A new antibiotic combination for frozen bovine semen

THERIOGENOLOGY

A NEW ANTIBIOTIC COMBINATION FOR FROZ~2~ BOVINE ~ , i. Control of Mycopla~m~s, Ureaplasmas, Campylobacter fetus subsp. venerealis and Haemophilus s c m m ~

S. J. Shin1

D. H. Lein I and V. H. Patten I H. L. l~lhnke2

iDiagnostic laboratory N.Y.S. College of Veterinary Medicine Cornell university Ithaca, NY 14853 2Ontario Ministry Agriculture and Food Veterinary laboratory Servioes Branch Guelph, Ontario, Canada Received for Publication : Februu-~j 16, 7887 Accepted : Sep%ember 25, 2982 ABSTRACT Systematic evaluations of new combinations of antibiotics for the control of bovine mycoplasmas, ureaplam~ls, Ca~oylobacter fetus subsp. venerealis and Haemophilus somnus in a bovine frozen semen process were made. These organisms were stardardized to 105 to 106 colony forming unit (CPU) and inoculated into each ml of raw semen. Antibiotics in a final volume of 0.02 ml were added to each ml of the raw semen and were contained at the same concentration in the nonglycerol portion of the extenders (whole milk, 20% egg yolk citrate, 20% egg yolk tris, Plus-X, and 28% egg yolk tris). •he combination of gentamicin (500 ug/ml) tylosin (i00 ug/ml) and Linco-Spectin (300/600 ug/ml) was more effective for the control of mycoplasmas and ureaplasmas and equally effective for the control of _C. fetus subsp, venerealis and Haemophilus s~,~lus than the standaz~ combination of penicillin, dihydrostreptcmycin and polymyxin B sulfate. Key words:

mycoplasmas, ureaplam~s, Campylobacter venerealis, bull semen, antibiotics

fetus

subsp.

Acknowledgements ~le authors deeply appreciate the support of this research project by members of National Association of Animal Breeders and Dr. Gordon Doak. We sincerely extend our appreciation to Mr. Bruce Bean and Mik~ Eaproth at Eastern Artificial Insemination Coop.; Drs. John Sullivan and Steve Lorton at American Breeders Service.) Mr. Howard Kellgren and Mr. Clifton Marshall at Select sires for their cgoperation in this project.

MARCH 1988 VOL. 29 NO. 3

577

THERIOGENOLOGY

INTR0~JCTION Bovine mycoplasmas, ureaplasmas, H. somnus and C. fetus subsp. venerealis all have been recently associated with infertility and abortion in cattle (1-7). Mycoplasmas and ureaplasmas appear to be frequent and insidious /nhabitants of mucous membranes of the lower urogenital ~nd upper respiratory tracts of cattle and at times are opportunistic pathogens (8ii). Recent reports by Canadian workers have associated these organisms, especially bovine ureaplasmas, with granular vulvitis and reduced conception rates (12-15). Experimental studies have shown that all of these organisms can cause transitory acute endometritis, vulvovaginitis and prolonged infective carrier states with organi~ns in the vulvovaginal areas (12-14). These agents have been isolated from raw and processed semen, but the incidence of seminal contamination and importance of semen in the spread of these organism~ is unknown. Other investigations (16-19) have provided useful insights into antibiotics which are useful additives to semen. However, when these scattered reports were coupled with the observation that activity of some antibiotics varied with different seminal extenders (20), it became clear that a cc~0rehensive study was required to clarify which combination of antibiotics and extenders would be the most effective in eliminating the aforementioned organisms from semen. Thus, the objective of our study was to determ/ne the most effective concentration of individual antibiotics or ccmbinations of antibiotics to control mycoplasmas, ureaplasmas, C. fetus subsp, yenerealis and H. somnus in bovine semen when extended and frozen in several co~nonly used seminal extenders. To meet this objective, a systematic evaluation was done to i) assess selected individual antibiotics in various seminal extenders for their efficacy against ureaplam~as and mycoplasmas, 2) evaluate combinations of these drugs for synergistic effects against these organisms, 3) evaluate other drugs individually for antimicrobial activity against H. somnus and C. fetus subsp, venerealis in various seminal extenders, 4) determine the optimal comb/nation of drugs for control of all organis~s studied and 5) ccmi0are the optimal drug combination with conventional seminal treatment (18) in a final confirmatory study. Parallel studies of seminal quality were performed by Lorton et al. (21). A preliminary report of this study has been previously presented (22).

