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Temperature and PH effects on rubber toxicity for equine spermatozoa
¢
~kT~,"~i~
Refereed
TEMPERATUREAND PH EFFECTSON RUBBERTOXICITY FOR EQUINESPERMATOZOA D.R. Colborn, MS,~ C.P. Merilan, PhD, 2 and W.E. Loch, PbD ~
SUMMARY Stallion spermatozoa collected in an artificial vagina with polyethylene liners had significantly higher (9<.01) percent living (unstained) cells and progressive motility after exposure to rubber for 0 to 10 m than did spermatozoa collected with rubber liners. However, the rate of decline, in both groups, following the timed exposure to rubber, was similar. In a second study, utilizing semen samples at pH 6.2, 6.6, 7.0 or 7.4 and temperatures of 12, 22, 32 or 42°C, the toxic effect of rubber was found to be greater at the higher temperature and pH levels.
live-dead staining of bovine sperm cells? Toxic effects of rubber materials, including syringe plungers and surgical gloves, for equine spermatozoa have been noted,3 and more recently, some artificial vagina liners were identified as being toxic to stallion spermatozoa.4 In the latter study, the maximum effects of the rubber toxicity were observable only after storage of the semen for several hours. Studies with bull spermatozoa have shown toxic effects to occur as a result of only brief exposure to rubber materials at 4°C during semen processing and handling procedures. ~ These experiments were undertaken to determine if temperature and pH of equine semen at time of exposure to rubber had any effects on the rubber toxicity.
MATERIALS AND METHODS INTRODUCTION The relative toxic effects of rubber vary from one species to another. These detrimental effects of rubber were reported to be detected sooner alter exposure with boar than with bull semen? Also, progressive motility has been found to be a more sensitive indicator of rubber toxicity than was Authors' present addresses: 'Department of Animal Science, Louisiana State University, Baton Rouge LA 70803, ~Departmentof Dairy Science and SDepartment of Animal Sciences, University of Missouri, Columbia, MO 65211. Acknowledgements: Gratitudeis expressed to J.F. Smith for technical assistance with the project. This project:was supported in part by the University of Missouri InstitutionalBiomedical Research Support Grant RR 07053 from the National institutes of Health. A contribution from the Missouri Agricultural Experiment Station. Journal Series No.: 10846.
Volume 10, Number 5, 1990
Two separate experiments were conducted to establish initially, the effect of rubber exposure time at room temperature (Exp. 1), and subsequently, the interactive effects of pH and temperature (Exp. 2) on rubber toxicity for equine spermatozoa.
Experiment 1 Semen was collected twice weekly from two stallions using Missouri model" artificial vaginas. One artificial vagina contained a standard rubber liner" while the second contained a liner made from polyethylene,b The type of Supply Co. Fort Atkinson, Wl 53538. bFlex-O-Glass, N Agricultural aInc.,tChicago, i oIL 60651. n a l
343
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80
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* Polyethylene L i n e r o Rubber Liner
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.... ---
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7.4
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pH
10
Figure 1.
Effect of time exposed to rubber at 22°C on progressive motility of equine spermatozoa collected in rubber or polyethylene liners.
liner used with a stallion was alternated with each successive collection. Both the rubber and the polyethylene linercollection cones were washed with a nonphosphate detergent,c rinsed five times each with tap and distilled water, and then air dried before use. The polyethylene linercollection cones were discarded after one use, while the rubber liner-collection cones were rewashed and reused. The ejaculates were collected into 250 ml polyethylene bottles enclosed in thermal protective jackets extending from the water-jacketed portion of the artificial vagina. Temperature inside the collection liner was maintained at approximately 45°C, and all liners were lubricated with HR Lubricating Jelly.d Following collection, the semen was filtered through eight layers of 100 mesh cheese cloth to remove the gel fraction. The ejaculates were cooled slowly to laboratory temperature, 22°C, then they were divided into 15 two ml aliquots and randomly allotted to five groups of three replicate tubes. Each of four groups .were exposed to 5 mm wide strips of rubber, from liners," for 1, 2, 5 or 10 m. The fifth group of tubes served as the zero exposure control. Semen quality evaluations were done initially after exposure to the rubber strips and again following storage of the semen samples for three hr at laboratory room temperature. Small aliquots of each semen sample were wanned to make progressive motility estimates at 35°C and 100 magnification. Aliquots of semen were used for live-dead staining with an eosin B:fast green FCF stain.8
Volume 10, Number 5, 1990
+
x
J
MINUTES EXPOSED TO RUBBER STRIPS
aNational Agricultural SupplyCo. Fort Atkinson, W153538. bFlex-O-Glass, Inc., Chicago, IL 60651. :Liqui-Nox, AIconox, Inc. New York, NY 10003. dyoung's Drug ProductCorporation, Piscataway, NJ 08854.
