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Pregnant Mares Grazing a Novel Endophyte-Infected Tall Fescue Foal Normally
Journal of Equine Veterinary Science 74 (2019) 56e64
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Original Research
Pregnant Mares Grazing a Novel Endophyte-Infected Tall Fescue Foal Normally Karen McDowell a, *, Victoria Taylor a, Tim Phillips b, Krista Lea b, Ray Smith b, Glen Aiken c, Michael Barrett b a b c
Department of Veterinary Science, University of Kentucky, Lexington, KY Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY USDA-ARS-FAPRU Lexcington, KY
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 July 2018 Received in revised form 4 October 2018 Accepted 6 December 2018 Available online 18 December 2018
These experiments were conducted to determine if pregnant mares grazing a novel endophyte tall fescue (NETF) pasture would have satisfactory foaling outcomes. The hypotheses tested were that mares grazing the NETF pasture or an orchardgrass/Kentucky bluegrass (OGBG) pasture would have statistically equivalent (1) serum progesterone, estradiol, and prolactin concentrations, (2) blood chemistry analyte concentrations, and (3) palmar artery sizes. In addition, foals born on the respective pastures would grow at comparable rates. Over 4.5 months, during each of two summers, 12 foaling mares were placed on either NETF or OGBG pastures (n ¼ 6 mares/pasture/year). There were no differences in the blood sample concentrations of progesterone, estradiol, prolactin, and chemistry analytes between the groups. Combined uterine placental thickness and placental weights were not different between the two groups of mares, and foals grew at equivalent rates. The diameter of the palmar artery of each mare was measured via Doppler ultrasonography as a bioassay for consumption of ergot alkaloids of fescue pastures. Diameters were not different between mares grazing NETF versus OGBG pastures. However, palmar artery diameters of nonpregnant mares grazing a pasture of toxic Kentucky 31 tall fescue were reduced compared to mares on the NETF or OGBG pastures. Pasture analyses demonstrated that the NETF was endophyte infected but produced no or trace amounts of ergovaline. In summary, there were no differences in any variable tested between mares grazing the new, NETF fescue pasture, compared with mares grazing a standard OGBG pasture. © 2019 Elsevier Inc. All rights reserved.
Keywords: Horses Fescue Novel endophyte Ergovaline Foals
1. Introduction Tall fescue (TF) (Lolium arundinaceum (Schreb.) Darbysh.) is one of the most widely grown perennial grasses in the world and covers approximately 15 million ha in the United States alone. It can be infected with an endophytic fungus, Epichlo€ e coenophiala (MorganJones and W. Gams) C.W. Bacon and Schardl, which, in a symbiotic relationship with the plant, produces a variety of potentially toxic alkaloids that may adversely affect grazing livestock [1,2].
Animal welfare/ethical statement: All experimental procedures were approved by the University of Kentucky Institutional Animal Care and Use Committee. Conflict of interest statement: The authors declare no conflict of interest. * Corresponding author at: Karen McDowell, Department of Veterinary Science, University of Kentucky, 108 Gluck Equine Research Center, Lexington, KY 405460099, USA. E-mail address: [email protected] (K. McDowell). https://doi.org/10.1016/j.jevs.2018.12.006 0737-0806/© 2019 Elsevier Inc. All rights reserved.
Ergovaline is the most abundant ergot alkaloid in TF [3]. In horses, the most frequently reported problems associated with endophyteinfected fescue consumption are in late pregnancy and include altered hormone concentrations, extended gestation, thickened placenta, placental retention, dystocia, birth of dysmature foals, increased foal and placental weights, and agalactia due primarily to decreased prolactin concentrations [4e6]. Although the alkaloids produced by TF are detrimental to grazing animals, they also confer benefit to the plant [7]. Hence TF cultivars containing a novel endophyte (NE) have been developed that continue to produce alkaloids that give the plant characteristics of drought tolerance, insect resistance, and increased vigor but that do not produce the alkaloids that are harmful to grazing animals. Therefore, the first objective of this experiment was to evaluate foaling mares grazing a new NETF pasture compared to grazing an orchardgrass/Kentucky bluegrass (OGBG) pasture. The second objective was to compare the sizes of the palmar arteries for mares
K. McDowell et al. / Journal of Equine Veterinary Science 74 (2019) 56e64
grazing NETF, OGBG, or toxic Kentucky 31 TF (KY31TF) pastures. Palmar artery constriction in horses has been shown to be a convenient and satisfactory bioassay for consumption of endophyte-infected fescue seed [8e11]. In addition, when pregnant and nonpregnant mares are exposed to toxic KY31TF seed, their palmar arteries constricted in a similar manner (KJ McDowell, unpublished). 2. Materials and Methods 2.1. Establishment of Pastures Four pastures at the University of Kentucky Agricultural Experiment Station in Lexington, KY, ranging from 2.7 to 3.2 ha, were used for these studies. The area where the pastures were established had previously been under a winter wheat-corn rotation for a number of years. The corn residue was mowed and then sprayed with glyphosate (4.7l per ha). The pastures were randomly assigned to be planted with either a mixture of “Potomac” orchardgrass (11 kg seed per ha) and Kentucky bluegrass (11 kg seed per ha), an NETF (22.5 kg seed per ha), or KY31TF infected with the common toxic endophyte (22.5 kg of seed per ha). Two pastures were planted with the OGBG, and one pasture each was planted with the NETF or KY31TF. The NETF, developed cooperatively by researchers in the Department of Plant and Soil Science at the University of Kentucky (TD Phillips) and at AgResearch in New Zealand, was an experimental cultivar of TF (KYFA9821), which contained an alternative strain of Epichlo€ e coenophiala, AR584. During the experiments, all pastures were mowed regularly to maintain the pasture height between 13 and 20 cm. This mowing would also prevent seed head development. Ambient temperature and rainfall during the experiments are shown in Fig. 1. 2.2. Experimental Procedures All experimental procedures were approved by the University of Kentucky Institutional Animal Care and Use Committee. The studies
57
were conducted over 2 years between March and September of each year. 2.2.1. Year 1 Light horse mares of mixed breeding, pregnant (n ¼ 12) and nonpregnant (n ¼ 4) were used. Pregnant mares had been pasturebred to foal during the summer, but exact breeding dates were unknown. In March, all the mares were placed on one of the OGBG pastures, used as a “holding pasture,” and supplemented with grass hay, free of ergovaline, as needed. While on the holding pasture, the mares were acclimated to all procedures, so that they would stand quietly in wooden stocks designed for mare handling. In the pregnant mares, fetal eye measurements were made via transrectal ultrasonography to estimate month of foaling [12], using a Sonosite Titan with a linear transducer, L52 10-5 Mz. The pregnant mares were assigned to be placed on either the NETF pasture or the second OGBG pasture such that the estimated foaling dates were equally distributed between the two pastures. The nonpregnant mares were assigned to be placed on the KY31TF pasture. These mares were used as a negative control group to document changes in palmar artery sizes when mares grazed a toxic endophyte infected KY31TF pasture. Mares were allowed to continuously graze these treatment pastures, except when they were in the sample collection pens, approximately 1 hour each day. They were not supplemented with hay and received only approximately 0.1e0.2 kg (a handful) of alfalfa-pelleted concentrate mix (approximately 1e3 times per week, depending on where they were in their estrous cycles) as an incentive to come to the collection pens for all procedures (Table 1). The left distal palmar artery of each mare was measured via pulsed-wave Doppler ultrasonography (Sonosite Titan with an 11 mm convex array 5/8 MHz transducer) during the first week of May, before being placed on the treatment pastures (May 5), and then during the first and third weeks of each month thereafter, as described by McDowell et al. [8e11]. Briefly, the lateral surface of the left foreleg, just proximal to the fetlock joint, was clipped free of hair. The transducer with coupling gel was placed between the suspensory
Year 1
PrecipitaƟon (cm)
PrecipitaƟon (cm)
Year 2
o
o
Air temperature ( C)
Air temperature ( C)
35 30 25 20 15 10 5 0 1
3
5
7
10
12
Week of Experiment o o
14
16
18
o
1 3 5 7
10 12 14 16 18 20 22
Week of Experiment o o
Max Temp C
Min C
o
Mean C
Fig. 1. Precipitation and ambient air temperature during the course of the experiment. The data were obtained from the University of Kentucky Research Farm Climate Data Center, Lexington KY, located approximately 0.4 km from the research station site.
58
K. McDowell et al. / Journal of Equine Veterinary Science 74 (2019) 56e64
Table 1 UK #3 Equine: University of Kentucky, custom mix alfalfa pellets. Production Formula Ingredient Name
Nutrient Composition Nutrient Name
Amount
Units
Pulverized oats Ground corn Soybean meal Dehydrated alfalfa Soy hulls Cane molasses Soy oil Copper sulfate Calcium carbonate White salt Zinc oxide Selenium Vitamin E
Digestible energy Crude protein Crude fat Crude fiber Ash Calcium Total phosphorus Salt Sodium Potassium Lysine Methionine Methionine þ cystine Threonine Sulfur Magnesium Chloride Manganese Zinc Iron Copper Selenium Cobalt Iodine Vitamin A Vitamin D Vitamin E Vitamin K menadione Vitamin B1 thiamine Vitamin B2 riboflavin Vitamin B3 niacin Vitamin B5 pantothenic acid Vitamin B6 pyridoxine Choline Vitamin B9 folic acid Biotin Vitamin B12 cyanocobalamin Dry matter Starch Vitamin C Nonstructural carbohydrates Acid detergent fiber Neutral detergent fiber Chromium Bulk density
2835.06 12.29 6.09 15.00 6.48 0.74 0.23 0.56 0.24 1.23 0.54 0.20 0.33 0.57 0.19 0.16 0.49 17.79 120.59 177.26 39.39 0.30 0.29 0.07 0.00 0.00 110.23 0.00 1.72 4.65 43.43 16.89 4.12 668.71 1.98 0.00 3.57 197.69 54.34 24.65 67.51 52.78 46.28 0.00 218.75
kcal/kg % % % % % % % % % % % % % % % % ng/g ng/g ng/g ng/g ng/g ng/g ng/g kIU/kg kIU/kg IU/kg mg/kg mg/kg mg/kg mg/kg mg/lb mg/kg mg/kg mg/kg mg/kg mcg/kg % % % mg/kg % % % kg/cu m
Makeup and nutrient content of the pellet mix offered to mares as an incentive to come from the pastures to the working pens.
ligaments and the flexor tendons to visualize the palmar artery and vein. Measurements of mean artery diameter (defined as the average of the longer and shorter axes of the elliptical shape of the vessel) were made at peak systole to reduce variation of measurements taken within and between horses. All machine settings were constant for all horses throughout the experiment. Three replicate scans were performed on each horse on each scan day. Jugular venous blood samples were collected during the first and third weeks of each month for hormone analyses and during the first week of each month for clinical chemistry analyses. Serum was stored frozen at 20 C until analyzed for estradiol, progesterone, and prolactin concentrations by the laboratory of Dr. Donald Thompson at Louisiana State University. Estradiol and progesterone were analyzed using PANTEX immunoassay
kits (Santa Monica, CA), and prolactin was analyzed as described by Colburn et al. [13]. The clinical chemistry panels were performed on fresh serum at the University of Kentucky Veterinary Diagnostic Laboratory (UKVDL). All the pregnant mares were allowed to foal in their respective treatment pastures. Mares and foals were weighed at the same time blood samples were obtained. Placentas were taken to the UKVDL for pathologic, bacteriologic, and virologic analyses. Palmar artery evaluations of the foaling mares were discontinued when the mares foaled. Ergovaline and its stereoisomer ergovalinine (referred together as ergovaline from here forward) concentrations in the NETF and the KY31TF pastures were determined on three occasions in June and on two occasions in July. Whole tillers clipped from 25 random plants from within each pasture were pooled, freeze-dried in a Botanique
Table 2 Evaluations for the novel endophyte tall fescue pasture, year 1. Date
6/3
6/23
6/30
7/7
7/23
Week on experimental pastures Ergovaline (ng/g)
4 ND
7 10
8 ND
9 ND
11 ND
Abbreviation: ND, no ergovaline was detected. Average ergovaline concentrations could not be calculated.
