in the outside dry lot.
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The RR was determined by observing flank movements of the cows where the time it took a
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cow to take 20 breaths was recorded and converted to breaths/min. A 0 to 5 PS was assigned to
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each cow (Gaughan et al., 2008) based on physical signs including degree of chest movement
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and presence of drool. Cows in two pens were simultaneously observed for heat stress behaviors
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by two individual scorers. Each scorer observed the same treatment and Control pen in each
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period. Scorers trained together prior to the study, and between periods, to eliminate scorer bias.
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To obtain RT, two small spots of ~40 x 60 mm each were shaved on each side of the rear
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flank, distal to where the udder meets the leg. Temperatures from the right and left flank were
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measured using an infrared gun (Fluke-561, Everett, WA, USA). 8 Page 33 of 53
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2.5. Analytical Methods
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2.5.1. Ingredient and total mixed ration chemical analysis All wet ingredients and TMR samples were dried at 55oC for 48 h and air equilibrated for 24
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h. Dried samples, with other ingredients which did not require drying, were analyzed at the UC
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Davis Analytical Laboratory (Davis, CA, USA). All samples were ground to pass a 0.4 mm
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screen on an Intermediate Wiley Mill or a 1 mm screen on a model 4 Wiley Mill (Thomas
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Scientific, Swedesboro, NJ, USA). Moisture was determined by gravimetric loss of free water by
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heating to 105°C in a forced air oven for 3 h, and ash was the gravimetric residue after heating to
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550°C for at least 3 h. Total N and N in ADF were determined with infrared detection and
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thermal conductivity (TruSpec CN Analyzers, St. Joseph, MI, USA) by AOAC (2006; #990.03).
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Analysis of aNDF used a heat stable amylase (AOAC, 2006; #2002-04), and aNDF and ADF are
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expressed exclusive of residual ash (i.e., aNDFom, ADFom) or with residual ash (i.e., aNDF).
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ADFom analysis was by boiling in acid detergent for 1 h (AOAC, 1997; #973.18).
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Crude fat, as ether extract (EE), was determined by extraction in ethyl ether (AOAC,
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2006; #2003.05). Free sugars are the sum of glucose, fructose and sucrose as determined by
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HPLC (Johansen et al., 1996). Enzymatic hydrolysis was used to determine the amount of total
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glucose, and free glucose was subtracted from total glucose, and the difference multiplied by 0.9
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to calculate starch (Smith, 1969). Lignin(sa) was determined by the sulfuric acid procedure
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(AOAC, 1997; #973.18). The Ca, Cu, Fe, Mg, Mn, P, K, Na, S and Zn contents were determined
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by microwave nitric acid/hydrogen peroxide digestion/dissolution by inductively coupled plasma
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atomic emission spectrometry (Meyer and Keliher, 1992, Sah and Miller, 1992). The Cl content
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was determined after water extraction and analysis by ion chromatography with conductivity
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detection (Jones, 2001). Total Se was extracted by nitric/perchloric acid digestion/dissolution
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and determined by vapor generation using ICP-AES (Tracy and Mueller, 1990).
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2.5.2. Fecal Fecal samples from the 18 cows/pen which had been sampled in each experimental period
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were divided into 3 subgroups according to ear tag number order. Samples were individually
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dried at 55oC for 48 h and then 150 g of each sample within a subgroup was pooled to create 3
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fecal sample groups/pen/period. The pooled samples were analyzed by the UC Davis Analytical
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Laboratory as described for the feed samples, as appropriate.
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2.5.3. Milk
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Fat, true protein and lactose, as well as SCC, were determined using infrared spectroscopy at the Dairy Herd Improvement Association laboratory in Hanford (CA, USA).
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2.6. Calculations
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2.6.1. Dry matter intake
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Intake of TMR for each pen was calculated during the collection week (i.e., day 22 through
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day 28, inclusive) of each period based on the total weight of TMR delivered to each pen for that
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period corrected for the orts which were removed prior to the first feeding of each day. The
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analyzed 105oC DM content of the TMR was used to determine DM intake. The number of
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cows/pen during the collection period was the average of the number of cows in the pen on the
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electronic record system (Dairy Comp 305, Valley Ag Systems, Tulare, CA, USA) on the first
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and last day of the final week of each period.
