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Antibiotics for Elimination of Mycoplasmas and Ureaplasma from Bovine Semen
A n t i b i o t i c s f o r E l i m i n a t i o n of M y c o p l a s m a s and U r e a p l a s m a f r o m Bovine Semen R. B. TRUSCOTT t and C. ABREO Oeloartment of Veterinary Microbiology University of Guelph Guelph, Ontario N1G 2W1 and United Breeders, inc. Guelph, Ontario
ABSTRACT
ductive tracts, abnormal milk, and nasal discharge of cattle (8). Lincospectin also was effective against strains of mycoplasma isolated from bovine semen and preputial washings but less effective against ureaplasmas from these same sources (14). This study was to establish a practical treatment for elimination of mycoplasmas and ureaplasma from bovine semen.
Minocin at 500 /~g/ml of semen extender eliminated ureaplasma from naturally or artificially infected bovine semen. Minocin with lincospectin eliminated
Acboleplasma laidlawii, Mycoplasma bovigenitalium, boris, canadense, and arginini from artificially infected semen. Stabilization times of 15 min at 35 C and 3 h at 4 C are important considerations to maximize antibiotic activity.
MATERIALS AND METHODS Test Strains
INTRODUCTION The role of mycoplasmas and ureaplasmas (T-Mycoplasmas) in pathology o f bovine reproductive tract has not been established (1, 5, 7). The shedding of mycoplasma and ureaplasma in bovine semen and their recovery from it and from preputial washings has been reported (3, 4, 10, t l ) . Furthermore, at least one species, Mycoplasma boris (M. agalactiae subsp, boris), was capable of surviving 18-mo storage at - 1 9 6 C in bovine semen (9), and survival of other possibly pathogenic species seems reasonable. Since these organisms are .potential pathogens in the female reproductive tract, to eliminate them from semen is desirable. Lincospectin was effective against M. bovigenitalium, M. boris, and A. laidlawii isolated from the repro-
Fifteen strains of bovine mycoplasmas and ureaplasma were used. They included Acboleplasma laidlawii (NTCC10116), Mycoplasma bovigenitalium (PG11), h4. canadense (Ontario milk isolate 466), M. boris (Ontario joint isolate 427), and M. arginini (Ontario lung isolate 108) and were obtained from L. Ruhnke2; ureaplasma strains T95, D48, A417, FS01, T315, T288, T44, Bu2 and MmB167 were obtained from C. Howard. 3 In addition, one strain of ureaplasma (SB-I) isolated during this study was used in a further experiment.
Received January 19, 1977. z Animal Pathology Division, Health of Animals Branch, Agriculture Canada, Atlantic Area Laboratory, P.O. Box 1410, Sackville, New Brunswick, EOA 3CO. Veterinary Services Laboratory, Ontario Ministry of Agriculture and Food, Guelph, Ontario. 3Compton, England. 4 Minocycline hydrochloride. Cyanamid of Canada Ltd., Montreal, Quebec. s Berenil, Farbwerke Hoechst, A.G. Frankfurt, Germany. 6Doxycycline hydate, Pfizer Company Ltd., Pointe-Clair-Dorval, Quebec.
Determination of Colony Forming Units (CFU)
Media The medium and techniques for isolation and growth of mycoplasmas were as described by Davies (2), and the cultural medium for ureaplasma was described (6, 12).
For counts serial dilutions were in broth, and .02 ml was deposited on appropriate agar plates in quadruplicate. Following incubation, the colonies were counted, averaged, and the number was multiplied by the appropriate dilution factor. Antibiotics
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Minocin 4, berenil s, or doxycycline 6 were
SEMEN ANTIBIOTIC TREATMENT dissolved in distilled w a t e r as 1% s o l u t i o n . F r e s h s o l u t i o n s were p r e p a r e d f o r e a c h experim e n t a n d were a d d e d t o g l y c e r a t e d e x t e n d e r to p r o v i d e t h e desired c o n c e n t r a t i o n . Lincospectin 7 s o l u t i o n was a d d e d d i r e c t l y to t h e semen. T h e e x t e n d e r c o n s i s t e d o f m i l k a n d glycerol a n d c o n t a i n e d 1 0 0 0 IU o f penicillin a n d 1 mg of streptomycin/ml. Antibiotic Safety
T h e a n t i b i o t i c s were t e s t e d for s a f e t y o n s p e r m cell livability as d e t e r m i n e d b y t h e p e r c e n t o f s p e r m viability a n d m o t i l i t y t h r o u g h o u t t h e processing p e r i o d a n d 14 days a f t e r freezing. Procedures for Test
Details were c h a n g e d as t h e s e studies progressed. In general, .1 t o .5 ml o f b r o t h c u l t u r e s w e r e a d d e d t o 1 m l of s e m e n t o w h i c h e x t e n d e r c o n t a i n i n g t h e test a n t i b i o t i c s was added. T h e s a m p l e was m i x e d well a n d allowed t o stabilize
7 IAncomycin-Spectinomycin, The Upjohn Company, Kalamazoo, MI.
955
at 35 C f o r 10 m i n (later e x t e n d e d t o 15 min). C o n t r o l s c o n s i s t e d o f s e m e n plus e x t e n d e r w i t h or w i t h o u t a d d e d b r o t h c u l t u r e s as a p p r o p r i a t e b u t w i t h o u t t h e a n t i b i o t i c u n d e r test. Following s t a b i l i z a t i o n , t h e samples were held at 4 C for 2 h (later increased to 3 h). A l o o p f u l t h e n was plated d i r e c t l y o n agar a n d a sample i n o c u l a t e d i n t o b r o t h t o p r o v i d e a 10% inocul u m . Samples c o n t a i n i n g a n t i b i o t i c s were diluted f u r t h e r t o 10 -2 a n d 10 -3 t o o v e r c o m e t h e possible stasis o f t h e agent. T h e b r o t h s were s u b c u l t u r e d t o fresh b r o t h at 6 days. B r o t h s were p l a t e d at 4 8 a n d 96 h a n d t h e plates e x a m i n e d for 7 days b e f o r e b e i n g discarded. S u b s e q u e n t l y , s o m e e x p e r i m e n t s were perf o r m e d b y c e n t r i f u g i n g t h e test samples f o r 8 m i n at 3 0 0 0 × g, d e c a n t i n g t h e s u p e r n a t a n t , a n d discarding t h e s e d i m e n t . T h e s u p e r n a t a n t was c e n t r i f u g e d for 2 0 m i n at 1 7 , 5 0 0 × g a n d t h e s u p e r n a t a n t discarded. T h e s e d i m e n t was r e s u s p e n d e d in b r o t h a n d t h e washing r e p e a t e d t w i c e w i t h b r o t h . T h e final s e d i m e n t was s u s p e n d e d in b r o t h a n d i n c u b a t e d .