Average
Table 3 Evaluations for the KY 31 tall fescue pasture, year 1. Date
6/3
6/23
6/30
7/7
7/23
Average
Week on experimental pastures Ergovaline (ng/g)
4 190
7 240
8 440
9 270
11 330
294
K. McDowell et al. / Journal of Equine Veterinary Science 74 (2019) 56e64
59
Table 4 Evaluations for the novel endophyte pasture, year 2.
Percentage of each type of plant identified
Tall fescue analyses
Calculations
Date
4/23
5/6
5/20
6/3
6/18
7/8
7/22
8/5
8/19
9/11
Week of Experiment
2
4
6
8
10
13
15
17
19
22
Tall fescue KY Bluegrass Orchardgrass White Clover
93 6 0 0
96 2 0 0
93 0 0 0
93 1 0 0
93 0 0 0
91 0 0 0
84 4 0 0
82 0 0 0
91 0 0 0
89 0 0 0
Weeds Bare soil Ergovaline (ng/g) Endophyte (%)
1 2 d 60
1 3 d 70
0 8
0 8
0 9
d 63
1 7 d 60
d 94
d 86
1 12 107 70
1 18 d 80
2 7 211 81
2 10 d 81
Available foragea Percentage of tall fescue in available forage Ergovaline concentration in tall fescue (ng/g)
99 94 d
98 98 d
93 100 d
94 99 d
93 100 d
91 100 d
88 95 102
82 100 d
91 100 MD
89 100 d
Avg.
91 1 0 0 1 8 b
74 92 99 b
Abbreviation: MD, missing data. The entry for each date represents the average of 10 sampling grids taken on that date. Additional forages of ryegrass, timothy, and/or red clover were present in these fields totaling <10% of the types of plants identified; therefore, percentages do not add to 100. d, represent that ergovaline concentrations were below the reliable quantitation (<100 ng/g) of the assay. a Available forage is the total of the tall fescue, KY bluegrass, orchardgrass, and white clover. b Cannot calculate an average with values less than the reliable quantitation of the assay.
Model 18DX485A freeze drier (Botanique Preservation Co, Peoria, AZ) and ground to pass through a 1-mm mesh screen (Cyclotec 1093 sample mill; FOSS North America, Eden Prairie, MN). These samples were analyzed by Dr. Lowell Bush, Department of Plant and Soil Science, University of Kentucky, for ergovaline using the highperformance liquid chromatography fluorescence procedure of Yates and Powell [14] as modified by Carter et al. [15]. The results were reported on a dry matter basis. The limit of detection was 10 ng/g. On September 3 (week 18 of the study), the mares from the KY31TF pasture were moved back to the original OGBG holding pasture. Foaling mares stayed on their respective pastures until the experiment ended on September 30. 2.2.2. Year 2 Twelve pregnant light horse mares of mixed breeding were used. All mares were initially placed on the OGBG holding pasture as described previously. Doppler ultrasound measurements of the palmar arteries were taken weekly, beginning three weeks before the mares being placed on the treatment pastures. Blood samples were collected during the first and third weeks of each month for hormone and clinical chemistry analyses, using the same methods as for Year 1. On May 6 (end of week 3), six of the mares were placed
on the NETF pasture and six on the second “test” OGBG pasture. Sampling and data collection were performed as described previously, except that combined uterine and placental thickness (CUPT) was also measured using the linear ultrasound transducer [16,17]. Mares and their foals remained on the treatment pastures until the experiment ended on September 16. During the first and third weeks of each month, all pastures were evaluated by members of the University of Kentucky Horse Pasture Evaluation Program for plant species present. In addition, for TF, the percentage of plants infected with endophyte was determined and ergovaline concentrations were measured. Evaluations were performed within a 2 2 foot square at 10 locations in each pasture. Two TF tillers were taken at each location, combined, and placed on ice in a cooler until delivered to the testing facilities according to the methods described by Lea et al. [18]. For characterization of the fescue tillers, the percentage with endophyte infection was determined by the University of Kentucky Division of Regulatory Services using a Phytoscreen Immunoblot Kit (Agrinostics, Watkinsville, GA). Ergovaline was quantitated at the UKVDL toxicology section using ultraperformance liquid chromatography and fluorimetric detection. Briefly, fresh forage was frozen with liquid nitrogen, milled and extracted with a 2-propanol/lactic acid
Table 5 Evaluations for the orchardgrass/KY bluegrass “test” pasture, year 2. Date
4/23
5/6
5/20
6/3
6/18
7/8
7/22
8/5
8/19
9/11
Week of Experiment
2
4
6
8
10
13
15
17
19
22
Avg.
Percentage of each type of plant identified
Tall fescue KY Bluegrass Orchardgrass White clover Weeds Bare soil
0 9 89 0 1 2
0 32 60 0 2 6
0 8 77 0 1 15
0 7 72 0 1 21
0 9 68 0 2 22
0 5 71 1 2 22
0 15 44 0 4 38
0 7 53 0 4 37
0 20 42 0 3 36
0 18 27 0 2 54
Tall fescue analyses
Endophyte (%) Ergovaline (ng/g)
Endophyte and ergovaline could not be reported because of lack of tall fescue
Calculations
Available foragea Percentage of tall fescue in available forage Ergovaline concentration in tall fescue (ng/g)
98 92 85 79 77 77 59 60 62 45 73 0 0 0 0 0 0 0 0 0 0 0 Ergovaline concentrations could not be calculated because of the lack of tall fescue.
0 13 60 0 2 25
The entry for each date represents the average of 10 sampling grids taken on that date. Additional forages of ryegrass, timothy, and/or red clover were present in these fields totaling <10% of the types of plants identified; therefore, percentages do not add to 100. a Available forage is the total of the tall fescue, KY bluegrass, orchardgrass, and white clover.
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K. McDowell et al. / Journal of Equine Veterinary Science 74 (2019) 56e64
Table 6 Evaluations for the KY 31 tall fescue pasture, year 2. Date
4/23
5/6
5/20
6/3
6/18
7/8
7/22
8/5
8/19
9/11
Week of Experiment
2
4
6
8
10
13
15
17
19
22
Percentage of each type of plant identified
Tall fescue KY Bluegrass Orchardgrass White clover Weeds Bare soil
74 1 23 0 0 2
75 1 24 0 0 1
87 1 3 0 1 10
77 0 8 0 0 15
84 0 5 0 1 11
81 0 3 0 1 16
77 1 5 0 0 18
85 1 5 0 1 9
78 3 7 0 2 11
82 0 2 0 1 15
80 1 9 0 1 11
Tall fescue analyses
Ergovaline (ng/g) Endophyte (%)
d 21
217 20
564 38
419 53
d 45
130 30
113 42
200 43
MD 55
755 68
42
Available foragea Percentage of tall fescue in available forage Ergovaline concentration in tall fescue (ng/g)
98 76 d
100 75 163
91 96 539
85 91 380
89 94 d
84 96 125
83 93 105
91 93 187
88 89 d
84 98 737
Calculations
Avg.
b
89 90 b
The entry for each date represents the average of 10 sampling grids taken on that date. Additional forages of ryegrass, timothy, and/or red clover were present in these fields totaling <10% of the types of plants identified; therefore, percentages do not add to 100. d, represent that ergovaline concentrations were below the reliable quantitation (<100 ng/g) of the assay. a Available forage is the total of the tall fescue, KY bluegrass, orchardgrass, and white clover. b Cannot calculate an average with values less than the reliable quantitation of the assay.
solution using procedures adapted from Spiering [19]. The results were reported on a dry matter basis and the minimum quantitative sensitivity was 100 ng/g. 2.3. Statistical Analyses Data analyses were performed using SAS [20]. Chemistry analytes, hormonal data, palmar artery diameter measurements, and CUPT were analyzed with the Mixed Procedure as completely randomized designs with measurements repeated over time. Placental weights were compared using a one-way classification in a completely randomized design. Foal growth rates were analyzed with the regression procedure and then compared via the general linear model procedure. Data are presented as least square means plus or minus standard error of the mean, and differences are considered significant when P < .05.
3.1. Pasture Analyses In year 1, ergovaline was undetectable in the NETF samples except on one occasion where it averaged 10 ng/g. In the KY31TF pasture, ergovaline averaged 294 ng/g and ranged from 190 to 440 ng/g over the 5-week sampling period (Tables 2 and 3). The results of the pasture evaluations for year 2 are shown in Tables 4e7. These results indicate that, although TF represented most of the available forage in the NETF pasture (98%), and most of the NETF samples (73%) were infected with endophyte, ergovaline concentrations were minimal or not quantifiable. By contrast, TF represented most of the available forage in the KY31TF pasture (80%) and only 42% was infected with endophytes. Ergovaline concentrations averaged to 319 ng/g, except on three occasions, when the concentrations were below the reliable quantitation of the assay.
3. Results
3.2. Foaling Outcomes, Hormone Concentrations, and Clinical Chemistry
Based on data collected during year 1, modifications were made in animal and pasture sampling protocols, as well as the differences in the ergovaline and endophyte assays; therefore, results for years 1 and 2 are reported separately.
In Year 1 all but one of the mares foaled normally between May 10 and September 7, producing healthy live foals. One mare from the OGBG pasture presented with an unexpected and severe dystocia and was subsequently euthanatized by the attending
Table 7 Evaluations for the orchardgrass/KY bluegrass “holding” pasture, year 2. Date
4/23
7/22
8/5
8/19
9/11
Week of Experiment
2
15
17
19
22
Percentage of each type of plant identified
Tall fescue KY Bluegrass Orchardgrass White clover Weeds Bare soil
1 20 44 0 3 33
0 3 19 4 12 63
0 1 7 3 13 78
0 0 1 1 10 89
0 0 1 1 18 81
Tall fescue analyses
Endophyte (%) Ergovaline (ng/g)
Endophyte and ergovaline could not be reported because of the lack of tall fescue.
Calculations
Available foragea Percentage of tall fescue in available forage Ergovaline concentration in tall fescue (ng/g)b
65 2 d
26 0 d
11 0 d
2 0 d
3 33 d
Avg.