Oe9. C * 12 o 22
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42
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I,.
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x
6.2
6.6
7.0
7.4
pH Figure 2. Effect of pH on differential staining of equine spermatozoa immediately following exposure to rubber (A) and after 3 hours storage (B).
Experiment 2 Two stallions were collected once weekly using an artificial vagina with a liner-collection cone made of polyethylene,b Following collection, removal of the gel fraction and slow cooling to 12°C, 10 ml of the sample was diluted with 30 ml 0.9% NaCI. This mixture was subdivided into four aliquots which were diluted with 3% sodium citrate solutions to obtain pH 6.2, 6.6, 7.0 or 7.4. Duplicate aliquots from each pH were then incubated in water baths at 12, 22, 32 or 42°C. One sample from each pair was exposed, by immersion, to a 5 mm wide strip of rubber for 1, 2, 5 or 10 m with the other pair member serving as the zero exposure control. All pH levels were represented in each trial; whereas, only one of the exposure times was used for each trial because of the practical time constraints associ-
345
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-- --Rubber Exp.
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Figure 3. (A) Effect of pH on progressive motility of diluted equine spermatozoa immediately following exposure to rubber.(B) Progressive motility of rubber exposed cells as a percent of unexposed control spermatozoa.
Figure4. (A) Effect of pH on progressive motility of diluted equine spermatozoa following 3 hours storage after exposure to rubber. (B) Progressive motility of rubber exposed cells as a percent of unexposed control spermatozoa.
ated with motility evaluations and stained slide preparation. Following exposure to rubber at the various temperatures, a small aliquot from each of the samples was evaluated for progressive motility at 35°C and for live-dead staining. The remainder of each of the samples was stored at 22°C for 3 hr'prior to re-evaluation for motility and live-dead staining:
with rubber liners. Following 3 hr storage at 22°C, the motility continued to show decreases as a result of the 1 to 10 m rubber exposure, but the decrease over time had become nonlinear. The motilityof spermatozoa collected in polyethyleneliners averaged 15% greater than for the cells collected in rubber liners. Although the number of live (unstained) cells decreased as rubber exposure time increased (p<.01), the fiends were similar across liner type, and the magnitude of difference increased markedly after 3 hr storage at 22°C. A similar effect was observed for pH. As pH increased, the number of unstained cells after exposure to rubber decreased (Fig. 2A). Larger decreases were apparent after 3 hr storage (Fig. 2B). Exposure time to rubber had an effect (p<.01) on the progressive motility and percent unstained cells of the
RESULTS
The average spermatozoalprogressive motilitydecreased as rubber exposure time increased (p<.O1) regardless of the type of collection liner used (Fig. 1). However, the progressive motility of spermatozoa collected with a polyethylene liner was consistently 5to 7% higher than for cells collected 346
EQUIN E VETERINARY SCIENCE
levels which this study has found to increase toxic effects. The typical stallion semen ejaculate with a pH of 7.2 to 7.87 and a liner temperature of 40-45°C is at substantial risk from rubber toxicity because temperatures above 32°C and a pH of 7.0 or greater appear to represent critical levels for enhancing rubber toxicity. The finding that subsequent 1 to 10 m exposure to rubber can further impair motility of equine spermatozoa collected in either rubber or polyethylene liners provides additional evidence favoring complete elimination of any contact between sperm cells and toxic rubber materials.
various groups; however, trends for all exposure times were similar. Thus, the results were averaged across exposure time within pH and temperature combinations. The effect of pI-I on the percent progressive motility of spermatozoa at a given temperature immediately following exposure to rubber is given in Figure 3A with both increased temperature and pH resulting in decreased progressive motility. Upon comparison of rubber exposed samples with controls (Fig. 3B), it was apparent that as temperature increased greater rubber toxicities occurred at the higher pH levels. Following 3 hr storage, further decreases in progressive motility were evident at all temperature and pH levels (Fig. 4A). However, comparison of rubber exposed samples with unexposed controls (Fig. 4B) indicated that the toxic effects of rubber exposure were markedly greater at pH 7.0 or higher and at temperatures of 32°C or higher.