Field Trial
S e m e n was c o l l e c t e d f r o m a Holstein bull,
TABLE 1. Effect of minocin and berenil on ureaplasma in bovine semen.
Exp.
Agent
Conc.a
Ureaplasma added
Ureaplasma recovered
1
Minocin Minocin Minocin Control
100 500 1000 0
... ... ... ...
+ (SB-I)
2
Minocin Minocin Minocin Control
100 500 1000 0
SBI b SBI SBI SBI
--+
3
Minocin Minocin Minocin Control Berenil Berenil Berenil
100 500 1000 0 100 500 1000
Pool c Pool Pool Pool Pool Pool Pool
+ -+ + ÷ +
a#g/ml of extender. b Estimated approximately 1 × IO s SB-I/ml extended semen. CEqual amounts of active cultures of the nine strains of ureaplasma obtained from Howard were pooled and .2 ml added/ml of semen. Journal of Dairy Science Vol. 60, No. 6
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TRUSCOTr AND ABREO
TABLE 2. Efficacy of minocina on semen infected with bovine mycoplasmas and ureaplasma (Exp. 4). Recovery Untreated
Treated
Strain addedb
Mycoplasma
Ureaplasma
Mycoplasma
None A. laidlawii M. bovigenitalium M. boris M. arginini M. canadense Pool of above Pool + ureaplasma T288
ND c ND ND ND ND ND +
+ ND ND ND ND ND ND +
-+ + + + +
Ureaplasma
al000/~glml extender. bDilution of culture in semen * extender, 1/50. CNot done.
1.5 yr o f age. It was divided equally, and o n e - h a l f was p r o c e s s e d with e x t e n d e r c o n t a i n ing penicillin and s t r e p t o m y c i n . The o t h e r o n e - h a l f was p r o c e s s e d w i t h t h e e x t e n d e r t o which l i n c o s p e c t i n and m i n o c i n w e r e a d d e d at 300/~g l i n c o m y c i n , 6 0 0 / l g s p e c t i n o m y c i n , a n d 500 p g / m l , respectively. T w o t h o u s a n d doses o f each were processed. T h e p r o c e s s e d samples were c o d e d and i n s e m i n a t i o n results t a b u l a t e d t o p r o v i d e a 6 0 / 9 0 n o n r e t u r n rate f o r each. RESULTS Experiments 1 and 2 S e m e n f r o m a hull k n o w n t o shed ureaplas-
m a was used f o r the first e x p e r i m e n t . Minocin was a d d e d at 100, 500, a n d 1000 /~g/ml o f s e m e n e x t e n d e r . The ureaplasma isolated (SBI) f r o m t h e c o n t r o l o f e x p e r i m e n t 1 was used f o r t h e s e c o n d e x p e r i m e n t , and .2 ml o f an active b r o t h culture was a d d e d per ml o f s e m e n . Minocin e l i m i n a t e d u r e a p l a s m a (Table 1). Experiment 3 Minocin a n d berenil were c o m p a r e d at t h e s a m e c o n c e n t r a t i o n s . Equal a m o u n t s o f active b r o t h c u l t u r e s o f t h e eight d e s i g n a t e d ureaplasm a strains f r o m H o w a r d w e r e p o o l e d , and 2 ml w e r e a d d e d t o 10 ml o f s e m e n ; t h e n e x t e n d e r c o n t a i n i n g t h e t w o agents was a d d e d t o a 1/25
TABLE 3. Effect of a combination of minocin a and lincospectinb on s e m e n i n f e c t e d with bovine mycoplasmas and ureaplasma (Exp. 5). Recovery Strain added
CFU/ml extended semen
Mycoplasma
Ureaplasma
Control semen A. laldlav~ii M. bovigenitalium M. boris M. arginini M. canadense Ureaplasma T288
7.2 X 103 1.4 X 10 ~ 4.4 X 104 4.8 × 104 2 × 104 4 X 102
-+ + -
+ ---
a500 t~g/ml extender. b Lmcomycm . 300/~g, spectinomycin 600 t~g/ml semen. Journal of Dairy Science Vol. 60, No. 6
SEMEN ANTIBIOTIC TREATMENT dilution. Results in T a b l e 1 i n d i c a t e t h a t minocin at 500 and 1000 pg/ml eliminated the t e s t strains while m i n o c i n at 1 0 0 ~tg/ml a n d berenil at t h e test c o n c e n t r a t i o n s did not. Experiment 4 To d e t e r m i n e t h e e f f e c t i v e n e s s o f m i n o c i n f o r e l i m i n a t i o n o f b o v i n e m y c o p l a s m a , .5 ml o f a n active 18 h c u l t u r e was a d d e d p e r ml of r a w s e m e n m i x e d w i t h 2 4 ml of e x t e n d e r t r e a t e d w i t h 1 0 0 0 / a g / m l o f m i n o c i n . Since p r e l i m i n a r y studies ( u n r e p o r t e d ) h a d i n d i c a t e d t h a t Ureap l a s m a s t r a i n T 2 8 8 was m o r e r e s i s t a n t t o m i n o c i n t h a n t h e o t h e r test strains, an active c u l t u r e o f t h i s s t r a i n was a d d e d t o a p o o l w i t h t h e m y c o p l a s m a s . T h e results in T a b l e 2 indicate t h a t M. bovigenitalium, M. canadense, T 2 8 8 , a n d a u r e a p l a s m a initially in t h e s e m e n were e l i m i n a t e d while A. laidlawii, M. boris, a n d M. arginini were n o t a f f e c t e d b y 1 0 0 0 / a g o f minocin. Experiment 5 T o d e t e r m i n e w h e t h e r m i n o c i n c o u l d be used in c o n j u n c t i o n w i t h l i n c o s p e c t i n , t h e e x p e r i m e n t was r e p e a t e d w i t h l i n c o m y c i n 3 0 0 gg/ml, spectinomycin 600/ag/ml of raw semen,