0 5 14 2 11 69
21 7 b
The entry for each date represents the average of 10 sampling grids taken on that date. Additional forages of ryegrass, timothy, and/or red clover were present in these fields totaling < 10% of the types of plants identified; therefore, percentages do not add to 100. d, represent that ergovaline concentrations were below the reliable quantitation (<100 ng/g) of the assay. a Available forage is the total of the tall fescue, KY bluegrass, orchardgrass and white clover. b Cannot calculate an average with values less than the reliable quantitation of the assay.
K. McDowell et al. / Journal of Equine Veterinary Science 74 (2019) 56e64
Year 2
120
70
100
60
Prolactin (ng/ml)
Prolactin (ng/ml)
Year 1
80 60 40 20 0 -6
-4
-2
40 30 20 10
0
-14
-12
-10
-8
-6
-14
-12
-10
-8
-6
-4
-2
0
4
8
Progesterone (ng/ml)
Progesterone (ng/ml)
-8
*
6 4
*
2 -14
-12
-10
-8
-6
-4
-2
3 2 1 0
0
300
-4
-2
0
-4
-2
0
400
250
Estradiol (pg/ml)
Estradiol (pg/ml)
50
0 -12 -10 -10 -14 -12
0
61
200 150 100 50
300 200 100 0
0 -14 14
-12 - -10 12 10
-8
-6
-4
-2
0
Weeks relative to foaling
-14
-12
-10
-8
-6
Weeks relative to foaling
Fig. 2. Serum prolactin, progesterone, and estradiol concentrations in pregnant mares grazing the novel endophyte (solid lines) or orchardgrass/bluegrass (dashed lines) pastures. As expected, serum prolactin and progesterone concentrations increased and estradiol concentrations decreased toward the end of gestation. There were no treatments by week of foaling interactions for any of the hormones for either year.
As expected, the CUPT measurements increased as gestation progressed (P < .0001; measured in year 2 only). However, they were not different between the mares grazing NETF or OGBG pastures (P ¼ .6243; Fig. 3). Placental weights were not different between mares foaling on NETF and OGBG pastures in either year (P ¼ .5975 year 1; P ¼ .6938 year 2) (Fig. 4). All placentas from both years were negative for pathogenic bacteria, leptospira, equine
1.2 1.0 CUPT (cm)
veterinarian. Mares on the NE pasture foaled between May 10 and September 7, giving them a mean of 57.5 days and a median of 54 days on the pasture. Mares on the OGBG pasture foaled between May 22 and July 29, with a mean of 57 days and a median of 64 days on their pasture. In Year 2, all of the mares produced live healthy foals between June 28 and September 14, giving them a mean of 79.5 days and a median of 67 days on their pastures. Mares on the NE pasture foaled between June 30 and August 19, with a mean of 75.1 days and a median of 65 days on their pasture, while mares on the OGBG pasture foaled between June 28 and September 14, with a mean of 84.8 and a median pf 72 days. Mare serum prolactin, progesterone, and estradiol concentrations are shown in Fig. 2 and all were within normal ranges for foaling mares. All three hormones in both years had significant effects of weeks relative to foaling: prolactin (P ¼ .0465 year 1; P ¼ .0017 year 2); progesterone (P ¼ .0655 year 1; P ¼ .0002 year 2); and estradiol (P ¼ .0017, P < .0001 year 2), as prolactin and progesterone concentrations increased and estradiol concentrations decreased toward the end of gestation. Progesterone was statically lower at weeks -2 and 0 in year 1 for mares grazing the OGBG pasture than for mares grazing the NETF pasture. None of the three hormones had a significant treatment by week of foaling interaction: prolactin (P ¼ .1349 year 1; P ¼ .7265 year 2); progesterone (P ¼ .5306 year 1; P ¼ .8183 year 2); and estradiol (P ¼ .7067 year 1; P ¼ .7063 year 2).
0.8 0.6 0.4 0.2 0.0 -18 -14 -12 -10 -8 -6 -4 -2 -1 Weeks relative to foaling
Fig. 3. Combined uterine and placental thickness (CUPT) in pregnant mares grazing the novel endophyte tall fescue (solid lines) or orchardgrass/bluegrass (dashed lines) pastures. There were no differences between the groups of mares grazing either pasture type.
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K. McDowell et al. / Journal of Equine Veterinary Science 74 (2019) 56e64
Year 1
4 3 2 1 0
5
Placental Weights (kg)
Placental Weights (kg)
5
NETF OGBG
Year 2
4 3
4. Discussion
2 1 0
the mares remained on the toxic fescue pasture (P < .0001). When mares were removed from that pasture, the palmar artery diameters returned to their pretreatment sizes.
NETF OGBG
Fig. 4. Placental weights for mares grazing and foaling on novel endophyte tall fescue (solid bars) or orchardgrass/bluegrass (striped bars) pastures. There were no differences in placental weights between the treatment groups for either year. NETF, novel endophyte tall fescue; OGBG, orchardgrass/bluegrass pasture.
herpesvirus, nocardioform bacteria, and equine arteritis virus (data not shown). No significant pathologic lesions were observed in any of the placentas. Foal growth rates, expressed as foal weight as a percentage of mare weight, are shown in Fig. 5. Foal growth rates increased with age (P < .0001 for each year) and were not different due to pasture type (P ¼ .4926 Year 1; P ¼ .6220). The serum analytes tested in the chemistry panel are shown in Table 8. In year 1, there were no differences for the serum analytes between mares grazing NETF versus OGBG pastures. In year 2, however, serum chloride was higher (P ¼ .0278) for the mares grazing NETF than for mares grazing OGBG. Examination of the data revealed that the differences were solely on one bleeding date (July 1). When the remaining aliquots of those samples were examined, it was observed that many of them had some degree of hemolysis, an apparent sample handling effect. When chloride data from that date were removed, there were no differences between mares on the NETF versus OGBG treatment pastures (P ¼ .1018). 3.3. Palmar Artery Diameters Palmar artery mean diameters are shown in Fig. 6. In each year, there were no differences in the mean luminal diameters of the mares grazing NETF versus OGBG and the artery sizes in those groups remained fairly constant throughout the experiments (treatment by week of experiment interactions: P ¼ .2423 year 1; P ¼ .1931 year 2). However, artery diameters of the mares grazing the KY31 toxic fescue in year 1 were significantly reduced during weeks 5 through 18 compared to weeks 1 and 3 or 20 and 22 and significantly reduced compared to the other two groups as long as
Pregnant mares consuming toxic KY31TF frequently have longer than normal gestation lengths, placentas that are heavier and thicker than normal and which may be retained, plus reduced foal viability [4e6,21]. Pregnancy may be prolonged by as much as 2e4 weeks [5,6,22]. The mares also typically have reduced serum estradiol and progesterone concentrations at foaling, as well as reduced serum prolactin concentration, causing lack of proper udder development and milk production [6,21,23e25]. In the present studies, all the pregnant mares grazing and foaling on the NETF pasture had satisfactory foaling outcomes, and all variables tested were similar between mares on the NETF pasture and on the OGBG pasture. Breeding dates were not known so gestation lengths could not be documented. However, all mares that foaled had normal placentas, and there were no retained placentas, red bags, or reduced foal viability. All the mares had udder development and foals were observed to nurse in a normal fashion throughout the experiments. The foals born to mares grazing the NETF pasture had similar growth rates to the foals born to mares grazing the OGBG pasture. Serum hormone concentrations were within normal ranges and changed as expected, with prolactin and progesterone increasing and estradiol decreasing as gestation progressed. However, mares grazing the NETF in year 1 had statistically elevated progesterone in weeks -2 and 0 relative to the mares grazing the OGBG pasture, which is likely due to normal variability rather than a specific treatment effect. The study was repeated 2 years in a row and the hypotheses and objectives did not change between years. However, after gathering and analyzing data in the first year, it was decided to modify the protocol slightly to gather additional data. In addition, the UKVDL had begun performing ergovaline analyses, and the University of Kentucky Horse Pasture Evaluation Program began surveying enough local farms that we thought it advantageous, both for us and for the local farms, to use the same laboratory and the same personnel as our surrounding commercial horse farms. During the experiments, all pastures were mowed regularly, demonstrating that the pastures were not overstocked and that there was more than adequate forage available for the animals to graze throughout the experiments without the need for supplemental hay or grain. In a prior study, McDowell et al. [10] showed that ambient temperature or relative humidity was not correlated with vasoconstriction, heart rate, respiration rate, or rectal
Fig. 5. Linear regression curves for foal growth, expressed as foal weight as a percentage of mare weight. Results for foals on the novel endophyte tall fescue pasture (NETF) are shown in black with diamonds and those on the orchardgrass/bluegrass pasture (OGBG) are in gray with squares. There were no differences in foal growth rates in either year due to NE or OGBG pastures.
K. McDowell et al. / Journal of Equine Veterinary Science 74 (2019) 56e64
63
Table 8 P values for serum analytes for pregnant mares grazing NE versus OGBG pastures.
Chloride Sodium Potassium Cholesterol SGOTAST Kinase
Year 1
Year 2
.3403 .6403 .1018 .6403 .1391 .6207
.1018 .4536 .4076 .4860 .0875 .5001
GGT Alkaline phosphatase Total protein Albumin Globulin AGRatio
Year 1
Year 2
.2943 .2035 .4852 .7920 .1033 .1376
.0278 .6656 .3407 .1187 .8944 .2891
BUN Calcium Glucose Phosphorus Creatinine Total bilirubin
Year 1
Year 2
.1005 .8566 .2703 .5145 .0796 .4152
.1729 .5865 .1534 .1850 .6249 .4000
Abbreviations: AGRatio, albumin:globulin ratio; BUN, blood urea nitrogen; GGT, gamma-glutamyl transpeptidase; orchardgrass/Kentucky bluegrass; SGOST, serum glutamicoxaloacetic transaminase; NE, novel endophyte; OGBG, orchardgrass/Kentucky gluegrass.
temperatures over an experiment that went from mid-October through mid-December. One of the major signs of fescue toxicosis in ruminants is vasoconstriction [26e29]. Likewise, McDowell and her students reported that consumption of toxic KY31TF seed [8e11] or pasture (KJ McDowell, unpublished) containing the common toxic strain of the endophyte Epichlo€ e coenophiala caused a marked constriction of the palmar arteries in horses, as also evidenced in this study in the mares grazing the toxic KY31TF pasture. However, such vasoconstriction was absent in all mares grazing either the NETF or the OGBG pasture. The NETF used in these studies contained a variant of Epichlo€ e coenophiala that produced little to no ergovaline. Ergovaline was the only alkaloid measured in these studies, and ergovaline makes up 80%e100% of the ergopeptines communally found in the endophytic fungus of Epichlo€ e [30], but other ergot alkaloids are known to have vasoactive properties [14,31]. Klotz and McDowell exposed equine palmar arteries and veins to increasing concentrations of various ergot alkaloids in vitro, including ergovaline, ergonovine, ergotamine, ergocryptine, ergocornine, and ergocristine [32]. All of those alkaloids caused constriction in both the arteries and veins. However, the alkaloids, other than ergovaline, are found almost exclusively in the ergots of Claviceps purpurea, which forms on the seed heads [30]. In the experiments described here, pastures were mowed regularly to prevent seed head development. That none of the horses grazing the NETF in these studies showed any signs of artery constriction indicates that the NETF was either not producing those alkaloids or at least not producing them in any substantial quantities, and did not have seed heads infected with Claviceps purpurea. Ergovaline was detected in the NETF pasture on a single forage sampling day in year 1 and on two days in year 2, albeit in relatively low concentrations. This could be due to limited wild-type fescue ingrowth in the pasture, particularly if sampling occurred close to the fence-line with the KY31TF pasture. Nevertheless, mares grazing the NETF in these studies exhibited none of the signs of equine fescue toxicosis.
