PROJECT PROTOCOL
INVESTIGATORS:
PI: Dr. Carissa Wickens
STUDENT: Jessie Weir(Ph.D.)
PROJECT TITLE:
Modeling ammonia emissions from horses fed different crude protein
concentrations
PROJECT ID:
WEIR and WICKENS/2014
SPONSORS:
IFAS ANIM CARE #:
FACILITIES
INVOLVED:
 ESC
 HTU
START DATE:
END DATE:
11/28/2014 (trial will begin 12/1)
01/11/2015 (= last day horses will be on study)
HORSES NEEDED:
NUMBER:
AGE:
BREED:
HORSES:
10 geldings (9 study horses + 1 extra)
Mature (3-16 years of age)
QH and TB
 9 UF owned geldings
FEEDING
REQUIREMENTS:
PASTURE:
ACCESS:
NONE
HAY:
TYPE/AMT:
WEIGHED BY:
FED BY:
Bermudagrass (treatments)
Investigators (Jessie)
Investigators
GRAIN:
TYPE/AMT:
WEIGHED BY:
FED BY:
None
SUPPLEMENT
TYPE/AMT:
WEIGHED BY:
FED BY:
Triple Crown Lite (Ration balancer)
Investigators (Jessie)
Investigators
OTHER:
 Investigators will provide 210 small bales of Bermudagrass hay and
ration balancer
 Bedding needed for stalls, except during feces/urine collections (10
stalls x 10 days/period x 3 periods = 300 “stall days”)
 All grass in pens attached to arena stalls needs killed/removed
 Animal Sci.
 10-20 stalls + runs in arena
 10 stalls in T-barn area (temporary use)
 Full access to feed room in arena (hay
and supplement storage, weighing)
 Livestock scale at arena
 Other
 Indoor wash-rack for weighing of
feces/urine during collections
 Refrigerator near classroom
 Hot walker and/or round-pen(s) for
intermittent
exercise
• Arena stalls
overhead fans for cooling
1
HORSES:
#
NAME
BREED
Y.O.B.
1996
1
Cure My Wish (“Zman”)
TB
2
Lease TB?
TB
3
Cadet
TB
2010
4
Go for Nick (“Vinnie”)
QH
2008
5
Boggie Fever (“Bucky”)
QH
1996
6
UF Smokin Tuff (Watson)
QH
2009
7
Potentially Dangerous (“Dan”)
Miss Roman Casey gelding
(“Horseradish”)
Billy-Lease?
QH
2003
QH
2009
QH
2005
At Great Expense (“Kip”)
TB
2003
8
9
Extra
SUMMARY
EXPERIMENTAL
PROCEDURE:
UF ID
 This project is a digestibility study involving the collection of feces and urine after a
period of dietary adaptation.
 Important: feed will be weighted and intake quantified by investigators
 Important: no treats are allowed
 Important: try to maintain a quiet environment during collections (see
“Experimental Timeline”) and leave stall lights off at night at all times.
 Objectives:
1) Determine the effects of dietary CP concentration on potential NH3-N losses
from feces and urine of horses fed an all forage diet.
2) Develop an ammonia emissions model from equine operations as affected by
feeding practices and bedding type.
 Hypotheses: increasing dietary CP concentrations will increase the potential NH3-N
losses from feces and urine.
 Experimental design: 3 X 3 Latin Square, 3 periods and 3 treatments
 3 treatments: n=3 geldings/treatment/period
1) Control = providing 100% NRC Crude Protein (Bahia hay)
2) Medium = providing 150% NRC Crude Protein (Bahia/Tifton-85 combo)
3) High = providing 200% NRC Crude Protein (Tifton-85 Bermudagrass hay)
All diets will include a ration balancer (Triple Crown Lite)
 3 Periods: each 14-day period will consist of:
– Day 1 – 11 = Diet Acclimation Phase
– Day 11 = all bedding removed from stall by evening; harnesses put on horse
at 11PM; feces and urine collected from 11PM to 7AM will be tossed out;
official collection begins 7AM on day 12.
– Day 12 – 14 = Collection phase; collection of all feces and urine voided
beginning 7AM on day 12 and ending 7AM day 15
– Day 15 = at 7AM, diets will be switched and start the next Period
 Daily rations will be prepared and fed by investigators 3x/d at: 7AM, 3PM, & 10PM.
 During the collection phase, harnesses will be emptied 3x/d at: 7AM, 3PM, & 10PM
 All bedding will be removed from stalls during each collection phase.