MA~E~/EALS AND METHODS Orgasms Organi~mls for these studies were selected from diverse isolates representing conditions ranging from the normal carrier state to clinical disease (Table I). The inoculum frc~ each of the isolates was prepared b~ harvesting organisms in the log-growth phase and standardizing them to i0~ to 106 CYJ/ml of raw semen. Inocula were pooled and introduced into raw semen.

578

MARCH 1988 VOL. 29 NO. 3

THERIOGENOLOGY

Table I.

Source of organisms used in this study

Species

Isolate

Source

3713-2 5501 7147-1 7147-2 7325

u ~ a ~ i o n i c fluid joint man~arygland pneummnic lung

Strain-i 4695 6085 6988 7075

uterus prepuce embryo flushing fluid raw semen embryo flushing fluid

A. laidlawii M. canadense _M. alkalescens M. bovirhinis Unidentified mycoplasma

serosa oviduct stomach of aborted fetus pneumonic lung pneumonic lung law

se~_21

4781-2 4708-12 4708-11 4870

raw raw raw raw

semen; semen; semen; semen;

M. bovis~a

_M. boviqenitalium-a

Mycoplasma smm. a

Ureaplasma a

4722

normal bull normal bull normal bull from bull with vesiculitis lung of aborted fetus

8025 Jerihico Bull 73 23542 7256

brain prepuce seminal vesicle aborted fetus pneumonic lung

H. somnusb

C. fetus subsp, venerealisb ATCC 9438 Strain 2 Strain 3 Strain 4 Strain 5

preputial washing lung of aborted fetus preputial washing preputial washing

aFrc~ Veterinary Laboratory Services, Ontario, Canada. bFrc~ New York state College of Veterinary Medicine, Ithaca, NY.

MARCH 1988 VOL. 29 NO. 3

579

THERIOGENOLOGY

Mycoplasma and ureaplasma were grown in Hayflick's broth medium (23) and Shepard's broth medium (24), respectively. Mycoplasma and ureaplasma strains in semen were detected by culturing on Hayflick's agar (23) supplemented with 15% horse or swine serum and in Shepard's agar (24,25), respectively. Viable organisms were counted by CFUs from serial dilutions prepared for inoculation of the semen. The five strains of H. somnus (Table I) were 9xown on chocolate agar (BBL, Cockeysville, MD) for 24 h, then standardized to 105 to 106 CFU/ml for inoculation into raw semen. The~e organisms were detected in extended semen by streaking on chooolate agar and 5% sheep blood ag-dr and incubating it 35.5°C in 5 to 8% 002 envirorm~nt with 100% humidity. C. fetus subsp, venerealis was grown on cystine heart cow blood agar plates. PM plates and Albimi broth media (26) in an atmosphere of 85% N~, 10% 002 and 5% 02 were used to detect C. fetus subsp, venerealis in extended semen. Antibiotics and Seminal Extenders Minocin (Sigma, Minocycline hydrochloride), Linco-Spectin (Upjohn, Linccmycin/Spectincmycin), tylosin (Sigma, tylosin tartrate), clindamycin (Upjohn) and gentamicin (Sigma, gentamicin sulfate) were screened at several concentrations, both individually and in various ocmbinations for their ability to control mycoplasmas and ~ p l a ~ l s in processed semen. Gentamicin and amikacin (Bristol labs, amikacin sulfate [Amiglyde-V]) were evaluated as possible replacements for penicillin, streptomycin, and polymyxin B, which have been used to control C. fetus subsp, venerealis in processed bovine semen ( 1 8 ) . Penicillin (500 units/ml), dihydrostreptc~ycin (2000 ug/ml) and polymyxin B sulfate (i000 units/ml) were used as the control treatments for these studies ( 2 1 ) . Antibiotic levels (active potency) per milliliter refer to both the raw semen and the nonglycerol portion of the extenders. Stock solutions of antibiotics, except Linco-Spectin, were prepared in sterile distilled water. Concentrations were such that antibiotics at the desired levels were added in a final volume of 0.02 ml per ml of raw semen and nonglycerolated portion of seminal extenders. Linco-Spectin was used as is from the manufacturer. Stocks were stored separately at 5°C for up to eight days or were stored in IN 2 vapor. Initial screenings of individual, two-way and three-way combinations of antibiotics were performed using heated whole milk, 20% egg yolk citrate 20% egg yolk tris extenders as described by Lorton et al. (21). An additional two extenders, 28% egg yolk tris (28% [v/v] egg yolk, 158.9 mM tris (hydroxymethyl amincmethane), 52.3 ~M citric acid monohydrate, 55 raM glucose and 7% [v/v] glycerol) and Plus-X (Edwards Agri Supply, Baraboo, WI, 7% [v/v] glycerol), were also included during evaluations of the final selected antibiotic combination.