Because the NETF still contains the alkaloids that give it a competitive advantage over most other pastures, it is not anticipated that the NETE will be overgrown with KY31 fescue, as happens with endophyte free tall fescue. Our NETF pasture has been in use for mare studies for over 6 years, with no evidence of increased ingrowth of KY31 fescue. While we do pick up the occasional sample that contains ergovaline, that has not increased over the years, and could be due to sampling close to the fence line. Although endophyte-free TF varieties have been developed, their agronomic performance has been disappointing, particularly if grazed heavily or used in stress-prone locations [33,34]. Therefore, NE fescues can be an option for replacing either the common toxic strain of endophyte-infected TF or endophyte-free TF pastures. Plants that contain NEs generally persist better than endophyte-free varieties due to their maintenance of the alkaloids that confer a competitive advantage to fescue but lacking the alkaloids that are harmful to grazing livestock [35,36]. For a short review of the development of NE-infected TFs, including their advantages and disadvantages; see Phillips and Aiken [34]. In conclusion, results of these studies indicate that the TF cultivar KYFA9821 containing the novel endophyte AR584 can be a satisfactory pasture forage for pregnant mares and their foals. Acknowledgments This work was funded by the USDAdAnimal Research ServicedForage Animal Production Unit in a Specific Cooperative Agreement with the University of Kentucky College of Agriculture [grant number 3210000239], the Departments of Plant and Soil Sciences and Veterinary Science, and AgResearch. This work was also supported by the National Institute of Food and Agriculture, United States Department of Agriculture [HATCH under 1002202], publication No. 18-14-076 of the Kentucky Agricultural Experiment Station, and is published with the approval of the Director.
Year 2 Mean diameter (cm)
Mean diameter (cm)
Year 1 0.6 0.5 0.4 0.3 0.2 0.1 0.0 1
3
5
7
10 12 14 16 18 20 22
Week of experiment
0.6 0.5 0.4 0.3 0.2 0.1 0.0 2
4
6
8
10 12 14 16 18
Week of experiment
Fig. 6. Mean diameter of the palmar arteries of mares grazing novel endophyte tall fescue (solid lines), orchardgrass/bluegrass (OGBG, dashed lines), or Kentucky 31 tall fescue (dotted line) pastures. Vertical lines represent weeks that mares were placed onto and then were removed from their respective treatment pastures. Artery diameters of mares grazing KY31TF were reduced during weeks 5 through 18 compared to weeks 1 and 3 or 20 and 22 and were reduced compared to the other two groups as long as the mares remained on the toxic fescue pasture (P < .0001).
64
K. McDowell et al. / Journal of Equine Veterinary Science 74 (2019) 56e64
The authors wish to thank Mr. Lynn Ennis for his assistance throughout the projects, Mr. Tracy Hamilton for pasture sampling in year 1 and Phillip Shine and student workers in the Departments of Plant and Soil Sciences and Veterinary Science for assistance with pasture management and animal handling in both years. The authors offer a special thanks to Tony Stratton of AgResearch for his advice, support, and encouragement throughout the experiments. References [1] Burns JC, Chamblee DS. Adaptation. In: Buckner RC, Bush LP, editors. Tall Fescue, 20. Madison, WI, USA: Agron Monogr; 1979. p. 9e30. [2] Hannaway DB, Daly C, Halbleib M, James D, West CP, Volence JJ, Chapman D, Li X, Cao W, Shi X, Johnson S. Development of suitability maps with examples for the United States and China. http://forages oregonstate edu/tallfescuemonograph/ 2009. [3] Bush LP, Fannin FFN. Alkaloids. In: Fribourg HA, Hannaway DB, West CP, editors. Tall Fescue for the Twenty-first Century, 53. Madison, WI, USA: Agron Monogr; 2009. p. 229e49. [4] Cross DL, Redmond LM, Strickland JR. Equine fescue toxicosis: signs and solutions. J Anim Sci 1995;73:899e908. [5] Monroe JL, Cross DL, Hudson LW, Hendricks DM, Kennedy SW, Bridges WC. Effect of selenium and endophyte-contaminated fescue on performance and reproduction in mares. J Equine Vet Sci 1988;8:148e53. [6] Putnam MR, Bransby DI, Schumacher J, Boosinger TR, Bush L, Shelby RA, Vaughan JT, Ball D, Brendemuehl JP. Effects of the fungal endophyte Acremonium coenophialum in fescue on pregnant mares and foal viability. Am J Vet Res 1991;52:2071e4. [7] Hoveland CS. Origin and history. In: Fribourg HA, Hannaway DB, West CP, editors. Tall fesccue for the twenty-first century, 53. Madison, WI: Agron Monogr; 2009. p. 3e10. [8] Hestad DA. Ingestion of endophyte-infected tall fescue seed induces peripheral vasoconstriction but does not affect cyclicity in non-pregnant mares, and a population of biogenic amine receptors relative to vasoconstriction is identified. 2012. MS Thesis, Department of Veterinary Science, University of Kentucky, Lexington, KY, USA, https://uknowledge.uky.edu/gluck_etds/2. [9] McDowell KJ, Moore ES, Parks AG, Lawrence LM. Use of Doppler ultrasonography on horses to detect vasoconstriction caused by endophyte infected tall fescue. J Equine Vet Sci 2009;29:338e9. [10] McDowell KJ, Moore ES, Parks AG, Bush LP, Horohov DW, Lawrence LM. Vasoconstriction in horses caused by endophyte-infected tall fescue seed is detected with Doppler ultrasonography. J Anim Sci 2013;91:1677e84. [11] Moore ES, Parks AG, Lawrence LM, McDowell KJ. Endophyte infected fescue seed causes vasoconstriction in horses as measured by Doppler ultrasonography. J Anim Sci 2008;86(Suppl 2):403. [12] Turner RM, McDonnell SM, Feit EM, Grogan EH, Foglia R. How to determine gestational age of an equine pregnancy in the field using transrectal ultrasonography measurement of the fetal eye. AAEP Proc 2006;52:250e5. [13] Colborn DR, Thompson DL, Roth TL, Capehart JS, White KL. Responses of cortisol and prolactin to sexual excitement and stress in stallions and geldings. J Anim Sci 1991;69:2556e62. [14] Yates SG, Powell RG. Analysis of ergopeptine alkaloids in endophyte-infected tall fescue. J Agric Food Chem 1988;36:337e40. [15] Carter JM, Aiken GE, Dougherty CT, Schrick FN. Steer responses to feeding soybean hulls and steroid hormone implantation on toxic tall fescue pasture. J Anim Sci 2010;88:3759e66.
[16] Bucca S, Fogarty U, Collins A, Small V. Assessment of feto-placental well-being in the mare from mid-gestation to term: transrectal and transabdominal ultrasonographic features. Theriogenology 2005;64:542e57. [17] Colon JL. Trans-rectal ultrasonographic appearance of abnormal combined utero-placental thickness in late-term gestation and its incidence during routine survey in a population of Thoroughbred maes (2005-2008). Theriogenology 2008;54:279e85. [18] Lea K, Smith L, Gaskill C, Coleman R, Smith SR. Ergovaline stability in tall fescue based on sample handling and storage methods. Front Chem 2014;2:1e6. [19] Spiering MJ, Davies E, Tapper BA, Schmid J, Lane GA. Simplified extraction of ergovaline and peramine for analysis of yissue distribution in endophyteinfected grass tillers. J Agric Food Chem 2002;50:5856e62. [20] SAS Institute I. SAS System for Microsoft windows, Release 9.3. Cary, N.C: SAS Institute, Inc; 2012. [21] Cross DL. Fescue toxicosis. In: McKinnon AO, Squires EL, Vaala WE, Varner DD, editors. Equine reproduction. 2nd. Aimes, IA: Wiley-Blackwell; 2011. p. 2418e27. [22] Boosinger TR, Brendemuehl JP, Bransby DL, Wright JC, Kemppainen RJ, Kee DD. Prolonged gestation, decreased triiodothyronine concentration, and thyroid gland histomorphologic features in newborn foals of mares grazing Acremonion coenophialum-infected fescue. Am J Vet Res 1995;56: 66e9. [23] Brendemuehl JP, Carson RL, Wenzel JGW, Boosinger TR, Shelby RA. Effects of grazing endophyte-infected tall fescue on eCG and progestogen concentrations from gestation days 21 to 300 in the mare. Theriogenology 1996;46:85e95. [24] Evans TJ. The endocrine disruptive effects of ergopeptine alkaloids on pregnant mares. Vet Clin Equine 2011;27:165e73. [25] McCann JS, Caudle AB, Thompson FN, Stuedemann JA, Heusner GL, Thompson Jr DL. Influence of endophyte-infected tall fescue on serum prolactin and progesterone in gravid mares. J Anim Sci 1992;70:217e23. [26] Aiken GE, John A, Britt JL, Miller MF, Adams SK, Duckett SK. Vasoconstrictive responses of the carotid artery in pregnant ewes to ergot alkaloid exposure. J Anim Sci 2017;95:236. [27] Foote AP, Harmon DL, Strickland JR, Bush LP, Klotz JL. Effect of ergot alkaloids on contractility of bovine right ruminal artery and vein. J Anim Sci 2011;89: 2944e9. [28] Klotz JL, Bush LP, Smith DL, Shafer WD, Smith LL, Arrington BC, Strickland JR. Ergovaline-induced vasoconstriction in an isolated bovine lateral saphenous vein bioassay. J Anim Sci 2007;85:2330e6. [29] Klotz JL, Kirch BH, Aiken GE, Bush LP, Strickland JR. Contractile response of fescue-naive bovine lateral saphenous veins to increasing concentrations of tall fescue alkaloids. J Anim Sci 2010;88:408e15. [30] Guerre P. Ergot alkaloids produced by thee endophytic fungi of the genus Epichol€ e. Toxins 2015:773e90. [31] Lyons PC, Plattner RD, Bacon CW. Occurrence of peptide and clavine ergot alkaloids in tall fescue grass. Science 1986;232:487e9. [32] Klotz JL, McDowell KJ. Tall fescue ergot alkaloids are vasoactive in equine vasculature. J Anim Sci 2017;95:5151e60. [33] Bouton JH, Latch GCM, Hill NS, Hoveland CS, McCann MA, Watson RH, Parish JA, Hawkins LL, Thompson FN. Reinfection of tall fescue cultivars with non-ergot alkaloid-producing endophytes. Agron J 2002;94: 567e74. [34] Phillips TD, Aiken GE. Novel endophyte-infected tall fescues. Forage and Grazinglands 2009;7. https://doi.org/10.1094/FG-2009-1102-01-RV. [35] Bacon CW, Siegel MR. Endophyte parasitism of tall fescue. J Prod Agric 1988;1: 45e55. [36] Burns JC, Fisher DS, Rottinghaus GE. Grazing influences on mass, nutritive value, and persistence of stockpiled Jesup tall fescue without and with novel and wild-type fungal endophytes. Crop Sci 2006;46: 1898e912.