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a n d m i n o c i n at 5 0 0 g g / m l final c o n c e n t r a t i o n in e x t e n d e r . B r o t h c u l t u r e s o f t h e t e s t strains, .2 m l / m l of s e m e n were a d d e d a n d t h e C F U ' s o f the cultures determined. T h e results in T a b l e 3 i n d i c a t e t h a t w i t h t h e n u m b e r s used, t h e t e s t strains were e l i m i n a t e d e x c e p t f o r M. bovis a n d M. arginini. Since t h e s e s t r a i n s were initially in g r e a t e r n u m b e r s , it appeared there might be a relationship between numbers added and recovery. Experiment 6 In t h i s e x p e r i m e n t , t h e s t a b i l i z a t i o n t i m e at 35 C was i n c r e a s e d t o 15 m i n while t h e h o l d i n g t i m e at 4 C was e x t e n d e d t o 3 h; A. laidlawii a n d M. canadense were o m i t t e d . M i n o c i n a n d l i n c o s p e c t i n e l i m i n a t e d all t h e s t r a i n s t e s t e d a t various d i l u t i o n s ( T a b l e 4). Experiment 7 E x p e r i m e n t 7 was similar to e x p e r i m e n t 6 e x c e p t t h a t u r e a p l a s m a s were n o t a d d e d , b u t all m u c o p l a s m a s were utilized. Active 18-h cult u r e s were d i l u t e d 1 / 1 0 a n d 1 / 1 0 0 , C F U ' s were d e t e r m i n e d , a n d .1 m l o f b r o t h c u l t u r e was a d d e d p e r ml o f s e m e n w h i c h t h e n was d i l u t e d with extender. The controls contained only the
TABLE 4. Recovery of mycoplasma and ureaplasma from infected semen treated with lincospectin a and minocin b (Exp. 6). Recovery Untreated Strain added
Dilution c
Ureaplasma
Mycoplasma
Ureaplasma
1/130 1/1300 1/1300 1/13000 1/1300 1/13000 1/130 1/1300 Low High
-+ + + + + + ND d ND + +
-ND ND ND ND ND ND + + + +
ND -----ND ND --
ND ND ND ND ND ND ND ----
Control semen
M. bovigenitalium g. boris M. arginini Ureaplasma T288 Pool of all of above
Treated
Mycoplasma
aLincomycin 300 #g, spectinomycin 600 tag/ml semen. b 500 #g/ml extender. CBroth in semen + extender. dNot done. Journal of Dairy Science Vol. 60, No. 6
9 58
TRUSCOTT AND ABREO
TABLE 5. Recovery of mycoplasma from infected semen treated with lincospectin a and minocin b (Exp. 7).
Strain added
Recovery of mycoplasma
CFU/ml extended semen
Untreated
Treated
ND d + ND + ND + ND
ND
1.5 X 10 4 1.5 X 10 3 4 X 10 s 4 X 10 4 1.5 X l 0 s 1.5 × 10 4 1.2 X l 0 s 1,2
+
--
+
-
Control semen A. laidlawii M. boris M. arginini M. canadense Pool
of above
X 10 4
c
- -
-
-
-- -
- -
aLincomycin 300/~g, spectinomycin 600 #g/ml semen. b500 #g/ml extender. CEqual amounts of 1/100 dilution of broth cultures pooled, .1 ml added to semen. dNot done.
1 / 1 0 0 d i l u t i o n o f c u l t u r e . All t h e strains t e s t e d were e l i m i n a t e d f r o m t h e i n f e c t e d s e m e n treated with a combination of minocin and l i n c o s p e c t i n (Table 5).
Experiment 8
D o x y c y c l i n e was a d d e d t o e x t e n d e d s e m e n t o p r o v i d e e i t h e r 500 o r 2 5 0 / s g / m l . A n active c u l t u r e o f T 2 8 8 was a d d e d t o each as a 10 or 1% i n o c u l u m . F o l l o w i n g a 15-min h o l d i n g at 35 C a n d 3 h at 4 C, t h e samples were c e n t r i f u g e d , a n d t h e s e d i m e n t was w a s h e d as i n d i c a t e d . T h e c o n t r o l c o n s i s t e d o f e x t e n d e d s e m e n w i t h 1% u r e a p l a s m a i n o c u l u m . T h e final s e d i m e n t was s u s p e n d e d in b r o t h w h i c h was o b s e r v e d f o r 14 days for c o l o r change. No c h a n g e o c c u r r e d w i t h 5 0 0 # g / m l d o x y c y c l i n e while g r o w t h o c c u r r e d in all o t h e r t u b e s i n d i c a t i n g t h a t u r e a p l a s m a s were in t h e c o n t r o l a n d h a d survived t h e test o f 250/ag/ml.
Experiment 9
S e m e n f r o m six bulls s h o w n to b e s h e d d e r s was e x t e n d e d with a n d w i t h o u t l i n c o s p e c t i n a n d m i n o c i n . T h e s a m p l e s were c u l t u r e d , a n d Table 6 s h o w s m y c o p l a s m a s were r e c o v e r e d from one untreated and ureaplasma from one o t h e r u n t r e a t e d sample. No recoveries were f r o m t h e t r e a t e d samples. Journal of Dairy Science Vol. 60, No. 6
Effect on Sperm
T h e results o f t e s t s o f t h e e f f e c t o f m i n o c i n a n d berenil o n s p e r m a c t i v i t y are s u m m a r i z e d in T a b l e 7. M i n o c i n at 1 0 m g m / m l o f e x t e n d e r was t o l e r a t e d b y s p e r m cells, b u t m i n o c i n a t 50 m g m / m l or b e r e n i l at 1 m g m / m l were t o x i c . While n o t t a b u l a t e d , t o x i c i t y was e v i d e n t f r o m d o x y c y c l i n e at 125 Vg/ml as s o o n as t h e extender with doxycycline had been added. The influence of the combination of minocin a n d l i n c o s p e c t i n o n s p e r m cell m o t i l i t y f r o m t r e a t e d s e m e n f r o m seven sires is t a b u l a t e d (Table 8).
TABLE 6. Effect of lincospectina and minocin b treatment on the recovery of mycoplasma and ureaplasma from semen. Untreated Myeoplasma
Sire 1 2
Ureaplasma
Treated Mycoplasma
Ureaplasma
+
3 4 5 6
--
+
aLincomycin 300 ttg, spectinomycin 600 #g/ml semen.
b500 tag/ml extender.
