Antibiotics for Bull Semen Frozen in Milk and Egg Yolk Extenders

K. AHMAO and R. H. FOOTE Department of Animal Science

Cornell University Ithaca, NY 14853

M. KAPROTH Eastern Artif icial Insemination Coop., Inc.,

Ithaca, NY 14850

ABSTRACT

tn six experiments, gentamicin, clin- damycin, amikacin, minocin, tylosin, and Linco-Spectin ® were tested for their effect on motility and fertility of frozen bull spermatozoa and all but clindamycin were used in fertility trials. Antibiotics were added to raw (unextended) semen and nonglycerol portions of the three commonly used semen extenders, whole milk, egg yolk-Tris, and egg yolk-citrate. Semen was frozen in .5-ml straws, stored in liquid nitrogen, and thawed at 37~C for 30 s. Postthaw percentage of motile spermatozoa in antibiotic-treated semen was different for individual bulls in each experiment. Percentage of motile sperm was slightly but significantly depressed at the higher concentrations of clindamycin and Linco-Spectin® tested in whole milk and with minocin in the two egg yolk extenders. In general, nonspermicidal concentrations of each antibiotic were established. Of five antibiotics tested for fertility, only gentamicin reduced fertility on the basis of 59-d nonreturn rates.

INTRODUCTION

The AI industry has relied upon the use of penicillin, streptomycin, and polymyxin B to control contaminating organisms in bull semen for the last 35 yr (4, 8). Although this regimen is still effective against certain organisms found in bull semen (16), its efficacy against some other isolates such as mycoplasma and urea- plasma (6, 11, 13) is questionable. In addition, the possible development of resistant organisms in bull semen necessitates continued testing

Received January 14, 1987. Accepted June 1, 1987.

of new antibiotics for their harmlessness to sperm and for control of contaminating or- ganisms.

Amikacin, a new aminoglycoside, did not affect motility or fertility of frozen bull sper- matozoa (2). Likewise, minocin was innocuous to bull spermatozoa processed in whole milk (WM) extender. In combination with Linco- Spectin®, it eliminated several infectious types of mycoplasmas and ureaplasmas (17). Clin- damycin and tylosin were effective against several mycoplasma and ureaplasma isolates of bovine origin (10, 18), and tylosin was used successfully in egg yolk extenders for bull semen (7). As a part of the artificial breeding industry's effort to evaluate safe use of several antibiotics (12, 15), gentamicin, clindamycin, amikacin, minocin, tylosin, and Linco-Spectin® were tested in this investigation for their effects on postthaw motility of bull sperm frozen and thawed in egg yolk-citrate (EYC), egg yolk-Tris (EYT) and WM extenders. Fertility of anti- biotic-treated sperm was tested with all an- tibiotics except clindamycin.

MATERIALS AND METHODS

Whole milk, EYT, and EYC extenders were prepared as nonglycerol- and glycerol-con- taining fractions. Whole milk and EYT were similar to those described previously (3). Egg yolk-citrate consisted of four parts of 2.9% sodium citrate buffer and one part of egg yolk. Final concentration of glycerol in all extenders was 7% (vol/vol). Gentamicin (gentamicin sulfate, Sigma, St. Louis, MO), clindamycin (clindamycin hydrochloride, Sigma), minocin (minocycline hydrochloride, Sigma), tylosin (tylosin tartrate, Sigma), and Linco-Spectin ® (lin comycin-spectinomycin, Up john, Kala- mazoo, MI) were obtained in powder form. Amikacin (amikacin sulfate 250 mg/ml, Bristol Laboratories, Syracuse, NY) was supplied in pure

1987 J Dairy Sci 70:2439-2443 2439

2440 AHMAD ET AL.

aqueous solution. Stock solutions of powdered antibiotics were prepared in deionized distilled water at 50 times the concentration desired in the extenders and stored at 5°C for use up to 1 wk. Raw semen and nonglycerol fractions of WM, EYT, and EYC received similar con- centrations of experimental antibiotic treat- ments not exceeding 500 /ag/ml of extender, except for Linco-Spectin® (Table 1). The con- trol contained no antibiotic in raw semen but included penicillin, streptomycin, and poly- myxin B in the nonglycerol portion of the extender. Glycerol-containing extenders were treated with penicillin (500 IU/ml) only.

Semen Processing

In each of six experiments wi th six an- tibiotics, 18 ejaculates of semen collected from a random group of six bulls over 3 collection d were processed. Semen was processed in one extender only on a given collection day. Im- mediately after collection, aliquots of raw semen received antibiotic treatments at 35°C in the same concentration included in extended semen. Semen samples were held at that tem- perature for 5 rain and extended 1:2 with antibiotic-treated nonglycerol extenders at the same temperature. These partially extended aliquots were cooled to 5°C in 2 h, followed by further extension with two parts of 5°C nonglycerol extenders. An equal volume (five parts) of glycerol-containing extender was then added slowly giving a concentration of 7% glycerol. Then the extended semen was stored at 5°C for 4 h while being packaged into .5-ml straws properly labeled. Straws were sealed by an ultrasonic sealer and suspended in liquid nitrogen vapor at - 1 3 0 ° C for 7 min. During that time, gaseous nitrogen was bubbled through the liquid nitrogen in order to achieve a vapor temperature of -140°C. Semen was frozen at facilities of Eastern Artificial Insemination Coop., Inc. Frozen semen was stored and transported in liquid nitrogen to Cornell University for postthaw evaluation of the percentage of motile sperm (MS).