 Total Study duration: ~6 weeks
2
EXPERIMENTAL
TIMELINE:
SUN
MON
TUES
WED
NOV 23
24
25
30
DEC 1
Start
Period 1
geldings in
arena stalls
2
8
*Collect BW
9
7
THURS
26
27
Ship geldings
from ESC; put Thanksgiving
in T-barn
*Collect BW
3
10
16
17
22
*Collect BW
23
24
28
29
COLLECTION Harness off
7AM
DAY 3
*Collect BW
30
31
21
4
SAT
28
29
5
6
Judging Clinic
classes end
14
15
COLLECTION Harness off
7AM
DAY 3
*Collect BW
Start
Period 2
FRI
11
12
13
Harness on COLLECTION COLLECTION
11PM
DAY 1
DAY 2
will toss out Begin 7AM
11pm-7am
sample
18
19
20
25
26
27
Christmas COLLECTION COLLECTION
DAY 1
DAY 2
Harness on
11PM
Begin 7AM
will toss out
11pm-7am
sample
JAN 1
2
3
New Year’s
Day
Start
Period 3
4
5
*Collect BW
6
7
13
14
classes start
11
12
COLLECTION Harness off
7AM
DAY 3
*Collect BW
8
9
10
Harness on COLLECTION COLLECTION
11PM
DAY 1
DAY 2
will toss out Begin 7AM
11pm-7am
sample
15
16
17
3
SUMMARY OF
NEEDS FROM HTU:
 Immediate needs:
– Storage for 210 small bales of Coastal Hay; – we will need room for 3 stacks
of 70 bales (feeding different amounts of CP, but with same day)
– NOTE: feed room will also need to accommodate a pallet of ration balancer,
and stainless steel shelf (for scales and other items).
– Supply of wood shavings bedding (10 stalls x 10 days/period x 3 periods = 300
“stall days”); Stalls will need bedding all days except during the collection
phases
– Grass in outdoor runs needs killed & removed; also no hay in runs or stalls
from previous occupants
– Livestock scale in arena barn
– 2 water buckets per stall (10 stalls; all 20 stalls if possible)
– 1 feed bucket per stall (20 stalls)
– Stainless steel table in wash rack area, table/shelf in feed room
 Horses should be up to date on vaccinations, deworming and farrier care
(investigators will coordinate with staff at ESC prior to geldings being shipped to
HTU)
 Ensure proper function/maintenance on walkers and round pens
 Assistance with stall cleaning and routine barn maintenance
 Regular emptying of manure spreader
 Most, if not all, feeding will be performed by investigators
 Monitor horses daily—report problems on UF-owned and Leased horses to Jessie or
Dr. Carissa Wickens
– Look for: unusual feed refusals, no drinking
– Any illness requiring hospitalization or treatment (e.g. colic)
– Follow HTU S.O.P. for any veterinary services required
– If problems notify Jessie (cell 772-766-3389) and/or Dr. Wickens (cell 352359-4550)
4
DIETS:
 Important to get a %DM on all hays prior to feeding in EACH period
– 3 days before period is to start, collect samples to dry in oven (see “SAMPLE
HANDLING” section for how to process feeds)
 Use first 4 days of the 11-day Diet Adaptation Phase to transition horses to new diet
(the hay part); gradual transition is NOT needed for the mineral or oats in the new diet
(that can be done immediately).
– Days 1 & 2: Feed 50% new hay + 50% of previous hay
– Days 3 & 4: Feed 75% of new hay + 25% of previous hay
– Days 5 – 14: Feed 100% of new hay
– NOTE: this gradual adaptation is only important when going from Coastal to the
Coastal/Bahia mix diet (not necessary for the Coastal to Coastal diets)
 Feed amounts according to table below (or Excel spreadsheet)
 Feed 3x/day at 7:00AM, 3:00PM, and 10:00PM
– Feed 1/3 of daily total amount at each feeding
 Record amounts provided at each feeding
 For hay and ration balancer, record weight to nearest 0.01 kg; can pre-weigh if
have enough hay nets/buckets.
Adding corn oil to increase DE
 Observe/collect and record ORTS at the 7AM feeding
– No need to save orts during the Adaptation Phase (just document quantity)
– Document orts during Collection Phase and SAVE if orts are ≥ 100 grams
 WATER:
– Check and refill water buckets at each feeding. Rinse water buckets and replace
with clean water 1x/d
FEED
Amounts (DM basis) per feeding (kg/ kg BW/ feeding)
Control
Medium
High
Bahia Hay
Tifton-85
Triple Crown Lite
Corn oil
5
TREATMENT
ASSIGNMENT:
TREATMENT
HORSE
Zman
1st PERIOD
2nd PERIOD
3rd PERIOD
CONTROL
MEDIUM
HIGH
Cadet
Horseradish
Bucky
Vinnie
Watson
Dan
Billy
?? Lease Horse
Kip
Note to self: get BW’s on these guys before assigning them to treatment. And/or group them by age and then
assign.