580

MARCH 1988 VOL. 29 NO. 3

THERIOGENOLOGY

s ~ t ~ 1 pn~e~ing Semen was collected by artificial vagina and held at 35"C in a water jacket for approximately I0 min while being transported to the laboratory. Semen was processed as outlined (Figure i) and detailed as follows: i) Ten microliters of raw semen was streaked on three plates each of cystine heart agar (C. fetus), Hayflick's agar (Mycopla~a sl0p.), Shephard's agar CJreaplasma spp.) and chocolate agar (H. scmnus) to determine initial microbial o ~ status. 2) Organisms were added to semen to a final concentration of 105 to 106/ml; mixing was by gentle inversion and shaking. 3) Ten microliters of the inoculated semen was streaked as in Step 1 above to determine the number of viable or~li~ns. 4) ~he inoculated semen was then split into l-ml aliquots to a ~ t e the number of antibiotics and concentrations to be tested, including an untreated control. 5) Antibiotic solutions of various concentrations were added in a 0.02 ml vol~ne to each milliliter of raw semen aliquot. 6) Each specimen was extended with 2 ml of nonglycerolated extender containing a concentration of antibiotic equal to that in the specimen. 7) Following mixing by rotation (four to five times), the specimens were placed in a 35°C water jacket (i00 ml volume) and transferred to a cold room at 5"C. 8) Approximately 2 h later, each sample was streaked as in Step 1 above. 9) An a~d~tior~l 2 ml of nonglycerolated extender containing the same concentration of antibiotic as in step 6 was added to each tube followed by 5 ml of extender containing 14% glycerol and no antibiotics, i0) All specimens were held at 5°C for 4 h; each of three l-ml replicates for each specimen was placed into 1 ml cryovials (Costar, Cambridge, MA) or 0.5 ml French straws (IMV, L'Aigle, France). ii) Samples were again cultured as in Step 1 above ~ ! a t e l y before freezing. 12) Sa~i01es were then suspended over liquid nitrogen (iN2) at 130*C until frozen (20 to 30 min) and stored in IN2. The effect of antibiotics and IN2 freezing on inoculated extended semen specimens was determined for samples which either had been treated with antibiotics or remained as untreated controls. Accordingly, thawed samples were processed as follows: i) Each of three replicates of a sample was cultured as in Step 3 above. 2) Samples were sedimented and washed three times with alternate centrifugation (35,000~g for mycopla~m~s and ureapla~m3s and 12,000Mg for _H. scmnus and C. fetus for 20 min.) and resuspended in sterile PBS (pH 7.2). 3) Following resuspension to their original volume in sterile PBS, they were again cultured. 4) Equal volumes of a n t i b i o t i c - ~ t e d and control samples were mixed and incubated for 30 min at 35°C to verify the removal of antibiotic residue by washing. 5) All sanTples were then cultured as above. Data Peduction The efficacy of the antibiotics was assessed by ccmparison of colony counts of organisms on antibiotic-treated vs untreated samples.

MARCH 1988 VOL. 29 NO. 3

581

THERIOGENOLOGY

Time (hour) i ml aliquoted semen collected

0

Cultured 105 to 106 or~ani~/ml ............. added Cull ~red

0.08

Split for treatm~ kt with antibiotics Added 0.02 ml of same co?centration of antibiotics !

2 ml nonglycerolated extender | with antibiotics added ..........--J

0.17

!