SEMEN ANTIBIOTIC T R E A T M E N T
959
T A B L E 7. Effect of minocin and berenil on sperm motility and viability at various stages of processing. Days after freezing
Hours after cooling Drug
Conc. a
3
5
7
20
1
14
Minocin
.1 .5 1.0 Control
50 b 50 50 50
50 50 50 50
50 50 50 50
ND ND ND ND
45 45 45 45
ND ND ND ND
Minocin
.1 .5 1.0 Control
50 50 50 50
50 50 50 50
50 50 50 50
50 50 50 50
45 45 45 45
45 45 45 45
Minocin
1.0 10.0 50.0 Control
50 40 0c 40
50 40 0 40
50 40 0 40
40 40 0 40
30 30 ND 30
ND ND ND ND
Berenil
.1 .5 1.0 Control
45 45 45 45
45 45 45 45
45 45 30 45
45 45 30 45
ND ND ND ND
ND ND ND ND
amg/ml extender. bpercent progressive motility. CSperm dead.
Field Trial
DISCUSSION
Results from insemination of semen without compared to semen treated with lincospectin a n d m i n o c i n a r e in T a b l e 9. L i n c o s p e c t i n , m i n o cin treatment did not have a deleterious effect o n c o n c e p t i o n r a t e b u t a p p e a r e d t o i m p r o v e it.
Minocin eliminated ureaplasma strains from both naturally and artificially infected semen. It is o u r e s t i m a t e t h a t e x c e p t f o r t h o s e e x p e r i ments with mycoplasma when the cultures were diluted prior to addition to semen, the numbers
T A B L E 8. S e m e n motility a in extender containing m i n o c i n b and lincospectin c. Frozen and t h a w e d 14 Days 4 C (h) Sire 1 2 3 4 5 6 7
3
5
7
20
1 Day
Oh
3h -32C
50 50 50 45 55 40 50
50 50 55 45 55 40 50
50 45 50 50 50 40 50
50 45 50 45 55 40 45
50 50 50 40 50 40 50
50 50 50 40 50 45 45
50 45 50 40 50 45 40
aExpressed as percent progressive motility. b s o 0 #g/ml extender. C
Lm c o m y c m 300 #g, s p e c t i n o m y c i n 600 #g/ml semen.
Journal of Dairy Science Vol. 60, No. 6
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TRUSCOTT AND ABREO
TABLE 9. Conception rates from field use of semen with and without lincospectin a and minocin b treatment. 60/90 non-return rate Treatment
1st services
% Conception
Linco-spectin and minocin Without above
345 388
6 5.5 55.2
aLincomycin 300 ~g, spectinomycin 600 tag/ml of extender. b500/ag/ml of extender.
been s h o w n for minocin since it appears to be effective at 500 p g / m l while still well tolerated at 10 mg/ml. On the o t h e r hand, berenil and d o x y c y c l i n e exhibited t o x i c i t y for sperm at 1 nag and 125 gg/ml, respectively. In addition, neither of these was as effective as m i n o c i n for elimination of ureaplasma. ACKNOWLEDGMENTS
This research was supported by the Ontario Ministry of Agriculture and F o o d . The technical assistance of 1. R. Patel and R. Sivendra was greatly appreciated. We t h a n k L. R u h n k e and C. Howard for providing the cultures. REFERENCES
of ureaplasma added w o u l d range f r o m 4 × 10 a to 4 × 104 C F U / m l w h e n diluted in e x t e n d e r . Since O n o v i r o n (11) had d e t e r m i n e d that t h e m a j o r i t y of raw semen samples containing ureaplasma c o n t a i n e d f r o m 1.1 to 5 X 10 ° C F U / m l o f raw semen, it appears likely t h a t m i n o c i n should be effective for elimination of ureaplasma f r o m such samples, particularly since a dilution w o u l d o c c u r w h e n e x t e n d e r was added. On the o t h e r hand, minocin eliminated M. bovigenitalium and M. canadense but was n o t effective against A. laidlawii, M. boris, or M. arginini with the n u m b e r s and concentrations. Minocin could be c o m b i n e d with lincospectin to eliminate these (Tables 4 and 5). The holding t i m e at 3 5 C appears to be significant. While it is not desirable to hold semen for a prolonged period at this t e m p e r a t u r e , it is likely that t h e antibiotics are most effective at this temperature. The time, therefore, should be as long as practical. Likewise 3 h at 4 C has been desirable in t h a t m y c o p l a s m a still recoverable at 2 h under these c o n d i t i o n s apparently have been eliminated by 3 h (unpublished). An additional consideration n o t investigated would be the effect of adding lincospectin to e x t e n d e r to provide 300 pg t i n c o m y c i n and 600 pg spect i n o m y c i n / m l of e x t e n d e r . Provided no interference b e t w e e n lincospectin and minocin with this increased c o n c e n t r a t i o n , a m o r e rapid elimination of m y c o p l a s m a might occur. Nonetheless, the final times were satisfactory and practical in t e r m s of semen preparation prior t o cooling and freezing in liquid nitrogen. A f u r t h e r r e q u i r e m e n t o f lack of t o x i c i t y with respect to sperm viability and activity has Journal of Dairy Science Vol. 60, No. 6
1 Afshar, A. 1975. Diseases of bovine reproduction associated with mycoplasma infections. Vet. Bull. 45:211. 2 Davies, G. 1967. Isolation of mycoplasma from calf lungs. J. Comp. Path. 77:353. 3 Emg[, H., W. N. Plastridge, and M. E. TourteUotte. 1967. Mycoplasma: isolation from prepuce and semen of bulls. Acta. Vet. Scan& 8:123. 4 Ern~b, H. 1975. Bovine mycoplasmas: identification of 100 strains isolated from semen of hulls. Acta. Vet. Scand. 16: 321. 5 Fabricant, J. 1973. The pathogenicity of bovine mycoplasmas. Ann. N.Y. Acad. Sci. 225:369. 6 Gourlay, R. N. 1968. The isolation of T-strains of mycoplasma from penumonic calf lungs. Res. Vet. Sci. 9:376. 7 Gourlay, R. N. 1973. Significance of mycoplasma infections in cattle. J. Amer. Vet. Med. Ass. 163:905. 8 Hamdy, A. H., and C. C. Miller. 1971. Antibiotics for bovine myeoplasmas. J. Dairy Sci. 54:1541. 9 Hirth, R. S., W. N. Plastridge, and M. E. Tourtellotte. 1967. Survival of a mycoplasma in frozen bovine semen. Amer. J. Vet. Res. 28:97. 10 Jasper, D. E., J. M. AbAubaidi, and J. Fabricam. 1974. Isolation of mycoplasma from preputial washings of hulls. Cornell Vet. 64:296. 11 Onoviran, O., R. B. Truscott, N. A. Fish, C. A. V. Barker, and H. L. Ruhnke. 1975. The recovery of mycoplasmas from the genital tracts of bulls in artificial breeding units in Ontario. Can. J. Comp. Met. 39:474. 12 Shephard, M. C. 1969. Fundamental biology of the T strains. The mycoplasmatales and L-phase of bacteria. L. Hayflick, 1st edition. Appleton-Century-Crofts. 49, 13 Stalheim, O. H. V., S. J. Proctor, and J. E. Gallagher. 1976. Growth and effects of ureaplasmas (T mycoplasmas) in bovine oviductal organ cukures. Inf. lmmun. 13:915. 14 Truscott, R. B., O. Onoviran, H. L. Ruhnke, N. A. Fish, and C. A. V. Barker. 1975. In vitro antimicrobial sensitivity of mycoplasmas isolated from the bovine genital tract. Can. J. Comp. Med. 39:416.