6
GENERAL
PROCEDURE:
GENERAL
PROCEDURE,
DATE:
DESCRIPTION:
BEFORE Nov 26  Get leased gelding lined up
 Have ESC/HTU verify all vaccines, deworming, farrier = up to date
 Get all geldings started on eating hay (if not doing so already)
 Order supplies, feeds, etc
 Label fecal bags, urine cups, etc.
 Organize other supplies needed for study (eg, scales, harnesses,
hay nets, buckets, etc)
Nov 26
 Ship geldings to HTU; house in arena stalls
 Get used to being in stalls, eating hay; can feed 2x/day
 Collect body weights
 Collect initial feed samples – determine %DM on hays
 Assign horses to treatment schedule
PERIOD 1
 Feed 3x/day on respective diets (1/3 of daily total per feeding)
Dec 1-14
 Follow acclimation schedule under “Diets” for first 4 days, if
Adaptation
needed)
Phase
 RECORD AMOUNTS of feed given
 RECORD AMOUNTS of ORTS left behind each day at 7AM
 Provide a minimum of 15-30 min/day turnout in stall runs (or
round pen/hot walker)
Dec 8
 Re-weigh horses; adjust diet if necessary
Dec 11
 Remove ALL bedding from stall in evening
 Put harnesses on at 11:00PM
 NOTE: will discard any feces and urine collected from 11PM on
Dec 11 to 7AM on Dec 12.
Dec 12- 14
 RECORD AMOUNTS of feed given
Collection
 RECORD AMOUNTS of ORTS left behind each day at 7AM
Phase
 Collect feces and urine @ 7:00AM, 3:00PM, and 10:00PM (NOTE:
each 24-hr composite will be 3PM + 10PM + next day’s 7AM)
 Collect grab samples of each feed when weighing diets (1
composite per feed ingredient, per period)
 See “SAMPLE HANDLING” for how to process feeds, feces, urine
 Provide a minimum of 15 min/day turnout in stall runs
 Take harness off so horses don’t roll/break them
 Have 2-3 people outside to monitor for excreta when
horses don’t have harnesses on
 HOW TO COLLECT FOR IN VITRO SAMPLE
Dec 10
 Collect hay & oat samples – perform DM analysis for Period 2
Dec 15
 Remove harnesses Dec 15 at 7:00AM (last collection)
 Collect body weights on horses (= end of period 1)
PERIOD 2
 Feed 3x/day on respective diets (1/3 of daily total per feeding)
Dec 15 – 25
 Follow acclimation schedule under “Diets” for first 4 days, if
Adaptation
needed)
Phase
 RECORD AMOUNTS of feed given
 RECORD AMOUNTS of ORTS left behind each day at 7AM
 Provide a minimum of 15-30 min/day turnout in stall runs (or
round pen/hot walker)
DATE:
DESCRIPTION:
Dec 22
 Re-weigh horses; adjust diet if necessary
7
continued…
Dec 24
 Collect hay & oat samples – perform DM analysis for Period 3
Dec 25
 Remove ALL bedding from stall in evening
 Put harnesses on at 11:00PM
 NOTE: will discard any feces and urine collected from 11PM on
Dec 25 to 7AM on Dec 26.
 RECORD AMOUNTS of feed given
 RECORD AMOUNTS of ORTS left behind each day at 7AM
 Collect feces and urine @ 7:00AM, 3:00PM, and 10:00PM (NOTE:
each 24-hr composite will be 3PM + 10PM + next day’s 7AM)
 Collect grab samples of each feed when weighing diets (1
composite per feed ingredient, per period)
 HOW TO COLLECT FOR IN VITRO SAMPLE
 See “SAMPLE HANDLING” for how to process feeds, feces, urine
 Provide a minimum of 15 min/day turnout in stall runs
 Take harness off so horses don’t roll/break them
 Have 2-3 people outside to monitor for excreta when
horses don’t have harnesses on
 Remove harnesses Dec 29 at 7:00AM (last collection)
 Collect body weights on horses (= end of period 2)
 Feed 3x/day on respective diets (1/3 of daily total per feeding)
 Follow acclimation schedule under “Diets” for first 4 days, if
needed)
 RECORD AMOUNTS of feed given
 RECORD AMOUNTS of ORTS left behind each day at 7AM
 Provide a minimum of 15-30 min/day turnout in stall runs (or
round pen/hot walker)
 Re-weigh horses; adjust diet if necessary
Dec 26 – 28
Collection
Phase
Dec 29
PERIOD 3
Dec 29 – Jan 8
Adaptation
Phase
Jan 5
Jan 8
Jan 9 – 11
Collection
Phase
Jan 12
SAMPLE
HANDLING:
1. Feces
 Remove ALL bedding from stall in evening
 Put harnesses on at 11:00PM
 NOTE: will discard any feces and urine collected from 11PM on Jan
8 to 7AM on Jan 9.