Cooled f r ~ 35@C to 5°C

ovi2 h C~ltured i /

2.0

2 ml nonglycerolated extender containing antibiotics:........... | .......... Held at 5"C 5 ml glycerolated extender with | no antibiotics added Cul"tnlrea j

6.0 6.1

forl>3 d

Samples Thawed Samples (containing ~ antibiotics) ~

~

Control sanloles (no antibiotic treatment)

Centrifuged and washed thr~e times

Centrifugled and washed ~ times

P ~ J

Resuspend(~ to original

ed to original

Cultured ~

~

Equal volumes mixed a n d ~ incubated 340 min at 35°C Cultured

Figure i. Protocol for processing of semen.

582

MARCH 1988 VOL. 29 NO. 3

THERIOGENOLOGY

RESULTS A N D D ~ C ~ S S I ~ There are very few antibiotics or chemical agents to control mycoplasma and ureaplasma due to their morphological, metabolic and nutritional differences from bacterial agents (27). For exaI~01e, the penicillin and cephalosporin groups, which interfere with cell wall formation, cannot be used to eliminate mycoplasmas and ureaplammas because both lack cell walls. Rifani0in , trimethroprim, sulfa drugs, nitrofurantoin, polymyxin and v~nccanycin are also ineffective against myooplasmas and ureaplasmas (28,29), while chloramphenicol, streptomycin and kanamycin are only slightly effective in vitro (30). Rosaramycin and tiamulin have proven to be effective in controlling mycopl~ and ureaplam~as but are toxic to spermatozoa (31). The tetracycline group (doxycycline, oxy- and chloro-tetracycline) are effective but show toxic effects to spermatozoa in relatively low concentrations (29,32). However, minocin is le~s toxic to spermatozoa (especially in milk extenders) and is very effective against both mycoplasmas and ureaplasmas (29,32). Although the miniman inhibitory concentration ~IIC) of minocin on these organism~ in vitro ranges from 1 to 6 ug/ml (28), the effective concentrations of minocin for bovine s~nen extended in milk has been determined to be 500 ug/ml by other investigators (19). Others (16) have found that Linco-Spectin is partially effective against mycoplasmas and ureaplam~as in semen, with ralatively low toxicity to spermatozoa. This drug combination has a broad spectrum of activity against gram positive bacterial contaminants in semen and thus should be more effective than penicillin which is currently used in bull seminal extenders. Tylosin has also been used effectively to eliminate mycoplasma infections. Low concentrations of this drug are relatively nontoxic to spermatozoa (33). This drug therefore warranted further investigation. In the our study, selected concentrations of minocin, tylosin and Linco-Spectin were all effective against m y c o p l ~ and ureaplam~as (Table 2). Although minocin and tylosin provided antimicrobial activity at high concentrations, they were toxic for spe/m~tozoa (21). Minocin at 85 ug/ml was highly effective for control of M. boviqenitalium and ureaplam~as, but slightly less effective against _M. bovis and equivocal in its effect on other Mycoplasma s p p . Concentrations of tylosin above i00 ug/ml were highly effective against mycoplasmas and ureaplam~as; but at concentrations above 125 ug/ml; it showed evidence of spermatozoa toxicity in selected extenders (21). Linoo-Spectin at concentrations of 300/600 ug/ml or higher was also highly effective against these organisn~ and was not toxic to sperlpatozoa (21). Clindamycin has MIC levels for UreaDlasma spp. of 12.5 ug/ml and 0.4 ug/ml for Mycoplasma spp. (28,34) ; however, its toxicity for spermatozoa has previously not been determined. This drug has a broad spectrum of activity against gram positive bacteria and is quite stable. However, Clindamycin was highly toxic for spermatozoa at all levels tested (21). It was thus excluded frcm further consideration in these studies.