ABSTRACT
ductive tracts, abnormal milk, and nasal discharge of cattle (8). Lincospectin also was effective against strains of mycoplasma isolated from bovine semen and preputial washings but less effective against ureaplasmas from these same sources (14). This study was to establish a practical treatment for elimination of mycoplasmas and ureaplasma from bovine semen.
Minocin at 500 /~g/ml of semen extender eliminated ureaplasma from naturally or artificially infected bovine semen. Minocin with lincospectin eliminated
Acboleplasma laidlawii, Mycoplasma bovigenitalium, boris, canadense, and arginini from artificially infected semen. Stabilization times of 15 min at 35 C and 3 h at 4 C are important considerations to maximize antibiotic activity.
MATERIALS AND METHODS Test Strains
INTRODUCTION The role of mycoplasmas and ureaplasmas (T-Mycoplasmas) in pathology o f bovine reproductive tract has not been established (1, 5, 7). The shedding of mycoplasma and ureaplasma in bovine semen and their recovery from it and from preputial washings has been reported (3, 4, 10, t l ) . Furthermore, at least one species, Mycoplasma boris (M. agalactiae subsp, boris), was capable of surviving 18-mo storage at - 1 9 6 C in bovine semen (9), and survival of other possibly pathogenic species seems reasonable. Since these organisms are .potential pathogens in the female reproductive tract, to eliminate them from semen is desirable. Lincospectin was effective against M. bovigenitalium, M. boris, and A. laidlawii isolated from the repro-
Fifteen strains of bovine mycoplasmas and ureaplasma were used. They included Acboleplasma laidlawii (NTCC10116), Mycoplasma bovigenitalium (PG11), h4. canadense (Ontario milk isolate 466), M. boris (Ontario joint isolate 427), and M. arginini (Ontario lung isolate 108) and were obtained from L. Ruhnke2; ureaplasma strains T95, D48, A417, FS01, T315, T288, T44, Bu2 and MmB167 were obtained from C. Howard. 3 In addition, one strain of ureaplasma (SB-I) isolated during this study was used in a further experiment.
Received January 19, 1977. z Animal Pathology Division, Health of Animals Branch, Agriculture Canada, Atlantic Area Laboratory, P.O. Box 1410, Sackville, New Brunswick, EOA 3CO. Veterinary Services Laboratory, Ontario Ministry of Agriculture and Food, Guelph, Ontario. 3Compton, England. 4 Minocycline hydrochloride. Cyanamid of Canada Ltd., Montreal, Quebec. s Berenil, Farbwerke Hoechst, A.G. Frankfurt, Germany. 6Doxycycline hydate, Pfizer Company Ltd., Pointe-Clair-Dorval, Quebec.
Determination of Colony Forming Units (CFU)
Media The medium and techniques for isolation and growth of mycoplasmas were as described by Davies (2), and the cultural medium for ureaplasma was described (6, 12).
For counts serial dilutions were in broth, and .02 ml was deposited on appropriate agar plates in quadruplicate. Following incubation, the colonies were counted, averaged, and the number was multiplied by the appropriate dilution factor. Antibiotics
954
Minocin 4, berenil s, or doxycycline 6 were
SEMEN ANTIBIOTIC TREATMENT dissolved in distilled w a t e r as 1% s o l u t i o n . F r e s h s o l u t i o n s were p r e p a r e d f o r e a c h experim e n t a n d were a d d e d t o g l y c e r a t e d e x t e n d e r to p r o v i d e t h e desired c o n c e n t r a t i o n . Lincospectin 7 s o l u t i o n was a d d e d d i r e c t l y to t h e semen. T h e e x t e n d e r c o n s i s t e d o f m i l k a n d glycerol a n d c o n t a i n e d 1 0 0 0 IU o f penicillin a n d 1 mg of streptomycin/ml. Antibiotic Safety
T h e a n t i b i o t i c s were t e s t e d for s a f e t y o n s p e r m cell livability as d e t e r m i n e d b y t h e p e r c e n t o f s p e r m viability a n d m o t i l i t y t h r o u g h o u t t h e processing p e r i o d a n d 14 days a f t e r freezing. Procedures for Test
Details were c h a n g e d as t h e s e studies progressed. In general, .1 t o .5 ml o f b r o t h c u l t u r e s w e r e a d d e d t o 1 m l of s e m e n t o w h i c h e x t e n d e r c o n t a i n i n g t h e test a n t i b i o t i c s was added. T h e s a m p l e was m i x e d well a n d allowed t o stabilize