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2.6.2. Energy calculations
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Milk energy (MJ/kg) was calculated for individual cows according to Tyrrell and Reid
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(1965) using milk fat, crude protein and lactose, and the energy (MJ/d) in BCS change was
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calculated by cow according to NRC (2001) as 1255.2 MJ/unit BCS. Maintenance energy
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(MJ/d) was calculated as: (BW0.75*0.08*4.184) according to NRC (2001) for all groups assuming
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an average body weight of 650 kg for all cows. Total energy output was calculated as the sum of 10 Page 35 of 53
milk energy (MJ/d), BCS change energy (MJ/d) and maintenance energy (MJ/d). The NEL
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density of the TMR were calculated by pen and period as: Total Energy Output (MJ/d) / DM
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intake (kg/d).
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2.6.3. Somatic cell count and linear conversion
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Because distribution of SCC in milk has a positive skewness with heterogeneous variance
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(Ali and Shook, 1980), log transformation (Schukken et al., 2003) was used to normalize the data
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to create a linear SCC as: LnS = log2(SCC/100)+3, where: SCC is expressed as cells/µL.
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2.6.4. Whole tract digestibility
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Apparent digestibility of dietary components was calculated by the proportion of feed
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components remaining in the feces from the diet, using lignin(sa) as the indigestible marker but
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assuming 0.95 fecal recovery of lignin(sa) (Stensig and Robinson, 1997).
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2.7. Statistical Analysis
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The chemical composition of the TMR fed was statistically analyzed using the GLM
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procedure of SAS (2012) with pen, period and treatment as effects. The two TMR samples
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collected from each pen at the start and end of each collection week (i.e., day 21 and 27 of each
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period) were combined prior to chemical analysis to be representative of the final week of the
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collection period. The DM intake data was analyzed using the GLM option of SAS (2012) with
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pen, period and treatment as fixed effects.
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The eligibility criteria for cows to be included in the statistical analysis was that they had to
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have been in her ≤6 lactation, and have remained in the same pen for the entire study (n=670).
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However, cows with milk or milk component values determined visually to be biological outliers
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were excluded from statistical analysis. Removal selection was completed blind to treatment and
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pen. A total of 16 cows were so removed, leaving 654 cows which met the criteria for inclusion
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and were included in the data set for statistical analysis. Milk yield and components, as well as 11 Page 36 of 53
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LS, were analyzed using the MIXED option of SAS with cow nested within pens as a random
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effect. The statistical model included fixed effects of pen, period and treatment. Data for cows included in the statistical analysis for BCS, which were a subset of the cows
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used for milk production data, had to meet all criteria required for milk analysis, as well as
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having been scored for BCS at the start of the study and at the end of both experimental periods.
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The BCS (n=168) data was analyzed with the MIXED model of SAS with cows, pen, period and
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treatment, as effects, as described above for analysis of milk parameters.
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Statistical inclusion criteria of cows for digestibility of feed components were the same as the
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milk criteria for each cow, and were a subset of these cows. Digestibility was analyzed with the
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MIXED model of SAS with fecal group, pen, period and treatment as effects, as described above
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for milk parameter statistical analysis. Fecal group was a random effect in this model.
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3. Results
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3.1. Environmental conditions
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Diurnal patterns of ambient temperature and THI were similar between experimental periods
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(Figure 1). While the daily temperature highs were similar to historical averages, the nighttime
280
lows were slightly cooler than normal. During the study period, highs were 36.5°C and 35.6°C
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with lows of 16.9°C and 15.8°C, for period 1 (July) and 2 (August), respectively.
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3.2. Chemical composition of feeds and total mixed ration
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The chemical composition of feeds in the TMR were generally consistent with NRC (2001)
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values, and the chemical profile of the TMR (Table 1) met or exceeded recommendations of the
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NRC (2001) for dairy cattle at similar production levels. There were no differences between the 12 Page 37 of 53
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chemical profile of the TMR fed to the Control and CED groups, except for a slightly higher
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level of Ca (P=0.02) of the CED diet.
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3.3. Feed intake, milk production and body condition score DM intake was not affected by feeding CE (Table 2), and milk and milk component yields
290
also did not differ between groups. Milk SCC (P=0.04) and linear score of SCC (P<0.01) were
291
lower for cows fed CE. The BCS and LS, as well change of BCS, did not differ between groups.