REFERENCES 1. Beseth L: Biologicaltesting of the toxicityof rubber used in artificial vaginas with boar semen. Nord Vet Med 14:689-701,1962. 2. Flick DL, Merilan CP: Toxicity evaluation with bovine spermatozoa. JAnim Sci 59(suppl 1):310 (abst),1984. 3. JonesWE, editor: Toxic agents in equine AI procedures. Equine Vet Data 5(14):219,1984. 4. MerilanCP, Loch WE: The effect of artificial vagina liners on livability of stallion spermatozoa. Equine Vet Sci 7(4):226228,1987. 5. Smith JF, Merilan CP: Effect of polymers at 5°C on post thaw motility of bovine spermatozoa. J Dairy Sci 71(suppl 1):192 (abst),1988. 6. Herman HA, Madden FW: The artificial insemination of dairy and beef cattle. 6th ed, Lucas Brothers, Columbia, Me, 1980. 7. SorensonAM: Animal reproduction - principles and practices. McGraw-Hill, New York, pp 15-16,1979.
DISCUSSION
These data confirm earlier suggestions 4 that even short term exposure to rubber can cause damage to equine spermatozoa, and the consequences of the toxicity are increasingly evident following storage of the sperm cells. The initial decreased motility for sperm cells collected with rubber liners is attributed to the brief exposure occurring during the collection process at temperatures and pH
VET PRO LABS INC.
AN OFFICIAL USDA 4720 E. 51st St. Suite E AGID EIA LABORATORY PO Box 35328-0328 Tulsa, OK 74135 • 18 Chemistry Panel (Small & Large Animal) $5.00 • CBC $4.00 • FELV $4.00 *TOXO $4.00 SAME DAY RESULTS • T3T4 RIA $8.00 - FTLV $10.00 ON THESE PANELS I ~ • Brucellosis (Canine) $4.00 • Occult Heartworm $4.00
1-800-999-VETS (8387}
•
1-918-492-1767
iiiiiiiiiiiiiiiiiii!ii!ii!!ii!!ii~i~~~i
~i~iiiili i!i}i!!!i!iiii!!ii!iiiii!~i}i~ii~}ii~i~ii!ii~i!ilii!!ii~i ii~i~ii i i i i i! i
Volume 10, Number 5, 1990
B
7 DAYSA WEEK J "ONCOGGINSTEST" IIBN B
n
mBII ~
~
n
IIBIB B
• Bleeder studies by four veterinarians on 209 horses gave 87.5% excellent results with no post race bleeding. • Pharyngitis studies by three veterinarians on 8 horses showed that 87.5% were healed within 48-72 hours after three treatments.
iiiiiiiii!ii!i!!i i i i i i i i i i i i i i i i !i
~i ii i i ilili!i!;ii!i ili ii i i i i i i i i i iii i ii!iiUii iii~i iii~iii~iiMiiiii:iiii~ii:!i!i i!i i!iiii ii i i i i iii~ !ili
Promune-E patent applied for
L
Distributedby PHOENIX PHARMACEUTICAL,INC P.O.,Box 7., St. Joseph, M e 64506 816-364-5777
347
~kT~,"~i~
Refereed
TEMPERATUREAND PH EFFECTSON RUBBERTOXICITY FOR EQUINESPERMATOZOA D.R. Colborn, MS,~ C.P. Merilan, PhD, 2 and W.E. Loch, PbD ~
SUMMARY Stallion spermatozoa collected in an artificial vagina with polyethylene liners had significantly higher (9<.01) percent living (unstained) cells and progressive motility after exposure to rubber for 0 to 10 m than did spermatozoa collected with rubber liners. However, the rate of decline, in both groups, following the timed exposure to rubber, was similar. In a second study, utilizing semen samples at pH 6.2, 6.6, 7.0 or 7.4 and temperatures of 12, 22, 32 or 42°C, the toxic effect of rubber was found to be greater at the higher temperature and pH levels.