7 IAncomycin-Spectinomycin, The Upjohn Company, Kalamazoo, MI.
955
at 35 C f o r 10 m i n (later e x t e n d e d t o 15 min). C o n t r o l s c o n s i s t e d o f s e m e n plus e x t e n d e r w i t h or w i t h o u t a d d e d b r o t h c u l t u r e s as a p p r o p r i a t e b u t w i t h o u t t h e a n t i b i o t i c u n d e r test. Following s t a b i l i z a t i o n , t h e samples were held at 4 C for 2 h (later increased to 3 h). A l o o p f u l t h e n was plated d i r e c t l y o n agar a n d a sample i n o c u l a t e d i n t o b r o t h t o p r o v i d e a 10% inocul u m . Samples c o n t a i n i n g a n t i b i o t i c s were diluted f u r t h e r t o 10 -2 a n d 10 -3 t o o v e r c o m e t h e possible stasis o f t h e agent. T h e b r o t h s were s u b c u l t u r e d t o fresh b r o t h at 6 days. B r o t h s were p l a t e d at 4 8 a n d 96 h a n d t h e plates e x a m i n e d for 7 days b e f o r e b e i n g discarded. S u b s e q u e n t l y , s o m e e x p e r i m e n t s were perf o r m e d b y c e n t r i f u g i n g t h e test samples f o r 8 m i n at 3 0 0 0 × g, d e c a n t i n g t h e s u p e r n a t a n t , a n d discarding t h e s e d i m e n t . T h e s u p e r n a t a n t was c e n t r i f u g e d for 2 0 m i n at 1 7 , 5 0 0 × g a n d t h e s u p e r n a t a n t discarded. T h e s e d i m e n t was r e s u s p e n d e d in b r o t h a n d t h e washing r e p e a t e d t w i c e w i t h b r o t h . T h e final s e d i m e n t was s u s p e n d e d in b r o t h a n d i n c u b a t e d .
Field Trial
S e m e n was c o l l e c t e d f r o m a Holstein bull,
TABLE 1. Effect of minocin and berenil on ureaplasma in bovine semen.
Exp.
Agent
Conc.a
Ureaplasma added
Ureaplasma recovered
1
Minocin Minocin Minocin Control
100 500 1000 0
... ... ... ...
+ (SB-I)
2
Minocin Minocin Minocin Control
100 500 1000 0
SBI b SBI SBI SBI
--+
3
Minocin Minocin Minocin Control Berenil Berenil Berenil
100 500 1000 0 100 500 1000
Pool c Pool Pool Pool Pool Pool Pool
+ -+ + ÷ +
a#g/ml of extender. b Estimated approximately 1 × IO s SB-I/ml extended semen. CEqual amounts of active cultures of the nine strains of ureaplasma obtained from Howard were pooled and .2 ml added/ml of semen. Journal of Dairy Science Vol. 60, No. 6
956
TRUSCOTr AND ABREO
TABLE 2. Efficacy of minocina on semen infected with bovine mycoplasmas and ureaplasma (Exp. 4). Recovery Untreated
Treated
Strain addedb
Mycoplasma
Ureaplasma
Mycoplasma
None A. laidlawii M. bovigenitalium M. boris M. arginini M. canadense Pool of above Pool + ureaplasma T288
ND c ND ND ND ND ND +
+ ND ND ND ND ND ND +
-+ + + + +
Ureaplasma
al000/~glml extender. bDilution of culture in semen * extender, 1/50. CNot done.
1.5 yr o f age. It was divided equally, and o n e - h a l f was p r o c e s s e d with e x t e n d e r c o n t a i n ing penicillin and s t r e p t o m y c i n . The o t h e r o n e - h a l f was p r o c e s s e d w i t h t h e e x t e n d e r t o which l i n c o s p e c t i n and m i n o c i n w e r e a d d e d at 300/~g l i n c o m y c i n , 6 0 0 / l g s p e c t i n o m y c i n , a n d 500 p g / m l , respectively. T w o t h o u s a n d doses o f each were processed. T h e p r o c e s s e d samples were c o d e d and i n s e m i n a t i o n results t a b u l a t e d t o p r o v i d e a 6 0 / 9 0 n o n r e t u r n rate f o r each. RESULTS Experiments 1 and 2 S e m e n f r o m a hull k n o w n t o shed ureaplas-
m a was used f o r the first e x p e r i m e n t . Minocin was a d d e d at 100, 500, a n d 1000 /~g/ml o f s e m e n e x t e n d e r . The ureaplasma isolated (SBI) f r o m t h e c o n t r o l o f e x p e r i m e n t 1 was used f o r t h e s e c o n d e x p e r i m e n t , and .2 ml o f an active b r o t h culture was a d d e d per ml o f s e m e n . Minocin e l i m i n a t e d u r e a p l a s m a (Table 1). Experiment 3 Minocin a n d berenil were c o m p a r e d at t h e s a m e c o n c e n t r a t i o n s . Equal a m o u n t s o f active b r o t h c u l t u r e s o f t h e eight d e s i g n a t e d ureaplasm a strains f r o m H o w a r d w e r e p o o l e d , and 2 ml w e r e a d d e d t o 10 ml o f s e m e n ; t h e n e x t e n d e r c o n t a i n i n g t h e t w o agents was a d d e d t o a 1/25
TABLE 3. Effect of a combination of minocin a and lincospectinb on s e m e n i n f e c t e d with bovine mycoplasmas and ureaplasma (Exp. 5). Recovery Strain added
CFU/ml extended semen
Mycoplasma
Ureaplasma
Control semen A. laldlav~ii M. bovigenitalium M. boris M. arginini M. canadense Ureaplasma T288
7.2 X 103 1.4 X 10 ~ 4.4 X 104 4.8 × 104 2 × 104 4 X 102
-+ + -
+ ---
a500 t~g/ml extender. b Lmcomycm . 300/~g, spectinomycin 600 t~g/ml semen. Journal of Dairy Science Vol. 60, No. 6