292
3.4. Heat stress parameters, body temperatures and locomotion score
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The RR, PS and RT (Table 3) did not differ between treatment groups at any observation
294
time. The proportion of cows lying versus standing was higher (P<0.01) for cows fed the CED
295
(68.6 versus 53.7 cows/100 cows) at 02:45 h, but did not differ at other times.
296
3.5. Whole tract apparent digestibility
M
ranges for cows with high DM intake (Colucci et al., 1982; NRC, 2001).
d
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Feeding the CE did not impact whole tract digestibility (Table 4), with values within normal
299
te
297
an
293
4. Discussion
301
4.1. Effect of citrus extract on heat stress, body temperature and locomotion
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300
302
Cows in the study were judged to have experienced mild heat stress because, even though
303
daytime high THI levels were often in the “alert” or “danger” levels, THI values of <74 (i.e.,
304
normal) occurred for ~10 h/d. This judgement is consistent with the measured RR of 58.7 to
305
64.0 breaths/min, which contrasts to RR of cattle of 100 breaths/min, or more, under extreme
306
heat stress (Turner, 1992). Likewise, the low PS of 0.89 to 1.27 of cows in both treatment groups
307
suggests that they were not substantively heat stressed, and rump temperatures were also within
308
the range of mildly heat stressed cows (Di Costanzo et al., 1997). This low level of observed
309
heat stress is probably because there was consistent night-time cooling, and because the cows 13 Page 38 of 53
310
were fully shaded from the sun with access to bunk line misters (West, 2003; Gaughan et al.,
311
2008). It is clear that feeding the CE had little or no effect on heat stress of our cows since RR, PS
313
and RT were not influenced. That the RR of both groups was lower at 17:30 than at 2:45 h
314
differed from expectations due to temperatures at those times of day (Di Costanzo et al., 1997).
315
In contrast, the PS pattern for both groups was highest at 17:30 h, the hottest time of the day, and
316
the lowest at 09:15 h probably due to cows having recovered during the cooler night. At 2:45 h
317
little TMR remained in the feed bunks and very few cows were eating, with the majority in the
318
open drylot where more CED cows were lying versus standing. As Hillman et al. (2005) found
319
that the body temperature of cows rises when they are lying, and that they tend to stand and seek
320
cooling when core body temperature rises above 38.9°C, the higher number of CED cows lying
321
at 02:45 h suggests that CE may have reduced core body temperatures and heat stress, at least at
322
this time.
323
4.2. Production responses to citrus extracts
te
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312
Lack of a treatment difference on DM intake was not unexpected because studies with
325
limonene fed in concert with other EO have shown no DM intake effect (Benchaar et al., 2006,
326
2007), or a slight trend to a reduction (Santos et al., 2010). Vitamin C supplementation also does
327
not appear to effect DM intake (Weiss, 2001; Chaiyotwittayakun et al., 2002; Weiss et al., 2004).
328
While there are no in vivo studies that have only fed limonene, there is little support from in
329
vitro studies that limonene affects microbial fermentation, feed efficiency or nutrient utilization
330
to support milk production (Dorman and Deans, 2000). This is consistent with our similar
331
dietary NEL density (7.18 versus 7.08 MJ/kg) of the Control and CED TMR (Table 4). In a
332
review of citrus by-product feeding, Bampidis and Robinson (2006) reported that adding citrus
333
by-products to diets of lactating dairy cows did not change milk yield or composition if the diets
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14 Page 39 of 53
334
had a similar nutrient profile, which is also consistent with our finding that there were no
335
production differences between the Control and CED groups.
336
4.3. Milk somatic cell count (SCC) response to citrus extracts Based on our SCC differences between treatments, the CED diet appears to have reduced the
338
incidence and/or extent of mastitis. Indeed Lund et al. (1994) found a high genetic correlation
339
between SCC and clinical mastitis (0.97), concluding that SCC is an indicator of clinical
340
mastitis. In addition, increased SCC has been shown to be positively correlated with increased
341
mastitis risk (Ward and Schultz, 1972; Kirk, 1984; Barkema et al., 1998), and Harmon (1994)
342
found that, in cows with higher SCC, lactose tends to leak out of the infected mammary gland
343
into the blood, resulting in less milk lactose, which is consistent with the slightly lower lactose
344
output of Control cows, which also had higher SCC.