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Original Research
Pregnant Mares Grazing a Novel Endophyte-Infected Tall Fescue Foal Normally Karen McDowell a, *, Victoria Taylor a, Tim Phillips b, Krista Lea b, Ray Smith b, Glen Aiken c, Michael Barrett b a b c
Department of Veterinary Science, University of Kentucky, Lexington, KY Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY USDA-ARS-FAPRU Lexcington, KY
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 July 2018 Received in revised form 4 October 2018 Accepted 6 December 2018 Available online 18 December 2018
These experiments were conducted to determine if pregnant mares grazing a novel endophyte tall fescue (NETF) pasture would have satisfactory foaling outcomes. The hypotheses tested were that mares grazing the NETF pasture or an orchardgrass/Kentucky bluegrass (OGBG) pasture would have statistically equivalent (1) serum progesterone, estradiol, and prolactin concentrations, (2) blood chemistry analyte concentrations, and (3) palmar artery sizes. In addition, foals born on the respective pastures would grow at comparable rates. Over 4.5 months, during each of two summers, 12 foaling mares were placed on either NETF or OGBG pastures (n ¼ 6 mares/pasture/year). There were no differences in the blood sample concentrations of progesterone, estradiol, prolactin, and chemistry analytes between the groups. Combined uterine placental thickness and placental weights were not different between the two groups of mares, and foals grew at equivalent rates. The diameter of the palmar artery of each mare was measured via Doppler ultrasonography as a bioassay for consumption of ergot alkaloids of fescue pastures. Diameters were not different between mares grazing NETF versus OGBG pastures. However, palmar artery diameters of nonpregnant mares grazing a pasture of toxic Kentucky 31 tall fescue were reduced compared to mares on the NETF or OGBG pastures. Pasture analyses demonstrated that the NETF was endophyte infected but produced no or trace amounts of ergovaline. In summary, there were no differences in any variable tested between mares grazing the new, NETF fescue pasture, compared with mares grazing a standard OGBG pasture. © 2019 Elsevier Inc. All rights reserved.
Keywords: Horses Fescue Novel endophyte Ergovaline Foals
1. Introduction Tall fescue (TF) (Lolium arundinaceum (Schreb.) Darbysh.) is one of the most widely grown perennial grasses in the world and covers approximately 15 million ha in the United States alone. It can be infected with an endophytic fungus, Epichlo€ e coenophiala (MorganJones and W. Gams) C.W. Bacon and Schardl, which, in a symbiotic relationship with the plant, produces a variety of potentially toxic alkaloids that may adversely affect grazing livestock [1,2].
Animal welfare/ethical statement: All experimental procedures were approved by the University of Kentucky Institutional Animal Care and Use Committee. Conflict of interest statement: The authors declare no conflict of interest. * Corresponding author at: Karen McDowell, Department of Veterinary Science, University of Kentucky, 108 Gluck Equine Research Center, Lexington, KY 405460099, USA. E-mail address: [email protected] (K. McDowell). https://doi.org/10.1016/j.jevs.2018.12.006 0737-0806/© 2019 Elsevier Inc. All rights reserved.
Ergovaline is the most abundant ergot alkaloid in TF [3]. In horses, the most frequently reported problems associated with endophyteinfected fescue consumption are in late pregnancy and include altered hormone concentrations, extended gestation, thickened placenta, placental retention, dystocia, birth of dysmature foals, increased foal and placental weights, and agalactia due primarily to decreased prolactin concentrations [4e6]. Although the alkaloids produced by TF are detrimental to grazing animals, they also confer benefit to the plant [7]. Hence TF cultivars containing a novel endophyte (NE) have been developed that continue to produce alkaloids that give the plant characteristics of drought tolerance, insect resistance, and increased vigor but that do not produce the alkaloids that are harmful to grazing animals. Therefore, the first objective of this experiment was to evaluate foaling mares grazing a new NETF pasture compared to grazing an orchardgrass/Kentucky bluegrass (OGBG) pasture. The second objective was to compare the sizes of the palmar arteries for mares
K. McDowell et al. / Journal of Equine Veterinary Science 74 (2019) 56e64
grazing NETF, OGBG, or toxic Kentucky 31 TF (KY31TF) pastures. Palmar artery constriction in horses has been shown to be a convenient and satisfactory bioassay for consumption of endophyte-infected fescue seed [8e11]. In addition, when pregnant and nonpregnant mares are exposed to toxic KY31TF seed, their palmar arteries constricted in a similar manner (KJ McDowell, unpublished). 2. Materials and Methods 2.1. Establishment of Pastures Four pastures at the University of Kentucky Agricultural Experiment Station in Lexington, KY, ranging from 2.7 to 3.2 ha, were used for these studies. The area where the pastures were established had previously been under a winter wheat-corn rotation for a number of years. The corn residue was mowed and then sprayed with glyphosate (4.7l per ha). The pastures were randomly assigned to be planted with either a mixture of “Potomac” orchardgrass (11 kg seed per ha) and Kentucky bluegrass (11 kg seed per ha), an NETF (22.5 kg seed per ha), or KY31TF infected with the common toxic endophyte (22.5 kg of seed per ha). Two pastures were planted with the OGBG, and one pasture each was planted with the NETF or KY31TF. The NETF, developed cooperatively by researchers in the Department of Plant and Soil Science at the University of Kentucky (TD Phillips) and at AgResearch in New Zealand, was an experimental cultivar of TF (KYFA9821), which contained an alternative strain of Epichlo€ e coenophiala, AR584. During the experiments, all pastures were mowed regularly to maintain the pasture height between 13 and 20 cm. This mowing would also prevent seed head development. Ambient temperature and rainfall during the experiments are shown in Fig. 1. 2.2. Experimental Procedures All experimental procedures were approved by the University of Kentucky Institutional Animal Care and Use Committee. The studies
57
were conducted over 2 years between March and September of each year. 2.2.1. Year 1 Light horse mares of mixed breeding, pregnant (n ¼ 12) and nonpregnant (n ¼ 4) were used. Pregnant mares had been pasturebred to foal during the summer, but exact breeding dates were unknown. In March, all the mares were placed on one of the OGBG pastures, used as a “holding pasture,” and supplemented with grass hay, free of ergovaline, as needed. While on the holding pasture, the mares were acclimated to all procedures, so that they would stand quietly in wooden stocks designed for mare handling. In the pregnant mares, fetal eye measurements were made via transrectal ultrasonography to estimate month of foaling [12], using a Sonosite Titan with a linear transducer, L52 10-5 Mz. The pregnant mares were assigned to be placed on either the NETF pasture or the second OGBG pasture such that the estimated foaling dates were equally distributed between the two pastures. The nonpregnant mares were assigned to be placed on the KY31TF pasture. These mares were used as a negative control group to document changes in palmar artery sizes when mares grazed a toxic endophyte infected KY31TF pasture. Mares were allowed to continuously graze these treatment pastures, except when they were in the sample collection pens, approximately 1 hour each day. They were not supplemented with hay and received only approximately 0.1e0.2 kg (a handful) of alfalfa-pelleted concentrate mix (approximately 1e3 times per week, depending on where they were in their estrous cycles) as an incentive to come to the collection pens for all procedures (Table 1). The left distal palmar artery of each mare was measured via pulsed-wave Doppler ultrasonography (Sonosite Titan with an 11 mm convex array 5/8 MHz transducer) during the first week of May, before being placed on the treatment pastures (May 5), and then during the first and third weeks of each month thereafter, as described by McDowell et al. [8e11]. Briefly, the lateral surface of the left foreleg, just proximal to the fetlock joint, was clipped free of hair. The transducer with coupling gel was placed between the suspensory
Year 1
PrecipitaƟon (cm)
PrecipitaƟon (cm)
Year 2
o
o
Air temperature ( C)
Air temperature ( C)
35 30 25 20 15 10 5 0 1
3
5
7
10
12
Week of Experiment o o
14
16
18
o
1 3 5 7
10 12 14 16 18 20 22
Week of Experiment o o
Max Temp C
Min C
o
Mean C
Fig. 1. Precipitation and ambient air temperature during the course of the experiment. The data were obtained from the University of Kentucky Research Farm Climate Data Center, Lexington KY, located approximately 0.4 km from the research station site.
58
K. McDowell et al. / Journal of Equine Veterinary Science 74 (2019) 56e64
Table 1 UK #3 Equine: University of Kentucky, custom mix alfalfa pellets. Production Formula Ingredient Name
Nutrient Composition Nutrient Name
Amount
Units
Pulverized oats Ground corn Soybean meal Dehydrated alfalfa Soy hulls Cane molasses Soy oil Copper sulfate Calcium carbonate White salt Zinc oxide Selenium Vitamin E
Digestible energy Crude protein Crude fat Crude fiber Ash Calcium Total phosphorus Salt Sodium Potassium Lysine Methionine Methionine þ cystine Threonine Sulfur Magnesium Chloride Manganese Zinc Iron Copper Selenium Cobalt Iodine Vitamin A Vitamin D Vitamin E Vitamin K menadione Vitamin B1 thiamine Vitamin B2 riboflavin Vitamin B3 niacin Vitamin B5 pantothenic acid Vitamin B6 pyridoxine Choline Vitamin B9 folic acid Biotin Vitamin B12 cyanocobalamin Dry matter Starch Vitamin C Nonstructural carbohydrates Acid detergent fiber Neutral detergent fiber Chromium Bulk density
2835.06 12.29 6.09 15.00 6.48 0.74 0.23 0.56 0.24 1.23 0.54 0.20 0.33 0.57 0.19 0.16 0.49 17.79 120.59 177.26 39.39 0.30 0.29 0.07 0.00 0.00 110.23 0.00 1.72 4.65 43.43 16.89 4.12 668.71 1.98 0.00 3.57 197.69 54.34 24.65 67.51 52.78 46.28 0.00 218.75
kcal/kg % % % % % % % % % % % % % % % % ng/g ng/g ng/g ng/g ng/g ng/g ng/g kIU/kg kIU/kg IU/kg mg/kg mg/kg mg/kg mg/kg mg/lb mg/kg mg/kg mg/kg mg/kg mcg/kg % % % mg/kg % % % kg/cu m
Makeup and nutrient content of the pellet mix offered to mares as an incentive to come from the pastures to the working pens.
ligaments and the flexor tendons to visualize the palmar artery and vein. Measurements of mean artery diameter (defined as the average of the longer and shorter axes of the elliptical shape of the vessel) were made at peak systole to reduce variation of measurements taken within and between horses. All machine settings were constant for all horses throughout the experiment. Three replicate scans were performed on each horse on each scan day. Jugular venous blood samples were collected during the first and third weeks of each month for hormone analyses and during the first week of each month for clinical chemistry analyses. Serum was stored frozen at 20 C until analyzed for estradiol, progesterone, and prolactin concentrations by the laboratory of Dr. Donald Thompson at Louisiana State University. Estradiol and progesterone were analyzed using PANTEX immunoassay
kits (Santa Monica, CA), and prolactin was analyzed as described by Colburn et al. [13]. The clinical chemistry panels were performed on fresh serum at the University of Kentucky Veterinary Diagnostic Laboratory (UKVDL). All the pregnant mares were allowed to foal in their respective treatment pastures. Mares and foals were weighed at the same time blood samples were obtained. Placentas were taken to the UKVDL for pathologic, bacteriologic, and virologic analyses. Palmar artery evaluations of the foaling mares were discontinued when the mares foaled. Ergovaline and its stereoisomer ergovalinine (referred together as ergovaline from here forward) concentrations in the NETF and the KY31TF pastures were determined on three occasions in June and on two occasions in July. Whole tillers clipped from 25 random plants from within each pasture were pooled, freeze-dried in a Botanique
Table 2 Evaluations for the novel endophyte tall fescue pasture, year 1. Date
6/3
6/23
6/30
7/7
7/23
Week on experimental pastures Ergovaline (ng/g)
4 ND
7 10
8 ND
9 ND
11 ND
Abbreviation: ND, no ergovaline was detected. Average ergovaline concentrations could not be calculated.