 RECORD AMOUNTS of feed given
 RECORD AMOUNTS of ORTS left behind each day at 7AM
 Collect feces and urine @ 7:00AM, 3:00PM, and 10:00PM (NOTE:
each 24-hr composite will be 3PM + 10PM + next day’s 7AM)
 Collect grab samples of each feed when weighing diets (1
composite per feed ingredient, per period)
 HOW TO COLLECT FOR IN VITRO SAMPLE
 See “SAMPLE HANDLING” for how to process feeds, feces, urine
 Provide a minimum of 15 min/day turnout in stall runs
 Take harness off so horses don’t roll/break them
 Have 2-3 people outside to monitor for excreta when
horses don’t have harnesses on
 Remove harnesses Jan 12 at 7:00AM (last collection)
 Collect body weights on horses (= end of period 3)
 Horses can go home!
 At each of the 3 daily collections: (perform at HTU)
1] Empty harness fecal compartment into plastic tub (tare the
tub before feces are added)
8
2]
3]
4]
5]
6]
Record total weight of feces (weigh to nearest 0.01 kg)
Mix/squish feces well.
Measure pH of feces
Save 10% (wet weight) subsample.
Place subsample in labeled whirlpak bag; Bags should be
labeled with project ID, horse, period #, day #, collection time,
and diet. For example: WEIR-2014
Zman
P1-D1-3PM
Control
*NOTE: no 7AM bag is needed – just put 7AM feces (10%)
directly in 24-h composite feces bag.
7] Store subsample at 4°C at HTU until 24-hr composite is created
 Daily (24-hr) composites: (perform at HTU)
1] After each 7:00AM collection, mix feces from previous day’s
3PM & 11PM subsamples with today’s 7AM subsample to
create 24-hr composite. Mix well.
2] Save ONE fecal composites (each ~10% wet weight) for
analysis (for nutrient evaluation)
3] Store at -20°C in ANS Lab. Use boxes to group by period.
 NOTE: any feces voided while harness is off need to be weighed
and recorded (but not saved).
SAMPLE
2. Urine
 At each of the 3 daily collections: (perform at HTU)
1] Empty harness urine compartment into plastic pitcher, then
into a graduated cylinder.
2] Record total volume AND weight of urine voided.
3] Mix urine well.
4] Measure pH of urine
5] Save max. 200 mL urine
6] Place subsample in labeled plastic snap-lid cup
– Label cups similar to feces bags (see above).
– Will need a snap-lid cup for 7AM, 3PM & 10PM
collections + 1 yellow screw-top cup for 24-h composite.
7] Add 20μL HCl (12 M) per mL urine (add slowly – foaming!)
– Each “pump” of HCl = 1 mL (leave pump set at 1 mL)
– 200 mL urine = add 4 mL HCl
– 150 mL urine = add 3 mL HCl
– 100 mL urine = add 2 mL HCl
8] Store subsample at 4°C at HTU until 24-hr composite is created
 Daily (24-hr) composites: (perform at HTU)
1] After each 7:00AM collection, mix urine from previous day’s
3PM & 11PM subsamples with today’s 7AM subsample to
create 24-hr composite. Mix well.
2] Save maximum 200-mL composite urine for analysis.
3] Store at -20°C in ANS Lab. Use boxes to group by period.
 NOTE: any urine voided while harness is off need to be estimated
and recorded (keep a plastic bucket handy!!)
3. Feeds
 Refer to “DIETS” section for information on feeding (page 5)
9
HANDLING
continued…
 BEFORE the start of the study: All feed ingredients should be
sampled and analyzed (by Dairy One) for nutrient content
– Hay: core 20 bales for each type of hay; submit ~1 Lb of each
hay for analysis & store the rest in the walk-in fridge in ANS Lab.