345
4.3.1. Effects of feeding citrus pulp or limonene in an EO blend on milk SCC
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337
When lactating ewes were fed whole citrus pulp at 100, 200 and 300 g/kg DM of their ration,
347
Jaramillo et al. (2009) reported lower milk SCC. Giannenas et al. (2011) fed an EO blend, which
348
included limonene, to dairy ewes at three levels. At day 50, there was no change in SCC but, at
349
day 100, ewes fed the highest EO treatment had lower SCC and, at day 150, all EO treatments
350
had a lower SCC, with ewes fed the highest EO level having a lower SCC than the other ewes.
351
This suggests a dose-dependent effect to the EO blend containing limonene which is magnified
352
with longer feeding time.
353
4.3.2. Effects of feeding vitamin C on milk SCC
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d
346
354
In contrast to EO, there has been substantial amount of research on impacts of dietary
355
Vitamin C supplementation on udder health, especially mastitis. When pathogenic
356
microorganisms enter the mammary gland through the streak canal an inflammatory response is
357
triggered in the infected gland, thereby increasing the number of reactive oxygen species (Ranjan 15 Page 40 of 53
et al, 2005). Since two lines of defense are occurring at this time (i.e., sequestering of
359
antioxidants and recruiting of leukocytes (Ranjan et al., 2005; Harmon, 1994)), antioxidants such
360
as vitamin C can be utilized in high quantities to inactivate reactive oxygen species to decrease
361
their concentrations in plasma and the infected mammary quarter (Harmon, 1994). Early studies
362
showed that vitamin C is quickly degraded in vitro and in vivo (Knight et al., 1941; Vavich et al.,
363
1945), presumably by rumen microbes. However Hidiroglou (1999) found that cows fed vitamin
364
C or ethyl cellulose coated vitamin C (i.e., ruminally protected), or had vitamin C infused
365
abomasally, all had elevated plasma amino acid (AA) concentrations, but cows fed uncoated
366
vitamin C had the smallest increase. This supports the probability that not all dietary vitamin C
367
is degraded in the rumen. Weiss et al. (2004) and Ranjan et al. (2005) showed that infected
368
mammary glands increase vitamin C utilization, and Weiss et al. (2004) also showed that
369
mammary quarters challenged with E. coli had decreased levels of vitamin C in milk and plasma
370
due to an inflammatory response which increased AA oxidation. This suggests that depletion of
371
vitamin C in plasma and milk occurs during infection, rather than a low vitamin C status per se,
372
which allows a more severe infection to occur.
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358
373
Although cattle are thought to synthesize adequate vitamin C to maintain healthy mammary
374
gland function, vitamin C synthesis is not up-regulated when cows are under immunological
375
stress (Weiss et al., 2004), but vitamin C uptake and oxidation is increased in effected tissues.
376
Thus it is possible that in a generally healthy mammary gland, such as was the case of cows in
377
our study based upon relatively low overall milk SCC counts, supplemental vitamin C may have
378
helped maintain a high antioxidant/pro-oxidant ratio, thereby aiding in prevention of oxidative
379
damage.
380 381
5. Conclusions 16 Page 41 of 53
382
Supplementing the diet of dairy cows with a commercially available citrus extract decreased
383
SCC in generally healthy dairy cows under mild heat stress conditions. However measures of
384
heat stress and animal production were not substantively treatment impacted.
386
ip t
385 Acknowledgements
The authors thank all who volunteered to help: Grace Cun, Stacy Wrinkle, Nadia Swanepoel,
388
Blanca Comacho and Emma Robinson. We are grateful to Stacy Wrinkle and Pablo Chilibroste
389
for sharing their knowledge on heat stress behaviors, and to William Van Die for allowing us to
390
conduct the study on his dairy.
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391 References
393
Ali, A.K.A., Shook, G.E., 1980. An optimum transformation for somatic cell concentration in
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AOAC, 1997. Official Methods of Analysis of AOAC International, 16th ed. Association of
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AOAC, 2006. Official Methods of Analysis of AOAC International, 18th ed. Association of Official Analytical Chemists, Arlington, VA, USA. Bampidis, V.A., Robinson P.H., 2006. Citrus by-products as ruminant feeds: A review. Anim. Feed Sci. Technol. 128, 175-217.