MARCH 1988 VOL. 29 NO. 3

583

THERIOGENOLOGY

Gentamicin is effective against many gram negative and gram positive bacteria as well as the mycoplasmas and ureaplasmas with MIC values of 1.6 to 12.5 ug/ml and 6.2 ug/ml, respectively (28,34). It is also effective against Campylobacter spp., Pseudcmonas spp., and Haemophilus sc~nus. Although gentamicin was highly effective against C. fetus subsp, venerealis and H. sc~mus at all concentrations tested, it was limited in its effect on mycopla~,as and ureapla~m~s (Table 2); even at the highest concentrations tested, it was not toxic to spermatozoa (21). similarly, amikacin in exterders was highly effective against H. sc~nus but less effective against C. fetus subsp, venerealis (Table 2). Following addition of antibiotics to raw semen (Figure I), incubation of the treated semen at 35°C for 15 rain prior to the cooling process was cc~Dared with 3 to 5 rain incubation prior to cooling. Antibiotics were equally effective irrespective of the incubation time, which is contrary to the suggestion made by other investigators (19). Spermatozoal motility, how-ever, was significantly reduced after the 15 min incubation (21). Combinations of antibiotics were then tested in all extenders for their additive effects on microorganisms and spermatozoa (Table 3). In contrast with all other ccmbinations of drugs, minocin combined with tylcsin was virtually ineffective against ureapla~nas. Mycoplasma Sl0p. were difficult to control with at least four cc~binations of drugs. However, when gentamicin was substituted for minocin and added to the tylcsin, Linco-Spectin combination (G/T/IS) increased control of these organisms (Table 3). The concentration of gentamicin 500 ug/ml showed a d d e d effect on mycoplasma spp and ureaplasma over 250 ug/ml. The G/T/IS combination (at conoentrations of 500, i00, 300/600 ug/ml, respectively) was also effective against all other or~/anisms tested; therefore, it was selected for further evaluation. Doubling the concentration of LincoSpectin did not increase effectiveness. In three replicates of the final experiment, semen samples were processed (Figure i) using the penicillin, dihydrostreptcmycin and polymyxin B sulfate (P/S/P) (18,21), and the G/T/IS (500, i00, 300/600 ug/ml, respectively) antibiotic combinations. Because three replicates of each sample were frozen during the processing steps, a total of nine observations were made for each antibiotic combination in each of five seminal extenders. Both antibiotic combinations were 100% efficacious against C. fetus subsp. venerealis and H. somnus (Figure 2). For the mycoplasmas and ureaplasmas, P/S/P combination was only partially effective with all extenders. On the other hand, G/T/IS was 100% effective against _M. boviqenitalium and the ureaplasmas. For M. bovis and MTcoplasma spp., t_hawed samples, which still contained the antibiotics, showed virtually no 9-~owth on culture. However, when the samples were washed to remove antibiotics and subsequently cultured, some growth was observed indicating that the G/T/IS combination was bactericstatic for a portion of the organisms (Figure 2). similar bacteriostatic effects were seen with P/S/P for these species. With _M. bovis, _M. boviqenitalit~ and ureaplasmas, bactericidal activity was significantly greater for G/T/IS combination than for P/S/P. However, these five miscellaneous Mycoplasma are not oonsidered as c c m ~ n pathogenic strains of mycopla~a in bovine reproductive problems. Also, a difference in resistance to antibiotic treatments among individual species of mycoplasma has been reported (19).

584

MARCH 1988 VOL. 29 NO. 3

THERIOGENOLOGY

2.

Effect of individual antibiotics on microorganisms in processed semen using whole milk, 20% egg y o l k citrate and 20% egg yolk tris extenders

Lotic

uq/ml

concentrations a

Ln

in

M.bq.

M. spp.

340 170

+++ ~

~ +++

4+ ±

85

Jr+

4-H-

±

~-~ +-F+ +4-F +4+ +

+++ ~4-F 4H+

~ +4-+ ++

500 250 125 i00 75

~spectin

Microoruanls.s ~ M.bv.

600/1200 300/600 150/300

U

C.f.H.s.

~1:

ND

ND

Ill Ill

ND ND

ND ND

tmt Ill III

,111 ~ Ill

~ ND ND

ND ND

+-F +

++Jr ~

ND ND

ND ND

+~ 4-++ ++

llt 4+ ±

~ +++ +++

~ ND ND

ND ND ND ND ND

amycin

250 150 50

+ + +

-I~ -H+

III III +

4-+ +Jr ~

ND ND ND

micin

500 250 150 50

ND + + +

ND ± ± ±

ND ± ± +

ND ++ ++ ++

+++ +++ +++ ++

500 250 150 50

ND ND ND ND

ND ND ND ND

ND ND ND ND

ND ND ND ND

4+ Jr+ 4-~ +

.cin

~_ntrations p e r 1 ml of raw semen and per ~V° X~.

-------

;pp.