– NOTE: all feed samples should be oven-dried before storage in
walk-in fridge (see DM determination below).
– Dairy One analysis should be conducted using wet chemistry
(not NIRS).
 4 Days Before the Start of Each PERIOD: Determine %DM on hays
for feeding purposes only
– Nov 26 (use for Period 1), Dec 10 (period 2), & Dec 24 (period
3)
– Collect grab sample (~ 1 Lb) of Bahia and Tifton-85
– DM determination: (perform in ANS Lab)
1] Record weight of a clean, empty paper bag.
2] Put feedstuff in bag and record “wet” weight + bag
3] Dry in 60°C oven for 3 days
4] Record dry weight of feed + bag.
5] Subtract weight of empty bag to get wet weight and dry
weight of feed.
6] Calculate %DM = [dry wt feed/wet wt feed]*100
 During Each COLLECTION PHASE: Obtain samples of both hays for
nutrient analysis
1] Take a grab sample (1-2 handfuls) of each hay on each day of
the 3-day collection phase.
2] Place in labeled paper bag (NOTE: the same bag can be used for
the entire 3-day collection)
3] At the end of the collection, store bag in walk-in cooler in ANS
Lab; sample will be sent to Dairy One for nutrient analysis.
4] Repeat for all 3 Periods.
4. Body Weight
SAMPLE
HANDLING
continued…
5. In vitro NH3
emissions
sampling
 Livestock scale in HTU arena (accurate ± 0.5 kg)
 Nov 26-29 – use BW for calculating feed offered during Period 1
 Dec 8 – reweigh to verify BW for period 1
 Dec 15 – use BW for calculating feed offered during Period 2
 Dec 22 – reweigh to verify BW for period 2
 Dec 29 – use BW for feed offered during Period 3
 Jan 5 – reweigh to verify BW for period 3
 Jan 12 – final BW
 Collect, weigh and record FRESH feces and urine once from each
horse
 Save X amount subsample in separate whirlpak bag (feces) or
Nalgene bottle (urine)
 Store subsample at 4°C at HTU until moved to -20°C in ANS Lab.
10
LIST OF SUPPLIES:

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


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

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

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Hay (Tifton-85)
Hay (Bahia)
Triple Crown Lite
Corn oil
Zip-lock bags, snack size?
Collection Harnesses (harness, chest strap,
blanket, extra pieces)
Trash bags, 3-mil, 42-gal (to line fecal
compartment)
Duct tape, heavy-duty (attach bag to fecal
compartment)
Ice bags (double-bagged, line urine
compartment)
Zip ties, large (8”) (for attaching urine
bags)
Zip ties, small (4”) (for temporary sealing
of urine bags)
Small nippers (for cutting zip ties on urine
bags; 4.5” diagonal pliers)
Scissors (for various)
Cheese cloth (filtering urine)
Rubber bands (bind cheese cloth to
graduated cylinder)
4-L plastic graduated cylinder (x 3; for
urine)
1-L plastic graduated cylinder (x 2; for
urine aliquoting)
Plastic pitchers (urine mixing)
Plastic spoons (urine mixing)
Plastic dish pans (feces weighing)
Palpation sleeves (hand-mixing feces)
Latex gloves (working with feces/urine)
Whirl pak bags (42-oz; feces subsamples)
Urine specimen cups (snap-top “milk”
cups for each collection)?
Urine specimen cups (500 ml Nalgene
bottles for 24-hr composites)
Sticky labels (label urine & fecal bags
during collections)
Magnetic clips (to hold sticky labels on
stall door)
 Baby butt wipes
 Paper towels
 Extension cord and multi-cord adapter (for
feces/urine scales)
 Feed scales (hay scale + scale for ration
balancer)
 Feces/urine scales (x 2)
 Large muck tubs (to weigh hay)
 Paper bags (feed samples, orts)
 White buckets/lids (for feeding oats)
 Feed tubs (1/horse in stall)
 Water buckets (2/horse in stall)
 Feed scoops
 Fly spray and spray bottle
 Hand brooms and dustpans
 Horse halters and lead ropes
 pH meter
 Clip boards
 Stall door labels
 Stuff to clean gelding sheaths
(Acepromazine, syringe + needle, cotton,
sheath wash or Ivory soap)?
 Cup to hold pens, sissors
 Data sheets – feed weighing (hay, oats,
minerals, salt)
 Data sheets – Orts
 Data sheets – Urine
 Data sheets – Feces
 Data sheets- pH of urine and feces
 Study diary (health observations)
11
2