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Barkema, H.W., Schukken, Y.H., Lam, T.J.G.M., Beiber, M.L., Wilmink, H., Benedictus G.,
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production, and milk composition in dairy cows. J. Dairy Sci. 89, 4352–4364. Benchaar, C., Petit H.V., Berthiaume, R., Ouellet, D.R., Chiquette, J., Chouinard, P.Y., 2007.
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Jones, J.B., 2001. Laboratory Guide for Conducting Soil Tests and Plant Analysis, CRC Press LLC, Boca Raton, FL, USA. pp. 227-228. Kirk, J.H., 1984. Programmable calculator program for linear somatic cell scores to estimate mastitis yield losses. J. Dairy Sci. 67, 441-443.
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in dairy cows through sprinkler and fan cooling. Appl. Eng. in Agric. 8, 251-256. Tyrrell, H.F., Reid, J.T., 1965. Prediction of the energy value of cow's milk. J. Dairy Sci. 48, 1215-1223. Vavich, M.G., Dutcher, R.A., Guerrant, N.B., 1945. Utilization and excretion of ingested ascorbic acid by the dairy cow. J. Dairy Sci. 28, 759-770.
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Ward, G.E., Schultz, L.H., 1972. Relationship of somatic cells in quarter milk to type of bacteria and production. J. Dairy Sci. 55, 1428-1431. Weiss, W.P., 2001. Effect of dietary vitamin C on concentrations of ascorbic acid in plasma and milk. J. Dairy Sci. 84, 2302-2307.
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Weiss, W.P., Hogan, J.S., Smith, K.L., 2004. Changes in vitamin C concentrations in plasma
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West, J.W., 2003. Effects of heat-stress on production in dairy cattle. J. Dairy Sci. 86, 2131-
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Table
Table 1 Ingredient and chemical composition of Control and orange extract (CED) total mixed rations. Control
CED
167 72 60 159 144 33 19 5 64 71 11 23 36 22 33 38 14 29
164 72 60 160 145 34 19 5 64 71 11 23 36 21 33 38 0.16 14 29
SEM
P
cr us
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d
M
Alfalfa, hay Cottonseed, whole uplandb Cottonseed, cracked pimab Almond, hullsb Canola, meal - solventb Wheat, strawb Mineral, premixb Limestone, crushedb DDGSb Corn gluten, pelletsb Fat, rumen inert b,c Molasses, liquidb Alfalfa, fresh chop Wheat, whole crop silage Corn, flaked grain Corn, whole crop silage “Cristalfeed® Gold Rush” d Tomato, pomace - wet Whey, liquid
ip t
Ingredient composition (g/kg DM) a
574
578
1.7
0.36
Crude protein ADICP (g/kg CP)e aNDF aNDFom ADFom Lignin(sa) Starch Sugarsf Crude Fat
172 64 335 316 230 5.0 125 37 54
170 66 334 317 231 5.2 144 37 55
1.5 1.5 2.3 2.0 3.3 0.4 7.3 0.4 0.4
0.37 0.50 0.88 0.69 0.82 0.09 0.25 0.51 0.31
Ash Calcium Phosphorus Magnesium Potassium Sulfur Sodium Chloride
94 9.3 5.4 3.0 19.3 2.9 2.8 6.5
93 9.8 5.5 3.1 19.2 2.9 2.8 6.6
0.2 0.05 0.06 0.02 0.15 0.04 0.04 0.11
0.07 0.02 0.63 0.27 0.66 0.77 0.92 0.75
Chemical composition (g/kg DM) Dry matter (g/kg)
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1 2 3
1 Page 48 of 53
71.5 39.4 212 13.6 0.41 0.42
0.311 0.336 17.1 0.08 0.018 0.005
a
TMR ingredients listed in the order in which they entered the mixer. Ingredients combined to create a premix which was added to create the TMR. c EngerG II, Virtus Nutrition, Corcoran, CA, USA. b
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Acid detergent insoluble CP. Free glucose, sucrose and fructose.