SEMEN ANTIBIOTIC TREATMENT dilution. Results in T a b l e 1 i n d i c a t e t h a t minocin at 500 and 1000 pg/ml eliminated the t e s t strains while m i n o c i n at 1 0 0 ~tg/ml a n d berenil at t h e test c o n c e n t r a t i o n s did not. Experiment 4 To d e t e r m i n e t h e e f f e c t i v e n e s s o f m i n o c i n f o r e l i m i n a t i o n o f b o v i n e m y c o p l a s m a , .5 ml o f a n active 18 h c u l t u r e was a d d e d p e r ml of r a w s e m e n m i x e d w i t h 2 4 ml of e x t e n d e r t r e a t e d w i t h 1 0 0 0 / a g / m l o f m i n o c i n . Since p r e l i m i n a r y studies ( u n r e p o r t e d ) h a d i n d i c a t e d t h a t Ureap l a s m a s t r a i n T 2 8 8 was m o r e r e s i s t a n t t o m i n o c i n t h a n t h e o t h e r test strains, an active c u l t u r e o f t h i s s t r a i n was a d d e d t o a p o o l w i t h t h e m y c o p l a s m a s . T h e results in T a b l e 2 indicate t h a t M. bovigenitalium, M. canadense, T 2 8 8 , a n d a u r e a p l a s m a initially in t h e s e m e n were e l i m i n a t e d while A. laidlawii, M. boris, a n d M. arginini were n o t a f f e c t e d b y 1 0 0 0 / a g o f minocin. Experiment 5 T o d e t e r m i n e w h e t h e r m i n o c i n c o u l d be used in c o n j u n c t i o n w i t h l i n c o s p e c t i n , t h e e x p e r i m e n t was r e p e a t e d w i t h l i n c o m y c i n 3 0 0 gg/ml, spectinomycin 600/ag/ml of raw semen,
957
a n d m i n o c i n at 5 0 0 g g / m l final c o n c e n t r a t i o n in e x t e n d e r . B r o t h c u l t u r e s o f t h e t e s t strains, .2 m l / m l of s e m e n were a d d e d a n d t h e C F U ' s o f the cultures determined. T h e results in T a b l e 3 i n d i c a t e t h a t w i t h t h e n u m b e r s used, t h e t e s t strains were e l i m i n a t e d e x c e p t f o r M. bovis a n d M. arginini. Since t h e s e s t r a i n s were initially in g r e a t e r n u m b e r s , it appeared there might be a relationship between numbers added and recovery. Experiment 6 In t h i s e x p e r i m e n t , t h e s t a b i l i z a t i o n t i m e at 35 C was i n c r e a s e d t o 15 m i n while t h e h o l d i n g t i m e at 4 C was e x t e n d e d t o 3 h; A. laidlawii a n d M. canadense were o m i t t e d . M i n o c i n a n d l i n c o s p e c t i n e l i m i n a t e d all t h e s t r a i n s t e s t e d a t various d i l u t i o n s ( T a b l e 4). Experiment 7 E x p e r i m e n t 7 was similar to e x p e r i m e n t 6 e x c e p t t h a t u r e a p l a s m a s were n o t a d d e d , b u t all m u c o p l a s m a s were utilized. Active 18-h cult u r e s were d i l u t e d 1 / 1 0 a n d 1 / 1 0 0 , C F U ' s were d e t e r m i n e d , a n d .1 m l o f b r o t h c u l t u r e was a d d e d p e r ml o f s e m e n w h i c h t h e n was d i l u t e d with extender. The controls contained only the
TABLE 4. Recovery of mycoplasma and ureaplasma from infected semen treated with lincospectin a and minocin b (Exp. 6). Recovery Untreated Strain added
Dilution c
Ureaplasma
Mycoplasma
Ureaplasma
1/130 1/1300 1/1300 1/13000 1/1300 1/13000 1/130 1/1300 Low High
-+ + + + + + ND d ND + +
-ND ND ND ND ND ND + + + +
ND -----ND ND --
ND ND ND ND ND ND ND ----
Control semen
M. bovigenitalium g. boris M. arginini Ureaplasma T288 Pool of all of above
Treated
Mycoplasma
aLincomycin 300 #g, spectinomycin 600 tag/ml semen. b 500 #g/ml extender. CBroth in semen + extender. dNot done. Journal of Dairy Science Vol. 60, No. 6
9 58
TRUSCOTT AND ABREO
TABLE 5. Recovery of mycoplasma from infected semen treated with lincospectin a and minocin b (Exp. 7).
Strain added
Recovery of mycoplasma
CFU/ml extended semen
Untreated
Treated
ND d + ND + ND + ND
ND
1.5 X 10 4 1.5 X 10 3 4 X 10 s 4 X 10 4 1.5 X l 0 s 1.5 × 10 4 1.2 X l 0 s 1,2
+
--
+
-
Control semen A. laidlawii M. boris M. arginini M. canadense Pool
of above
X 10 4
c
- -
-
-
-- -
- -
aLincomycin 300/~g, spectinomycin 600 #g/ml semen. b500 #g/ml extender. CEqual amounts of 1/100 dilution of broth cultures pooled, .1 ml added to semen. dNot done.
1 / 1 0 0 d i l u t i o n o f c u l t u r e . All t h e strains t e s t e d were e l i m i n a t e d f r o m t h e i n f e c t e d s e m e n treated with a combination of minocin and l i n c o s p e c t i n (Table 5).
Experiment 8
D o x y c y c l i n e was a d d e d t o e x t e n d e d s e m e n t o p r o v i d e e i t h e r 500 o r 2 5 0 / s g / m l . A n active c u l t u r e o f T 2 8 8 was a d d e d t o each as a 10 or 1% i n o c u l u m . F o l l o w i n g a 15-min h o l d i n g at 35 C a n d 3 h at 4 C, t h e samples were c e n t r i f u g e d , a n d t h e s e d i m e n t was w a s h e d as i n d i c a t e d . T h e c o n t r o l c o n s i s t e d o f e x t e n d e d s e m e n w i t h 1% u r e a p l a s m a i n o c u l u m . T h e final s e d i m e n t was s u s p e n d e d in b r o t h w h i c h was o b s e r v e d f o r 14 days for c o l o r change. No c h a n g e o c c u r r e d w i t h 5 0 0 # g / m l d o x y c y c l i n e while g r o w t h o c c u r r e d in all o t h e r t u b e s i n d i c a t i n g t h a t u r e a p l a s m a s were in t h e c o n t r o l a n d h a d survived t h e test o f 250/ag/ml.