Average
Table 3 Evaluations for the KY 31 tall fescue pasture, year 1. Date
6/3
6/23
6/30
7/7
7/23
Average
Week on experimental pastures Ergovaline (ng/g)
4 190
7 240
8 440
9 270
11 330
294
K. McDowell et al. / Journal of Equine Veterinary Science 74 (2019) 56e64
59
Table 4 Evaluations for the novel endophyte pasture, year 2.
Percentage of each type of plant identified
Tall fescue analyses
Calculations
Date
4/23
5/6
5/20
6/3
6/18
7/8
7/22
8/5
8/19
9/11
Week of Experiment
2
4
6
8
10
13
15
17
19
22
Tall fescue KY Bluegrass Orchardgrass White Clover
93 6 0 0
96 2 0 0
93 0 0 0
93 1 0 0
93 0 0 0
91 0 0 0
84 4 0 0
82 0 0 0
91 0 0 0
89 0 0 0
Weeds Bare soil Ergovaline (ng/g) Endophyte (%)
1 2 d 60
1 3 d 70
0 8
0 8
0 9
d 63
1 7 d 60
d 94
d 86
1 12 107 70
1 18 d 80
2 7 211 81
2 10 d 81
Available foragea Percentage of tall fescue in available forage Ergovaline concentration in tall fescue (ng/g)
99 94 d
98 98 d
93 100 d
94 99 d
93 100 d
91 100 d
88 95 102
82 100 d
91 100 MD
89 100 d
Avg.
91 1 0 0 1 8 b
74 92 99 b
Abbreviation: MD, missing data. The entry for each date represents the average of 10 sampling grids taken on that date. Additional forages of ryegrass, timothy, and/or red clover were present in these fields totaling <10% of the types of plants identified; therefore, percentages do not add to 100. d, represent that ergovaline concentrations were below the reliable quantitation (<100 ng/g) of the assay. a Available forage is the total of the tall fescue, KY bluegrass, orchardgrass, and white clover. b Cannot calculate an average with values less than the reliable quantitation of the assay.
Model 18DX485A freeze drier (Botanique Preservation Co, Peoria, AZ) and ground to pass through a 1-mm mesh screen (Cyclotec 1093 sample mill; FOSS North America, Eden Prairie, MN). These samples were analyzed by Dr. Lowell Bush, Department of Plant and Soil Science, University of Kentucky, for ergovaline using the highperformance liquid chromatography fluorescence procedure of Yates and Powell [14] as modified by Carter et al. [15]. The results were reported on a dry matter basis. The limit of detection was 10 ng/g. On September 3 (week 18 of the study), the mares from the KY31TF pasture were moved back to the original OGBG holding pasture. Foaling mares stayed on their respective pastures until the experiment ended on September 30. 2.2.2. Year 2 Twelve pregnant light horse mares of mixed breeding were used. All mares were initially placed on the OGBG holding pasture as described previously. Doppler ultrasound measurements of the palmar arteries were taken weekly, beginning three weeks before the mares being placed on the treatment pastures. Blood samples were collected during the first and third weeks of each month for hormone and clinical chemistry analyses, using the same methods as for Year 1. On May 6 (end of week 3), six of the mares were placed
on the NETF pasture and six on the second “test” OGBG pasture. Sampling and data collection were performed as described previously, except that combined uterine and placental thickness (CUPT) was also measured using the linear ultrasound transducer [16,17]. Mares and their foals remained on the treatment pastures until the experiment ended on September 16. During the first and third weeks of each month, all pastures were evaluated by members of the University of Kentucky Horse Pasture Evaluation Program for plant species present. In addition, for TF, the percentage of plants infected with endophyte was determined and ergovaline concentrations were measured. Evaluations were performed within a 2 2 foot square at 10 locations in each pasture. Two TF tillers were taken at each location, combined, and placed on ice in a cooler until delivered to the testing facilities according to the methods described by Lea et al. [18]. For characterization of the fescue tillers, the percentage with endophyte infection was determined by the University of Kentucky Division of Regulatory Services using a Phytoscreen Immunoblot Kit (Agrinostics, Watkinsville, GA). Ergovaline was quantitated at the UKVDL toxicology section using ultraperformance liquid chromatography and fluorimetric detection. Briefly, fresh forage was frozen with liquid nitrogen, milled and extracted with a 2-propanol/lactic acid
Table 5 Evaluations for the orchardgrass/KY bluegrass “test” pasture, year 2. Date
4/23
5/6
5/20
6/3
6/18
7/8
7/22
8/5
8/19
9/11
Week of Experiment
2
4
6
8
10
13
15
17
19
22
Avg.
Percentage of each type of plant identified
Tall fescue KY Bluegrass Orchardgrass White clover Weeds Bare soil
0 9 89 0 1 2
0 32 60 0 2 6
0 8 77 0 1 15
0 7 72 0 1 21
0 9 68 0 2 22
0 5 71 1 2 22
0 15 44 0 4 38
0 7 53 0 4 37
0 20 42 0 3 36
0 18 27 0 2 54
Tall fescue analyses
Endophyte (%) Ergovaline (ng/g)
Endophyte and ergovaline could not be reported because of lack of tall fescue
Calculations
Available foragea Percentage of tall fescue in available forage Ergovaline concentration in tall fescue (ng/g)
98 92 85 79 77 77 59 60 62 45 73 0 0 0 0 0 0 0 0 0 0 0 Ergovaline concentrations could not be calculated because of the lack of tall fescue.
0 13 60 0 2 25
The entry for each date represents the average of 10 sampling grids taken on that date. Additional forages of ryegrass, timothy, and/or red clover were present in these fields totaling <10% of the types of plants identified; therefore, percentages do not add to 100. a Available forage is the total of the tall fescue, KY bluegrass, orchardgrass, and white clover.
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K. McDowell et al. / Journal of Equine Veterinary Science 74 (2019) 56e64
Table 6 Evaluations for the KY 31 tall fescue pasture, year 2. Date
4/23
5/6
5/20
6/3
6/18
7/8
7/22
8/5
8/19
9/11
Week of Experiment
2
4
6
8
10
13
15
17
19
22
Percentage of each type of plant identified
Tall fescue KY Bluegrass Orchardgrass White clover Weeds Bare soil
74 1 23 0 0 2
75 1 24 0 0 1
87 1 3 0 1 10
77 0 8 0 0 15
84 0 5 0 1 11
81 0 3 0 1 16
77 1 5 0 0 18
85 1 5 0 1 9
78 3 7 0 2 11
82 0 2 0 1 15
80 1 9 0 1 11
Tall fescue analyses
Ergovaline (ng/g) Endophyte (%)
d 21
217 20
564 38
419 53
d 45
130 30
113 42
200 43
MD 55
755 68
42
Available foragea Percentage of tall fescue in available forage Ergovaline concentration in tall fescue (ng/g)
98 76 d
100 75 163
91 96 539
85 91 380
89 94 d
84 96 125
83 93 105
91 93 187
88 89 d
84 98 737
Calculations
Avg.
b
89 90 b
The entry for each date represents the average of 10 sampling grids taken on that date. Additional forages of ryegrass, timothy, and/or red clover were present in these fields totaling <10% of the types of plants identified; therefore, percentages do not add to 100. d, represent that ergovaline concentrations were below the reliable quantitation (<100 ng/g) of the assay. a Available forage is the total of the tall fescue, KY bluegrass, orchardgrass, and white clover. b Cannot calculate an average with values less than the reliable quantitation of the assay.
solution using procedures adapted from Spiering [19]. The results were reported on a dry matter basis and the minimum quantitative sensitivity was 100 ng/g. 2.3. Statistical Analyses Data analyses were performed using SAS [20]. Chemistry analytes, hormonal data, palmar artery diameter measurements, and CUPT were analyzed with the Mixed Procedure as completely randomized designs with measurements repeated over time. Placental weights were compared using a one-way classification in a completely randomized design. Foal growth rates were analyzed with the regression procedure and then compared via the general linear model procedure. Data are presented as least square means plus or minus standard error of the mean, and differences are considered significant when P < .05.
3.1. Pasture Analyses In year 1, ergovaline was undetectable in the NETF samples except on one occasion where it averaged 10 ng/g. In the KY31TF pasture, ergovaline averaged 294 ng/g and ranged from 190 to 440 ng/g over the 5-week sampling period (Tables 2 and 3). The results of the pasture evaluations for year 2 are shown in Tables 4e7. These results indicate that, although TF represented most of the available forage in the NETF pasture (98%), and most of the NETF samples (73%) were infected with endophyte, ergovaline concentrations were minimal or not quantifiable. By contrast, TF represented most of the available forage in the KY31TF pasture (80%) and only 42% was infected with endophytes. Ergovaline concentrations averaged to 319 ng/g, except on three occasions, when the concentrations were below the reliable quantitation of the assay.
3. Results
3.2. Foaling Outcomes, Hormone Concentrations, and Clinical Chemistry
Based on data collected during year 1, modifications were made in animal and pasture sampling protocols, as well as the differences in the ergovaline and endophyte assays; therefore, results for years 1 and 2 are reported separately.
In Year 1 all but one of the mares foaled normally between May 10 and September 7, producing healthy live foals. One mare from the OGBG pasture presented with an unexpected and severe dystocia and was subsequently euthanatized by the attending
Table 7 Evaluations for the orchardgrass/KY bluegrass “holding” pasture, year 2. Date
4/23
7/22
8/5
8/19
9/11
Week of Experiment
2
15
17
19
22
Percentage of each type of plant identified
Tall fescue KY Bluegrass Orchardgrass White clover Weeds Bare soil
1 20 44 0 3 33
0 3 19 4 12 63
0 1 7 3 13 78
0 0 1 1 10 89
0 0 1 1 18 81
Tall fescue analyses
Endophyte (%) Ergovaline (ng/g)
Endophyte and ergovaline could not be reported because of the lack of tall fescue.
Calculations
Available foragea Percentage of tall fescue in available forage Ergovaline concentration in tall fescue (ng/g)b
65 2 d
26 0 d
11 0 d
2 0 d
3 33 d
Avg.
0 5 14 2 11 69
21 7 b
The entry for each date represents the average of 10 sampling grids taken on that date. Additional forages of ryegrass, timothy, and/or red clover were present in these fields totaling < 10% of the types of plants identified; therefore, percentages do not add to 100. d, represent that ergovaline concentrations were below the reliable quantitation (<100 ng/g) of the assay. a Available forage is the total of the tall fescue, KY bluegrass, orchardgrass and white clover. b Cannot calculate an average with values less than the reliable quantitation of the assay.