live-dead staining of bovine sperm cells? Toxic effects of rubber materials, including syringe plungers and surgical gloves, for equine spermatozoa have been noted,3 and more recently, some artificial vagina liners were identified as being toxic to stallion spermatozoa.4 In the latter study, the maximum effects of the rubber toxicity were observable only after storage of the semen for several hours. Studies with bull spermatozoa have shown toxic effects to occur as a result of only brief exposure to rubber materials at 4°C during semen processing and handling procedures. ~ These experiments were undertaken to determine if temperature and pH of equine semen at time of exposure to rubber had any effects on the rubber toxicity.
MATERIALS AND METHODS INTRODUCTION The relative toxic effects of rubber vary from one species to another. These detrimental effects of rubber were reported to be detected sooner alter exposure with boar than with bull semen? Also, progressive motility has been found to be a more sensitive indicator of rubber toxicity than was Authors' present addresses: 'Department of Animal Science, Louisiana State University, Baton Rouge LA 70803, ~Departmentof Dairy Science and SDepartment of Animal Sciences, University of Missouri, Columbia, MO 65211. Acknowledgements: Gratitudeis expressed to J.F. Smith for technical assistance with the project. This project:was supported in part by the University of Missouri InstitutionalBiomedical Research Support Grant RR 07053 from the National institutes of Health. A contribution from the Missouri Agricultural Experiment Station. Journal Series No.: 10846.
Volume 10, Number 5, 1990
Two separate experiments were conducted to establish initially, the effect of rubber exposure time at room temperature (Exp. 1), and subsequently, the interactive effects of pH and temperature (Exp. 2) on rubber toxicity for equine spermatozoa.
Experiment 1 Semen was collected twice weekly from two stallions using Missouri model" artificial vaginas. One artificial vagina contained a standard rubber liner" while the second contained a liner made from polyethylene,b The type of Supply Co. Fort Atkinson, Wl 53538. bFlex-O-Glass, N Agricultural aInc.,tChicago, i oIL 60651. n a l
343
oo| A
:,p- go/--
80
..J I-0 X 70 I--4
t.Jo or) I.iJ n. {.3
n LU Z
* Polyethylene L i n e r o Rubber Liner
50
x
o
o~ oL I--
z bJ U n." W 0..
.... ---
50
40
Initiol
ED
~:
N
I--
7060-
(:3 or) I-- Z
Raodlng=
50-
After 3 Hr. Storogm
n- I-LU Z n LU L0 (..) 0~ OJ 0.
x%
30' 20
v
10~"
403020
0 0
I
I
1
2
I
f
I
I
,
I
I
5
!
X
32 42
I
I
I
I
7.4
6.26.67.0
pH
10
Figure 1.
Effect of time exposed to rubber at 22°C on progressive motility of equine spermatozoa collected in rubber or polyethylene liners.
liner used with a stallion was alternated with each successive collection. Both the rubber and the polyethylene linercollection cones were washed with a nonphosphate detergent,c rinsed five times each with tap and distilled water, and then air dried before use. The polyethylene linercollection cones were discarded after one use, while the rubber liner-collection cones were rewashed and reused. The ejaculates were collected into 250 ml polyethylene bottles enclosed in thermal protective jackets extending from the water-jacketed portion of the artificial vagina. Temperature inside the collection liner was maintained at approximately 45°C, and all liners were lubricated with HR Lubricating Jelly.d Following collection, the semen was filtered through eight layers of 100 mesh cheese cloth to remove the gel fraction. The ejaculates were cooled slowly to laboratory temperature, 22°C, then they were divided into 15 two ml aliquots and randomly allotted to five groups of three replicate tubes. Each of four groups .were exposed to 5 mm wide strips of rubber, from liners," for 1, 2, 5 or 10 m. The fifth group of tubes served as the zero exposure control. Semen quality evaluations were done initially after exposure to the rubber strips and again following storage of the semen samples for three hr at laboratory room temperature. Small aliquots of each semen sample were wanned to make progressive motility estimates at 35°C and 100 magnification. Aliquots of semen were used for live-dead staining with an eosin B:fast green FCF stain.8
Volume 10, Number 5, 1990
+
x
J
MINUTES EXPOSED TO RUBBER STRIPS
aNational Agricultural SupplyCo. Fort Atkinson, W153538. bFlex-O-Glass, Inc., Chicago, IL 60651. :Liqui-Nox, AIconox, Inc. New York, NY 10003. dyoung's Drug ProductCorporation, Piscataway, NJ 08854.