Experiment 9
S e m e n f r o m six bulls s h o w n to b e s h e d d e r s was e x t e n d e d with a n d w i t h o u t l i n c o s p e c t i n a n d m i n o c i n . T h e s a m p l e s were c u l t u r e d , a n d Table 6 s h o w s m y c o p l a s m a s were r e c o v e r e d from one untreated and ureaplasma from one o t h e r u n t r e a t e d sample. No recoveries were f r o m t h e t r e a t e d samples. Journal of Dairy Science Vol. 60, No. 6
Effect on Sperm
T h e results o f t e s t s o f t h e e f f e c t o f m i n o c i n a n d berenil o n s p e r m a c t i v i t y are s u m m a r i z e d in T a b l e 7. M i n o c i n at 1 0 m g m / m l o f e x t e n d e r was t o l e r a t e d b y s p e r m cells, b u t m i n o c i n a t 50 m g m / m l or b e r e n i l at 1 m g m / m l were t o x i c . While n o t t a b u l a t e d , t o x i c i t y was e v i d e n t f r o m d o x y c y c l i n e at 125 Vg/ml as s o o n as t h e extender with doxycycline had been added. The influence of the combination of minocin a n d l i n c o s p e c t i n o n s p e r m cell m o t i l i t y f r o m t r e a t e d s e m e n f r o m seven sires is t a b u l a t e d (Table 8).
TABLE 6. Effect of lincospectina and minocin b treatment on the recovery of mycoplasma and ureaplasma from semen. Untreated Myeoplasma
Sire 1 2
Ureaplasma
Treated Mycoplasma
Ureaplasma
+
3 4 5 6
--
+
aLincomycin 300 ttg, spectinomycin 600 #g/ml semen.
b500 tag/ml extender.
SEMEN ANTIBIOTIC T R E A T M E N T
959
T A B L E 7. Effect of minocin and berenil on sperm motility and viability at various stages of processing. Days after freezing
Hours after cooling Drug
Conc. a
3
5
7
20
1
14
Minocin
.1 .5 1.0 Control
50 b 50 50 50
50 50 50 50
50 50 50 50
ND ND ND ND
45 45 45 45
ND ND ND ND
Minocin
.1 .5 1.0 Control
50 50 50 50
50 50 50 50
50 50 50 50
50 50 50 50
45 45 45 45
45 45 45 45
Minocin
1.0 10.0 50.0 Control
50 40 0c 40
50 40 0 40
50 40 0 40
40 40 0 40
30 30 ND 30
ND ND ND ND
Berenil
.1 .5 1.0 Control
45 45 45 45
45 45 45 45
45 45 30 45
45 45 30 45
ND ND ND ND
ND ND ND ND
amg/ml extender. bpercent progressive motility. CSperm dead.
Field Trial
DISCUSSION
Results from insemination of semen without compared to semen treated with lincospectin a n d m i n o c i n a r e in T a b l e 9. L i n c o s p e c t i n , m i n o cin treatment did not have a deleterious effect o n c o n c e p t i o n r a t e b u t a p p e a r e d t o i m p r o v e it.
Minocin eliminated ureaplasma strains from both naturally and artificially infected semen. It is o u r e s t i m a t e t h a t e x c e p t f o r t h o s e e x p e r i ments with mycoplasma when the cultures were diluted prior to addition to semen, the numbers
T A B L E 8. S e m e n motility a in extender containing m i n o c i n b and lincospectin c. Frozen and t h a w e d 14 Days 4 C (h) Sire 1 2 3 4 5 6 7
3
5
7
20
1 Day
Oh
3h -32C
50 50 50 45 55 40 50
50 50 55 45 55 40 50
50 45 50 50 50 40 50
50 45 50 45 55 40 45
50 50 50 40 50 40 50
50 50 50 40 50 45 45
50 45 50 40 50 45 40
aExpressed as percent progressive motility. b s o 0 #g/ml extender. C
Lm c o m y c m 300 #g, s p e c t i n o m y c i n 600 #g/ml semen.
Journal of Dairy Science Vol. 60, No. 6
960
TRUSCOTT AND ABREO
TABLE 9. Conception rates from field use of semen with and without lincospectin a and minocin b treatment. 60/90 non-return rate Treatment
1st services
% Conception
Linco-spectin and minocin Without above
345 388
6 5.5 55.2
aLincomycin 300 ~g, spectinomycin 600 tag/ml of extender. b500/ag/ml of extender.
been s h o w n for minocin since it appears to be effective at 500 p g / m l while still well tolerated at 10 mg/ml. On the o t h e r hand, berenil and d o x y c y c l i n e exhibited t o x i c i t y for sperm at 1 nag and 125 gg/ml, respectively. In addition, neither of these was as effective as m i n o c i n for elimination of ureaplasma. ACKNOWLEDGMENTS
This research was supported by the Ontario Ministry of Agriculture and F o o d . The technical assistance of 1. R. Patel and R. Sivendra was greatly appreciated. We t h a n k L. R u h n k e and C. Howard for providing the cultures. REFERENCES
of ureaplasma added w o u l d range f r o m 4 × 10 a to 4 × 104 C F U / m l w h e n diluted in e x t e n d e r . Since O n o v i r o n (11) had d e t e r m i n e d that t h e m a j o r i t y of raw semen samples containing ureaplasma c o n t a i n e d f r o m 1.1 to 5 X 10 ° C F U / m l o f raw semen, it appears likely t h a t m i n o c i n should be effective for elimination of ureaplasma f r o m such samples, particularly since a dilution w o u l d o c c u r w h e n e x t e n d e r was added. On the o t h e r hand, minocin eliminated M. bovigenitalium and M. canadense but was n o t effective against A. laidlawii, M. boris, or M. arginini with the n u m b e r s and concentrations. Minocin could be c o m b i n e d with lincospectin to eliminate these (Tables 4 and 5). The holding t i m e at 3 5 C appears to be significant. While it is not desirable to hold semen for a prolonged period at this t e m p e r a t u r e , it is likely that t h e antibiotics are most effective at this temperature. The time, therefore, should be as long as practical. Likewise 3 h at 4 C has been desirable in t h a t m y c o p l a s m a still recoverable at 2 h under these c o n d i t i o n s apparently have been eliminated by 3 h (unpublished). An additional consideration n o t investigated would be the effect of adding lincospectin to e x t e n d e r to provide 300 pg t i n c o m y c i n and 600 pg spect i n o m y c i n / m l of e x t e n d e r . Provided no interference b e t w e e n lincospectin and minocin with this increased c o n c e n t r a t i o n , a m o r e rapid elimination of m y c o p l a s m a might occur. Nonetheless, the final times were satisfactory and practical in t e r m s of semen preparation prior t o cooling and freezing in liquid nitrogen. A f u r t h e r r e q u i r e m e n t o f lack of t o x i c i t y with respect to sperm viability and activity has Journal of Dairy Science Vol. 60, No. 6
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