K. McDowell et al. / Journal of Equine Veterinary Science 74 (2019) 56e64
Year 2
120
70
100
60
Prolactin (ng/ml)
Prolactin (ng/ml)
Year 1
80 60 40 20 0 -6
-4
-2
40 30 20 10
0
-14
-12
-10
-8
-6
-14
-12
-10
-8
-6
-4
-2
0
4
8
Progesterone (ng/ml)
Progesterone (ng/ml)
-8
*
6 4
*
2 -14
-12
-10
-8
-6
-4
-2
3 2 1 0
0
300
-4
-2
0
-4
-2
0
400
250
Estradiol (pg/ml)
Estradiol (pg/ml)
50
0 -12 -10 -10 -14 -12
0
61
200 150 100 50
300 200 100 0
0 -14 14
-12 - -10 12 10
-8
-6
-4
-2
0
Weeks relative to foaling
-14
-12
-10
-8
-6
Weeks relative to foaling
Fig. 2. Serum prolactin, progesterone, and estradiol concentrations in pregnant mares grazing the novel endophyte (solid lines) or orchardgrass/bluegrass (dashed lines) pastures. As expected, serum prolactin and progesterone concentrations increased and estradiol concentrations decreased toward the end of gestation. There were no treatments by week of foaling interactions for any of the hormones for either year.
As expected, the CUPT measurements increased as gestation progressed (P < .0001; measured in year 2 only). However, they were not different between the mares grazing NETF or OGBG pastures (P ¼ .6243; Fig. 3). Placental weights were not different between mares foaling on NETF and OGBG pastures in either year (P ¼ .5975 year 1; P ¼ .6938 year 2) (Fig. 4). All placentas from both years were negative for pathogenic bacteria, leptospira, equine
1.2 1.0 CUPT (cm)
veterinarian. Mares on the NE pasture foaled between May 10 and September 7, giving them a mean of 57.5 days and a median of 54 days on the pasture. Mares on the OGBG pasture foaled between May 22 and July 29, with a mean of 57 days and a median of 64 days on their pasture. In Year 2, all of the mares produced live healthy foals between June 28 and September 14, giving them a mean of 79.5 days and a median of 67 days on their pastures. Mares on the NE pasture foaled between June 30 and August 19, with a mean of 75.1 days and a median of 65 days on their pasture, while mares on the OGBG pasture foaled between June 28 and September 14, with a mean of 84.8 and a median pf 72 days. Mare serum prolactin, progesterone, and estradiol concentrations are shown in Fig. 2 and all were within normal ranges for foaling mares. All three hormones in both years had significant effects of weeks relative to foaling: prolactin (P ¼ .0465 year 1; P ¼ .0017 year 2); progesterone (P ¼ .0655 year 1; P ¼ .0002 year 2); and estradiol (P ¼ .0017, P < .0001 year 2), as prolactin and progesterone concentrations increased and estradiol concentrations decreased toward the end of gestation. Progesterone was statically lower at weeks -2 and 0 in year 1 for mares grazing the OGBG pasture than for mares grazing the NETF pasture. None of the three hormones had a significant treatment by week of foaling interaction: prolactin (P ¼ .1349 year 1; P ¼ .7265 year 2); progesterone (P ¼ .5306 year 1; P ¼ .8183 year 2); and estradiol (P ¼ .7067 year 1; P ¼ .7063 year 2).
0.8 0.6 0.4 0.2 0.0 -18 -14 -12 -10 -8 -6 -4 -2 -1 Weeks relative to foaling
Fig. 3. Combined uterine and placental thickness (CUPT) in pregnant mares grazing the novel endophyte tall fescue (solid lines) or orchardgrass/bluegrass (dashed lines) pastures. There were no differences between the groups of mares grazing either pasture type.
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K. McDowell et al. / Journal of Equine Veterinary Science 74 (2019) 56e64
Year 1
4 3 2 1 0
5
Placental Weights (kg)
Placental Weights (kg)
5
NETF OGBG
Year 2
4 3
4. Discussion
2 1 0
the mares remained on the toxic fescue pasture (P < .0001). When mares were removed from that pasture, the palmar artery diameters returned to their pretreatment sizes.
NETF OGBG
Fig. 4. Placental weights for mares grazing and foaling on novel endophyte tall fescue (solid bars) or orchardgrass/bluegrass (striped bars) pastures. There were no differences in placental weights between the treatment groups for either year. NETF, novel endophyte tall fescue; OGBG, orchardgrass/bluegrass pasture.
herpesvirus, nocardioform bacteria, and equine arteritis virus (data not shown). No significant pathologic lesions were observed in any of the placentas. Foal growth rates, expressed as foal weight as a percentage of mare weight, are shown in Fig. 5. Foal growth rates increased with age (P < .0001 for each year) and were not different due to pasture type (P ¼ .4926 Year 1; P ¼ .6220). The serum analytes tested in the chemistry panel are shown in Table 8. In year 1, there were no differences for the serum analytes between mares grazing NETF versus OGBG pastures. In year 2, however, serum chloride was higher (P ¼ .0278) for the mares grazing NETF than for mares grazing OGBG. Examination of the data revealed that the differences were solely on one bleeding date (July 1). When the remaining aliquots of those samples were examined, it was observed that many of them had some degree of hemolysis, an apparent sample handling effect. When chloride data from that date were removed, there were no differences between mares on the NETF versus OGBG treatment pastures (P ¼ .1018). 3.3. Palmar Artery Diameters Palmar artery mean diameters are shown in Fig. 6. In each year, there were no differences in the mean luminal diameters of the mares grazing NETF versus OGBG and the artery sizes in those groups remained fairly constant throughout the experiments (treatment by week of experiment interactions: P ¼ .2423 year 1; P ¼ .1931 year 2). However, artery diameters of the mares grazing the KY31 toxic fescue in year 1 were significantly reduced during weeks 5 through 18 compared to weeks 1 and 3 or 20 and 22 and significantly reduced compared to the other two groups as long as
Pregnant mares consuming toxic KY31TF frequently have longer than normal gestation lengths, placentas that are heavier and thicker than normal and which may be retained, plus reduced foal viability [4e6,21]. Pregnancy may be prolonged by as much as 2e4 weeks [5,6,22]. The mares also typically have reduced serum estradiol and progesterone concentrations at foaling, as well as reduced serum prolactin concentration, causing lack of proper udder development and milk production [6,21,23e25]. In the present studies, all the pregnant mares grazing and foaling on the NETF pasture had satisfactory foaling outcomes, and all variables tested were similar between mares on the NETF pasture and on the OGBG pasture. Breeding dates were not known so gestation lengths could not be documented. However, all mares that foaled had normal placentas, and there were no retained placentas, red bags, or reduced foal viability. All the mares had udder development and foals were observed to nurse in a normal fashion throughout the experiments. The foals born to mares grazing the NETF pasture had similar growth rates to the foals born to mares grazing the OGBG pasture. Serum hormone concentrations were within normal ranges and changed as expected, with prolactin and progesterone increasing and estradiol decreasing as gestation progressed. However, mares grazing the NETF in year 1 had statistically elevated progesterone in weeks -2 and 0 relative to the mares grazing the OGBG pasture, which is likely due to normal variability rather than a specific treatment effect. The study was repeated 2 years in a row and the hypotheses and objectives did not change between years. However, after gathering and analyzing data in the first year, it was decided to modify the protocol slightly to gather additional data. In addition, the UKVDL had begun performing ergovaline analyses, and the University of Kentucky Horse Pasture Evaluation Program began surveying enough local farms that we thought it advantageous, both for us and for the local farms, to use the same laboratory and the same personnel as our surrounding commercial horse farms. During the experiments, all pastures were mowed regularly, demonstrating that the pastures were not overstocked and that there was more than adequate forage available for the animals to graze throughout the experiments without the need for supplemental hay or grain. In a prior study, McDowell et al. [10] showed that ambient temperature or relative humidity was not correlated with vasoconstriction, heart rate, respiration rate, or rectal
Fig. 5. Linear regression curves for foal growth, expressed as foal weight as a percentage of mare weight. Results for foals on the novel endophyte tall fescue pasture (NETF) are shown in black with diamonds and those on the orchardgrass/bluegrass pasture (OGBG) are in gray with squares. There were no differences in foal growth rates in either year due to NE or OGBG pastures.
K. McDowell et al. / Journal of Equine Veterinary Science 74 (2019) 56e64
63
Table 8 P values for serum analytes for pregnant mares grazing NE versus OGBG pastures.
Chloride Sodium Potassium Cholesterol SGOTAST Kinase
Year 1
Year 2
.3403 .6403 .1018 .6403 .1391 .6207
.1018 .4536 .4076 .4860 .0875 .5001
GGT Alkaline phosphatase Total protein Albumin Globulin AGRatio
Year 1
Year 2
.2943 .2035 .4852 .7920 .1033 .1376
.0278 .6656 .3407 .1187 .8944 .2891
BUN Calcium Glucose Phosphorus Creatinine Total bilirubin
Year 1
Year 2
.1005 .8566 .2703 .5145 .0796 .4152
.1729 .5865 .1534 .1850 .6249 .4000
Abbreviations: AGRatio, albumin:globulin ratio; BUN, blood urea nitrogen; GGT, gamma-glutamyl transpeptidase; orchardgrass/Kentucky bluegrass; SGOST, serum glutamicoxaloacetic transaminase; NE, novel endophyte; OGBG, orchardgrass/Kentucky gluegrass.
temperatures over an experiment that went from mid-October through mid-December. One of the major signs of fescue toxicosis in ruminants is vasoconstriction [26e29]. Likewise, McDowell and her students reported that consumption of toxic KY31TF seed [8e11] or pasture (KJ McDowell, unpublished) containing the common toxic strain of the endophyte Epichlo€ e coenophiala caused a marked constriction of the palmar arteries in horses, as also evidenced in this study in the mares grazing the toxic KY31TF pasture. However, such vasoconstriction was absent in all mares grazing either the NETF or the OGBG pasture. The NETF used in these studies contained a variant of Epichlo€ e coenophiala that produced little to no ergovaline. Ergovaline was the only alkaloid measured in these studies, and ergovaline makes up 80%e100% of the ergopeptines communally found in the endophytic fungus of Epichlo€ e [30], but other ergot alkaloids are known to have vasoactive properties [14,31]. Klotz and McDowell exposed equine palmar arteries and veins to increasing concentrations of various ergot alkaloids in vitro, including ergovaline, ergonovine, ergotamine, ergocryptine, ergocornine, and ergocristine [32]. All of those alkaloids caused constriction in both the arteries and veins. However, the alkaloids, other than ergovaline, are found almost exclusively in the ergots of Claviceps purpurea, which forms on the seed heads [30]. In the experiments described here, pastures were mowed regularly to prevent seed head development. That none of the horses grazing the NETF in these studies showed any signs of artery constriction indicates that the NETF was either not producing those alkaloids or at least not producing them in any substantial quantities, and did not have seed heads infected with Claviceps purpurea. Ergovaline was detected in the NETF pasture on a single forage sampling day in year 1 and on two days in year 2, albeit in relatively low concentrations. This could be due to limited wild-type fescue ingrowth in the pasture, particularly if sampling occurred close to the fence-line with the KY31TF pasture. Nevertheless, mares grazing the NETF in these studies exhibited none of the signs of equine fescue toxicosis.