=

= = = = =

i ml of nonglycerolated

til

4++ III

+~+

extender.

Mycoplasma bovis. Mycoplasma boviqenitalium. Mycoplasma species. Ureapl~. Campylobacter fetus subsp, venerealis. Haemophilus sonmus. highly effective. moderately effective. partially effective. equivocal (effective in some cases and not in others). not determined.

MARCH 1988 VOL. 29 NO. 3

585

+~+ III +++ i11

THERIOGENOLOGY

Effects of combinations of antibiotics on microorganisms in p r o ~ semen using whole milk, 20% egg yolk citrate and 20% egg yolk t r : extenders

Fable 3.

Ccncentrations a uq/ml

kntibiotic Linco-Spectin and Minocin

600,/1200

iinco-Spectin and Tylosin

600/1200

Microorc~nismsb M. spp. U

M.bv.

M.bq.

C.f.

~

++

_+

+++

ND

++

++

+

++

ND

+++

++

+

+

ND

-I-I-I-

lit

-H-

-HI-

-HI-

"HI-

III

-H-

~

I,II

-H-k

+

4-}-

85

125

[inocin and .~{losin

85

~inco-Spectin plus

600/1200

125

T~losin

and Gentamicin

i00 500

/no~s~in

300/600

and Tylosin and ~ntamicin

i00 500

600/1200 plus Minocin and

85

ylcsin

125

Concentrations per 1 ml of raw se~en and per 1 ml of nonglycerolated extender. M, bv.

~-~ MVcoplasma bovis.

M. bg. M. spp. U C.f. H.s.

= = = = =

Mycoplasma boviqenitalium. Mycoplasma species. Ureaplasmas. Can~ylobacter fetus subsp, venerealis. Haemophilus somnus.

= = = = =

highly effective. raoderately effective. partially effective. equivocal (effective in some cases and not in others). not done.

÷

D

586

MARCH 1988 VOL. 29 NO. 3

ND

THERIOGENOLOGY

M. b o v i g e n i t a l i u m

M. bovis

2

3

4

1

5

2

3

4

Mycoplasma

1

5

Seminal Extender

Seminal Extender Ureaplasma

C. f e t u s

90

90-

70

70 -

~. 5 0

spp.

2 3 4 Seminal Extender

5

& H. s o m n u s

_u 5 0 -

30

30 -

101

2

3

4

5

Seminal Extender

1

2

3

4

5

Seminal Extender

.~. Conloarative

effects of Gentamicin/Tylosin/Linco-Spectin (G/T/IS) and Penicillin/dihydrostreptomycin/Polymyxin B sulfate (P/S/P) antibiotic treatment on microorganisms in processed semen. Solid bars (P/S/P) and striped bars (G/T/IS) represent bactericidal effect; open extensions of bars indicate bacteriostatic effect. Extenders: 1 = whole milk; 2 = 20% egg yolk citrate; 3 = 20% egg yolk tris; 4 = Plus X; 5 = 28% egg yolk tris. Each bar represents the percent efficacy for the mean of nine observations.

% efficacy

=

control - treated X i00, control

where "treated" and "control" refer to the number of CFU's for samples treated with antibiotics or left untreated, respectively.

MARCH 1988 VOL. 29 NO. 3

587

THERIOGENOLOGY

Generally, within a species or group of organisms, extenders tended to affect the activity of P/S/P to a greater extent than they did G/T/IS (Figure 2). Extenders generally had a limited effect on the efficacy of G/T/IS. The largest extender difference found was an 18% reduction of bactericidal effect on Mycoplau_om~ bovis in 28% egg yolk tris. Although the reduction in number of challenging organisms has been very significant, 100% bactericidal effect has not been obtained and may be imlx2ss_ible to achieve. The number of challenging organisms of each species (105 to 106 orgar~sm/ml of raw semen) which was used in the~e experiments are the maximum expected number of organisms in clinical or natural carrier states of bulls. When considering the high number of challenging organisms used and that very few of them were recoverable, we conclude that these treatments provided very effective control of microbial pathogens in semen. These studies showed that effective control of MTcoplasma bovis, Mycoplasma boviqenitalium, ureaplasma, Haemophilus scmnus, Campylobacter fetus subsp, veneraelis in bovine processed semen was achieved by the use of the combinations of 500 ug of gentamicin, i00 ug of tylosin, and 300/600 ug of Linco-Spectin, in each milliliter of raw semen and in each ml of nonglycerolated whole milk, 20% egg yolk citrate, 20% egg yolk tris, 28% egg yolk tris and Plus-X seminal extenders. Parallel studies (21,35) have concluded that this antibiotic combination is safe to spermatozoal viability and fertility.