M
f
VéO Premium (Cristalfeed® Gold Rush; Reference ST 232 P2; Phodé, Terssac, France).
d
e
te
d
0.86 0.63 0.35 0.73 0.70 0.31
Ac ce p
4 5 6 7 8 9 10
71.6 39.7 245 13.6 0.43 0.43
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Zinc (mg/kg DM) Manganese (mg/kg DM) Iron (mg/kg DM) Copper (mg/kg DM) Cobalt (mg/kg DM) Selenium (mg/kg DM)
2 Page 49 of 53
Table 2 Effects of feeding citrus extract (CE) on DM intake, milk yield, milk components and body condition score. SEM
DM intake (kg/d; n=8)
25.39
25.18
0.390
Yield (kg/d; n=654) Milk Fat True Protein Lactose Energy (MJ/d)
47.04 1.66 1.34 2.25 135.3
47.46 1.67 1.35 2.27 136.6
0.270 0.013 0.007 0.013 0.826
Components (g/kg milk) Fat True protein Lactose SCC (cells/µL) SCC (linear score)
35.4 28.6 47.8 196 2.30
0.13 0.46 0.08 0.06 0.19
us
an 35.3 28.6 47.9 160 2.12
0.20 0.08 0.06 16.5 0.795
0.79 0.49 0.14 0.04 <0.01
2.59 0.027
0.016 0.0133
0.53 0.93
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d
0.62
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Body Condition Score (n=168) Average (units) 2.59 Change (units/28 d) 0.028
P
ip t
CED
cr
Control
3 Page 50 of 53
CED
SEM
Respiration Rate (breaths/min) 2:45 h 9:15 h 17:30 h
63.2 62.9 59.8
61.6 63.9 58.7
1.18 1.34 1.14
Panting Score (unit)b 2:45 h 9:15 h 17:30 h
1.09 0.89 1.27
1.12 0.92 1.22
0.037 0.038 0.034
0.55 0.46 0.28
68.6 39.7 27.3
0.038 0.038 0.034
< 0.01 0.79 0.18
34.1
34.1
0.076
0.50
1.15
1.15
0.015
0.88
te
Rump temperature (°C)
a
0.36 0.59 0.50
Ac ce p
Locomotion score (unit; n=654)
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M
Lying versus standing (cow/100 cows)c 2:45 h 53.7 9:15 h 41.1 17:30 h 33.7
P
ip t
Control
d
Table 3 Effects of feeding citrus extract (CE) on respiration rates, panting scores, lying versus standing behavior at three times of the daya, as well as skin temperature and locomotion scores in early lactation cows (n=168).
Midpoint of the period starting 75 min prior and ending 75 min after the listed time. A measurement on a scale of 0 to 5 of the extent that a cow was is panting (Figure 2). c The proportion of cows scored as lying versus standing based on the position in which they were first found. b
4 Page 51 of 53
Table 4 Effects of feeding citrus extract (CE) on whole tract digestibility (g/kg) of some dietary nutrients (n=24), and partial energy balance (MJ/d). Control
CED
SEM
P
724
715
4.7
0.18
513
508
6.3
0.62
aNeutral detergent fiberb
501
492
Crude Protein
711
695
135.3
136.6
0.83
0.19
1.3
1.2
0.60
0.93
179.7
180.9
-d
-
7.18
-
-
Partial energy balance
an
Milk Change in BCS
M
NELc NEL densitye (MJ/kg DM)
7.08
a
cr
aNeutral detergent
fiberoma
7.5
0.41
7.3
0.14
us
Organic matter
ip t
Whole tract digestibility
Ac ce p
te
d
aNDFom expressed exclusive of residual ash. aNDF expressed inclusive of residual ash. c Net energy of lactation; calculated by summing maintenance, milk and change in BCS energy, where maintenance energy is 41.8 MJ/d assuming cow average body weight was 650 kg. d Net energy of lactation divided by measured DM intake. e Reported as listed values with arithmetic means of treatment means. b
5 Page 52 of 53
Figure
Figure 1. The 24 h pattern of ambient temperature and temperature humidity index (THI) during the 3 day period of behavioral observations in periods 1 and 2. Weather data was
Ac ce p
te
d
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recorded every 30 min. Graphs show the averages of each weather data point.
1 Page 53 of 53