Because the NETF still contains the alkaloids that give it a competitive advantage over most other pastures, it is not anticipated that the NETE will be overgrown with KY31 fescue, as happens with endophyte free tall fescue. Our NETF pasture has been in use for mare studies for over 6 years, with no evidence of increased ingrowth of KY31 fescue. While we do pick up the occasional sample that contains ergovaline, that has not increased over the years, and could be due to sampling close to the fence line. Although endophyte-free TF varieties have been developed, their agronomic performance has been disappointing, particularly if grazed heavily or used in stress-prone locations [33,34]. Therefore, NE fescues can be an option for replacing either the common toxic strain of endophyte-infected TF or endophyte-free TF pastures. Plants that contain NEs generally persist better than endophyte-free varieties due to their maintenance of the alkaloids that confer a competitive advantage to fescue but lacking the alkaloids that are harmful to grazing livestock [35,36]. For a short review of the development of NE-infected TFs, including their advantages and disadvantages; see Phillips and Aiken [34]. In conclusion, results of these studies indicate that the TF cultivar KYFA9821 containing the novel endophyte AR584 can be a satisfactory pasture forage for pregnant mares and their foals. Acknowledgments This work was funded by the USDAdAnimal Research ServicedForage Animal Production Unit in a Specific Cooperative Agreement with the University of Kentucky College of Agriculture [grant number 3210000239], the Departments of Plant and Soil Sciences and Veterinary Science, and AgResearch. This work was also supported by the National Institute of Food and Agriculture, United States Department of Agriculture [HATCH under 1002202], publication No. 18-14-076 of the Kentucky Agricultural Experiment Station, and is published with the approval of the Director.
Year 2 Mean diameter (cm)
Mean diameter (cm)
Year 1 0.6 0.5 0.4 0.3 0.2 0.1 0.0 1
3
5
7
10 12 14 16 18 20 22
Week of experiment
0.6 0.5 0.4 0.3 0.2 0.1 0.0 2
4
6
8
10 12 14 16 18
Week of experiment
Fig. 6. Mean diameter of the palmar arteries of mares grazing novel endophyte tall fescue (solid lines), orchardgrass/bluegrass (OGBG, dashed lines), or Kentucky 31 tall fescue (dotted line) pastures. Vertical lines represent weeks that mares were placed onto and then were removed from their respective treatment pastures. Artery diameters of mares grazing KY31TF were reduced during weeks 5 through 18 compared to weeks 1 and 3 or 20 and 22 and were reduced compared to the other two groups as long as the mares remained on the toxic fescue pasture (P < .0001).
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K. McDowell et al. / Journal of Equine Veterinary Science 74 (2019) 56e64
The authors wish to thank Mr. Lynn Ennis for his assistance throughout the projects, Mr. Tracy Hamilton for pasture sampling in year 1 and Phillip Shine and student workers in the Departments of Plant and Soil Sciences and Veterinary Science for assistance with pasture management and animal handling in both years. The authors offer a special thanks to Tony Stratton of AgResearch for his advice, support, and encouragement throughout the experiments. References [1] Burns JC, Chamblee DS. Adaptation. In: Buckner RC, Bush LP, editors. Tall Fescue, 20. Madison, WI, USA: Agron Monogr; 1979. p. 9e30. [2] Hannaway DB, Daly C, Halbleib M, James D, West CP, Volence JJ, Chapman D, Li X, Cao W, Shi X, Johnson S. Development of suitability maps with examples for the United States and China. http://forages oregonstate edu/tallfescuemonograph/ 2009. [3] Bush LP, Fannin FFN. Alkaloids. In: Fribourg HA, Hannaway DB, West CP, editors. Tall Fescue for the Twenty-first Century, 53. Madison, WI, USA: Agron Monogr; 2009. p. 229e49. [4] Cross DL, Redmond LM, Strickland JR. Equine fescue toxicosis: signs and solutions. J Anim Sci 1995;73:899e908. [5] Monroe JL, Cross DL, Hudson LW, Hendricks DM, Kennedy SW, Bridges WC. Effect of selenium and endophyte-contaminated fescue on performance and reproduction in mares. J Equine Vet Sci 1988;8:148e53. [6] Putnam MR, Bransby DI, Schumacher J, Boosinger TR, Bush L, Shelby RA, Vaughan JT, Ball D, Brendemuehl JP. Effects of the fungal endophyte Acremonium coenophialum in fescue on pregnant mares and foal viability. Am J Vet Res 1991;52:2071e4. [7] Hoveland CS. Origin and history. In: Fribourg HA, Hannaway DB, West CP, editors. Tall fesccue for the twenty-first century, 53. Madison, WI: Agron Monogr; 2009. p. 3e10. [8] Hestad DA. Ingestion of endophyte-infected tall fescue seed induces peripheral vasoconstriction but does not affect cyclicity in non-pregnant mares, and a population of biogenic amine receptors relative to vasoconstriction is identified. 2012. MS Thesis, Department of Veterinary Science, University of Kentucky, Lexington, KY, USA, https://uknowledge.uky.edu/gluck_etds/2. [9] McDowell KJ, Moore ES, Parks AG, Lawrence LM. Use of Doppler ultrasonography on horses to detect vasoconstriction caused by endophyte infected tall fescue. J Equine Vet Sci 2009;29:338e9. [10] McDowell KJ, Moore ES, Parks AG, Bush LP, Horohov DW, Lawrence LM. Vasoconstriction in horses caused by endophyte-infected tall fescue seed is detected with Doppler ultrasonography. J Anim Sci 2013;91:1677e84. [11] Moore ES, Parks AG, Lawrence LM, McDowell KJ. Endophyte infected fescue seed causes vasoconstriction in horses as measured by Doppler ultrasonography. J Anim Sci 2008;86(Suppl 2):403. [12] Turner RM, McDonnell SM, Feit EM, Grogan EH, Foglia R. How to determine gestational age of an equine pregnancy in the field using transrectal ultrasonography measurement of the fetal eye. AAEP Proc 2006;52:250e5. [13] Colborn DR, Thompson DL, Roth TL, Capehart JS, White KL. Responses of cortisol and prolactin to sexual excitement and stress in stallions and geldings. J Anim Sci 1991;69:2556e62. [14] Yates SG, Powell RG. Analysis of ergopeptine alkaloids in endophyte-infected tall fescue. J Agric Food Chem 1988;36:337e40. [15] Carter JM, Aiken GE, Dougherty CT, Schrick FN. Steer responses to feeding soybean hulls and steroid hormone implantation on toxic tall fescue pasture. J Anim Sci 2010;88:3759e66.
[16] Bucca S, Fogarty U, Collins A, Small V. Assessment of feto-placental well-being in the mare from mid-gestation to term: transrectal and transabdominal ultrasonographic features. Theriogenology 2005;64:542e57. [17] Colon JL. Trans-rectal ultrasonographic appearance of abnormal combined utero-placental thickness in late-term gestation and its incidence during routine survey in a population of Thoroughbred maes (2005-2008). Theriogenology 2008;54:279e85. [18] Lea K, Smith L, Gaskill C, Coleman R, Smith SR. Ergovaline stability in tall fescue based on sample handling and storage methods. Front Chem 2014;2:1e6. [19] Spiering MJ, Davies E, Tapper BA, Schmid J, Lane GA. Simplified extraction of ergovaline and peramine for analysis of yissue distribution in endophyteinfected grass tillers. J Agric Food Chem 2002;50:5856e62. [20] SAS Institute I. SAS System for Microsoft windows, Release 9.3. Cary, N.C: SAS Institute, Inc; 2012. [21] Cross DL. Fescue toxicosis. In: McKinnon AO, Squires EL, Vaala WE, Varner DD, editors. Equine reproduction. 2nd. Aimes, IA: Wiley-Blackwell; 2011. p. 2418e27. [22] Boosinger TR, Brendemuehl JP, Bransby DL, Wright JC, Kemppainen RJ, Kee DD. Prolonged gestation, decreased triiodothyronine concentration, and thyroid gland histomorphologic features in newborn foals of mares grazing Acremonion coenophialum-infected fescue. Am J Vet Res 1995;56: 66e9. [23] Brendemuehl JP, Carson RL, Wenzel JGW, Boosinger TR, Shelby RA. Effects of grazing endophyte-infected tall fescue on eCG and progestogen concentrations from gestation days 21 to 300 in the mare. Theriogenology 1996;46:85e95. [24] Evans TJ. The endocrine disruptive effects of ergopeptine alkaloids on pregnant mares. Vet Clin Equine 2011;27:165e73. [25] McCann JS, Caudle AB, Thompson FN, Stuedemann JA, Heusner GL, Thompson Jr DL. Influence of endophyte-infected tall fescue on serum prolactin and progesterone in gravid mares. J Anim Sci 1992;70:217e23. [26] Aiken GE, John A, Britt JL, Miller MF, Adams SK, Duckett SK. Vasoconstrictive responses of the carotid artery in pregnant ewes to ergot alkaloid exposure. J Anim Sci 2017;95:236. [27] Foote AP, Harmon DL, Strickland JR, Bush LP, Klotz JL. Effect of ergot alkaloids on contractility of bovine right ruminal artery and vein. J Anim Sci 2011;89: 2944e9. [28] Klotz JL, Bush LP, Smith DL, Shafer WD, Smith LL, Arrington BC, Strickland JR. Ergovaline-induced vasoconstriction in an isolated bovine lateral saphenous vein bioassay. J Anim Sci 2007;85:2330e6. [29] Klotz JL, Kirch BH, Aiken GE, Bush LP, Strickland JR. Contractile response of fescue-naive bovine lateral saphenous veins to increasing concentrations of tall fescue alkaloids. J Anim Sci 2010;88:408e15. [30] Guerre P. Ergot alkaloids produced by thee endophytic fungi of the genus Epichol€ e. Toxins 2015:773e90. [31] Lyons PC, Plattner RD, Bacon CW. Occurrence of peptide and clavine ergot alkaloids in tall fescue grass. Science 1986;232:487e9. [32] Klotz JL, McDowell KJ. Tall fescue ergot alkaloids are vasoactive in equine vasculature. J Anim Sci 2017;95:5151e60. [33] Bouton JH, Latch GCM, Hill NS, Hoveland CS, McCann MA, Watson RH, Parish JA, Hawkins LL, Thompson FN. Reinfection of tall fescue cultivars with non-ergot alkaloid-producing endophytes. Agron J 2002;94: 567e74. [34] Phillips TD, Aiken GE. Novel endophyte-infected tall fescues. Forage and Grazinglands 2009;7. https://doi.org/10.1094/FG-2009-1102-01-RV. [35] Bacon CW, Siegel MR. Endophyte parasitism of tall fescue. J Prod Agric 1988;1: 45e55. [36] Burns JC, Fisher DS, Rottinghaus GE. Grazing influences on mass, nutritive value, and persistence of stockpiled Jesup tall fescue without and with novel and wild-type fungal endophytes. Crop Sci 2006;46: 1898e912.