588

MARCH 1988 VOL. 29 NO. 3

THERIOGENOLOGY

R~CES i.

Doig, P.A., Rnhnke, H.L., Waelchi-Suter, R., Palmer, N.C. and Miller, R.D. The Role of ureaplasma infection in bovine reproductive disease. The Ccmpe/idi~ on Continuing Education 3:324-330 (1981).

2.

Friberg, J. MycoplaEm~s and ~ p l a ~ a s in infertility and abortion. Fertility and Sterility 33__/3:351-359 (1980).

3.

Klavano, G.G. Observation of Haemophilus scmnus infection on an agent producing reproductive diseases: infertility and abortion. Society for Theriogenology, Annual Proceedings 139-149 (1980).

4.

Lein, D.H. Bovine reproductive disorders associated with ureaplasma, mycoplasm~, Haemophilus so~mus and chlamydia. Proceedings at the Annual Meeting, Society for Theriogenology pp. 118-131 (1982).

5.

Miller, R.B. An investigation into the prevalence, pathogenesis and pathology of Hae~ophilus somnus infections of the reproductive tract in cows. Ph.D. Thesis. Cornell University, Ithaca, NY, (1980).

6.

IAuhnke, H.L., Palmer, N.C., Doig, P.A. and Miller, R.B. Bovine abortion and neonate death associated with Ureaplasma diversum. Theriogenology 2_!1:295-301 (1984).

7.

Vandeplassche, M., Florent, A., Bouters, R., Huysman, A., Brone, A. and DeKeyser, P. The pathogenesis, epidemiology, and treatment of vibrio fetus infection in cattle. C.R. Rech. 2__9:46 (1963).

8.

Doig, P.A. (1981).

9.

Hodges, R.T. and Holland, J.T.S. The recovery of ureaplasmas from the semen and prepuce of bulls. N.Z. Vet. J. 2--8:89-90 (1980).

Bovine genital mycoplasmosis.

Can. Vet. J. 2--2:339-343

I0.

Lein, D.H. Male bovine urogenital m y c o p l ~ i s University of Connecticut, Storrs, CT, (1974).

II.

Onoviran, O., Truscott, R.B., Fish, N.A., Barker, C.A.V. and Ruhnke, H.L. The Recovery of mycoplasmas from the genital tracts of bulls in artificial breeding units in Ontario. Canad. J. Cnmp. Med. 39:474475 (1975).

12.

Doig, P.A., Ruhnke, H.L., MacFay, A.L. and Palmer, N.C. Bovine granular vulvitis associated with ureaplasma infection. Can. Vet. J. 2__QO:89-94 (1979).

13.

Doig, P.A., Ruhnke, H.L. and Palmer, N.C. Experimental bovine genital ureaplasmosis I. Granular vulvitis following vulvar inoculation. Can. J. Cc~. Med. 4_~4:252-258 (1980).

MARCH 1988 VOL. 29 NO. 3

Ph.D.

Thesis,

589

THERIOGENOLOGY

14.

Doig, P.A., Ruhnke, H.L. and Palmer, N.C. Experimental bovine genital ureaplasmosis II. Granular vulvitis, endometritis and salpingitis following uterine inoculation. Can. J. Comp. Med. 4_44:259266

(198o).

15.

Ruhnk~, H.L., Doig, P.A. MacKay, A.L., Gargon, A. and Kierstead, M. Isolation of ureaplasma from bovine granular vulvitis. Can. J. Conio. Med. 4-2:151-155 (1978).

16.

Hamdy, A.H. Linco-Spectin as a bovine semen additive. Proc. IV Nat'l. Assoc. Anim. Breeders Tech. Conf. Artif. Insem. Reprod., pp. 54-55 (1972).

17.

Ahmad, K. and Focte, R.H. Postthaw survival and frozen bull spermatozoa treated with antibiotics and detergent. J. Dairy Sc. 69."

535-541 (1986). 18.

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