Oe9. C * 12 o 22
B
80 --
/-%
O ILl Z ,-, < I--
< O N O (Jr) t-- Z X 0~ k-W Z n Ld 00 LI rv" LU OL
70-
- - R u b b e r Exp. .Control
u
60...
50-
+
~
x
32
42
403020
I
I,.
I
x
6.2
6.6
7.0
7.4
pH Figure 2. Effect of pH on differential staining of equine spermatozoa immediately following exposure to rubber (A) and after 3 hours storage (B).
Experiment 2 Two stallions were collected once weekly using an artificial vagina with a liner-collection cone made of polyethylene,b Following collection, removal of the gel fraction and slow cooling to 12°C, 10 ml of the sample was diluted with 30 ml 0.9% NaCI. This mixture was subdivided into four aliquots which were diluted with 3% sodium citrate solutions to obtain pH 6.2, 6.6, 7.0 or 7.4. Duplicate aliquots from each pH were then incubated in water baths at 12, 22, 32 or 42°C. One sample from each pair was exposed, by immersion, to a 5 mm wide strip of rubber for 1, 2, 5 or 10 m with the other pair member serving as the zero exposure control. All pH levels were represented in each trial; whereas, only one of the exposure times was used for each trial because of the practical time constraints associ-
345
^
A
30
50
Rubber
-- --Rubber Exp.
¢::~
,,
Contro De 9.
Control
4o
>= == ==
2x \\
* o
-\\ ~XX~~~+ •
2O
x
Exp. 1 C
"
12 22 32 42
V
10
n,, O.
o_
0
I 6,2
I 6.6
I 7,0
I 7,4
0
6.2
6.6
pH
7.0
7.4
pH
B 100
100 >" I-- "..I
90
0=
eo
z ~" u ta o.
70 60 50
m =--
40 30 20 10
ol ,,, 0E o nQ.
Z ,,, U n, uJ vo_
0
>" I.j ,-, o
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I 7.4
pH
70
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20
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60
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10
0
m
D
x i 6.2
42 I 6,6
,
I 7,0
I,, 7,4
pH
Figure 3. (A) Effect of pH on progressive motility of diluted equine spermatozoa immediately following exposure to rubber.(B) Progressive motility of rubber exposed cells as a percent of unexposed control spermatozoa.
Figure4. (A) Effect of pH on progressive motility of diluted equine spermatozoa following 3 hours storage after exposure to rubber. (B) Progressive motility of rubber exposed cells as a percent of unexposed control spermatozoa.
ated with motility evaluations and stained slide preparation. Following exposure to rubber at the various temperatures, a small aliquot from each of the samples was evaluated for progressive motility at 35°C and for live-dead staining. The remainder of each of the samples was stored at 22°C for 3 hr'prior to re-evaluation for motility and live-dead staining:
with rubber liners. Following 3 hr storage at 22°C, the motility continued to show decreases as a result of the 1 to 10 m rubber exposure, but the decrease over time had become nonlinear. The motilityof spermatozoa collected in polyethyleneliners averaged 15% greater than for the cells collected in rubber liners. Although the number of live (unstained) cells decreased as rubber exposure time increased (p<.01), the fiends were similar across liner type, and the magnitude of difference increased markedly after 3 hr storage at 22°C. A similar effect was observed for pH. As pH increased, the number of unstained cells after exposure to rubber decreased (Fig. 2A). Larger decreases were apparent after 3 hr storage (Fig. 2B). Exposure time to rubber had an effect (p<.01) on the progressive motility and percent unstained cells of the
RESULTS
The average spermatozoalprogressive motilitydecreased as rubber exposure time increased (p<.O1) regardless of the type of collection liner used (Fig. 1). However, the progressive motility of spermatozoa collected with a polyethylene liner was consistently 5to 7% higher than for cells collected 346
EQUIN E VETERINARY SCIENCE
levels which this study has found to increase toxic effects. The typical stallion semen ejaculate with a pH of 7.2 to 7.87 and a liner temperature of 40-45°C is at substantial risk from rubber toxicity because temperatures above 32°C and a pH of 7.0 or greater appear to represent critical levels for enhancing rubber toxicity. The finding that subsequent 1 to 10 m exposure to rubber can further impair motility of equine spermatozoa collected in either rubber or polyethylene liners provides additional evidence favoring complete elimination of any contact between sperm cells and toxic rubber materials.
various groups; however, trends for all exposure times were similar. Thus, the results were averaged across exposure time within pH and temperature combinations. The effect of pI-I on the percent progressive motility of spermatozoa at a given temperature immediately following exposure to rubber is given in Figure 3A with both increased temperature and pH resulting in decreased progressive motility. Upon comparison of rubber exposed samples with controls (Fig. 3B), it was apparent that as temperature increased greater rubber toxicities occurred at the higher pH levels. Following 3 hr storage, further decreases in progressive motility were evident at all temperature and pH levels (Fig. 4A). However, comparison of rubber exposed samples with unexposed controls (Fig. 4B) indicated that the toxic effects of rubber exposure were markedly greater at pH 7.0 or higher and at temperatures of 32°C or higher.
REFERENCES 1. Beseth L: Biologicaltesting of the toxicityof rubber used in artificial vaginas with boar semen. Nord Vet Med 14:689-701,1962. 2. Flick DL, Merilan CP: Toxicity evaluation with bovine spermatozoa. JAnim Sci 59(suppl 1):310 (abst),1984. 3. JonesWE, editor: Toxic agents in equine AI procedures. Equine Vet Data 5(14):219,1984. 4. MerilanCP, Loch WE: The effect of artificial vagina liners on livability of stallion spermatozoa. Equine Vet Sci 7(4):226228,1987. 5. Smith JF, Merilan CP: Effect of polymers at 5°C on post thaw motility of bovine spermatozoa. J Dairy Sci 71(suppl 1):192 (abst),1988. 6. Herman HA, Madden FW: The artificial insemination of dairy and beef cattle. 6th ed, Lucas Brothers, Columbia, Me, 1980. 7. SorensonAM: Animal reproduction - principles and practices. McGraw-Hill, New York, pp 15-16,1979.
DISCUSSION
These data confirm earlier suggestions 4 that even short term exposure to rubber can cause damage to equine spermatozoa, and the consequences of the toxicity are increasingly evident following storage of the sperm cells. The initial decreased motility for sperm cells collected with rubber liners is attributed to the brief exposure occurring during the collection process at temperatures and pH
VET PRO LABS INC.
AN OFFICIAL USDA 4720 E. 51st St. Suite E AGID EIA LABORATORY PO Box 35328-0328 Tulsa, OK 74135 • 18 Chemistry Panel (Small & Large Animal) $5.00 • CBC $4.00 • FELV $4.00 *TOXO $4.00 SAME DAY RESULTS • T3T4 RIA $8.00 - FTLV $10.00 ON THESE PANELS I ~ • Brucellosis (Canine) $4.00 • Occult Heartworm $4.00
1-800-999-VETS (8387}
•
1-918-492-1767
iiiiiiiiiiiiiiiiiii!ii!ii!!ii!!ii~i~~~i
~i~iiiili i!i}i!!!i!iiii!!ii!iiiii!~i}i~ii~}ii~i~ii!ii~i!ilii!!ii~i ii~i~ii i i i i i! i
Volume 10, Number 5, 1990
B
7 DAYSA WEEK J "ONCOGGINSTEST" IIBN B
n
mBII ~
~
n
IIBIB B
• Bleeder studies by four veterinarians on 209 horses gave 87.5% excellent results with no post race bleeding. • Pharyngitis studies by three veterinarians on 8 horses showed that 87.5% were healed within 48-72 hours after three treatments.
iiiiiiiii!ii!i!!i i i i i i i i i i i i i i i i !i
~i ii i i ilili!i!;ii!i ili ii i i i i i i i i i iii i ii!iiUii iii~i iii~iii~iiMiiiii:iiii~ii:!i!i i!i i!iiii ii i i i i iii~ !ili
Promune-E patent applied for
L
Distributedby PHOENIX PHARMACEUTICAL,INC P.O.,Box 7., St. Joseph, M e 64506 816-364-5777
347