Evaluation of Antibiotic Treatments

Antibiotic treatments were evaluated on the basis of postthaw percentage of MS. Two straws per bull- treatment were thawed at random for

30 s at 37°C, and two people independently estimated the percentage of MS from a smear prepared from each straw following procedures described previously (3). There were 4968 estimates of percent MS.

Fertility of semen treated with experimental antibiotics added to WM extender was tested, one antibiotic per field trial. Ejaculates of semen were equally divided to receive either control antibiotics or control plus an experi- mental antibiotic. The concentration of anti- biotic tested was based on the studies of the percentage of MS and the concentrations that might be useful in controlling microorganisms. In each trial, semen from two or three bulls was processed, frozen, and distributed to insemi- nating technicians in coded form. Frozen straws of semen were thawed in an inside pocket at the time of insemination. Fertility was evalu- ated on the basis of 59-d nonreturn (NR) rate to first insemination.

Statistical Analysis

A completely randomized design with factorial arrangement was used to evaluate the percentage of MS in each antibiotic experiment. Because semen was processed only in one extender on a given collection day, data within each extender were analyzed separately. Bulls were random and served as blocks, Antibiotic treatments were fixed effects. Duplicate straws were treated as part of the error term in the analysis of variance (14). Treatment means were compared by Duncan's new multiple range test. With the extensive replications, differences of 2% MS generally were statistically significant (P<,05). To analyze fertility data, NR rates for all bulls in each field trial were pooled for the control and experimental semen. The dif- ferences between two groups were tested by Chi-square.

RESULTS AND DISCUSSION

Postthaw percent MS after freezing and thawing in EYC, EYT, and WM in the six antibiotic experiments arc summarized in Table 1. The results of fertility trials are in Table 2.

Experiment 1: Gentamicin

Increasing concentrations of gentamicin in EYC- and WM-processed bull semen did not

Journal of Dairy Science Vol. 70, No. 11, 1987

OUR INDUSTRY TODAY 2 4 4 1

TABLE 1. Post thaw percentage of mot i le spermatozoa in egg yolk-ci trate (EYC), egg yolk-Tris (EYT), and whole milk (WM) with raw semen and extenders t reated with different antibiotics.

Exper iment number Ant ib io t ic t reatments, gg/ml and ant ibiot ic Extenders 0 50 150 250

(% moti le sperm)

1. Gentamicin EYC 39 40 38 39 EYT 39 a 37 ab 38 ab 37 b WM 37 37 37 37

2. Clindamycin EYC 37 a 36 ab 36 ab 34 b EYT 39 a 36 b 38 ab 36 b WM 35 a 33 b 33 b 31 c

0 100 250 500

3. Amikacin EYC 43 43 43 42 EYT 42 a 42 ab 39 b 42 ab WM 40 a 40 ab 38 ab 38 b

4. Minocin EYC 36 a 36 a 32 b 23 c EYT 37 a 35 ab 34 b 28 c WM 35 36 34 34

5. Tylosin EYC 41 a . . . 40 ab 39 b EYT 43 . . . 43 42 WM 4O . . . 39 39

0 150/300 300/600 600 /1200

6. Linco-Spectin ® EYC 40 ab 38 b 42 a 38 b EYT 41 40 41 41 WM 38 a 36 ab 36ab 34 b

a'b'CMeans with different superscripts within each row differ (P<.05). For individual means, n = 72.

TABLE 2. Nonreturn rates with ant ibiot ics added to whole milk extender.

Trial no. Ant ib io t ic 1 t rea tments No. of services Nonreturn

(/~g/ml) (%)

1 Control 2 748 76.2 a Gentamicin, 250 857 71.2 b

2 Control 1504 70.5 Amikacin, 250 1209 70.5

3 Control 2423 71.2 Minocin, 3403 1720 70.9

4 Control 895 67.6 Tylosin, 175 1091 66.3

5 Control 1095 71.8 Linco-Spectin ®, 600 /1200 1035 70.3

a b . . . ' Nonreturn within rea l with different superscripts differ (P<.05).

1 Control plus experimental antibiotics.

2 Control = Penicillin (500 IU/ml), s t rep tomycin (1000 ~zg/ml), and po lymyxin B (500 IU/ml).

3 Minocin was the commercial product marketed by Lederle Laboratories.

Journal o f Dairy Science Vol. 70, No. 11, 1987

2442 AHMAD ET AL.

affect post thaw percent MS (Table 1), which is consistent with an earlier report that gentamicin was innocuous to post thaw percent MS and percent sperm with intact acrosomes (3). However, 250 /.tg/ml of gentamicin in EYT slightly depressed percent MS in frozen-thawed semen (P<.05). This is thought to represent a chance occurrence, with the many comparisons, because in previous studies, pure gentamicin (2000 /ag/ml) was innocuous to bull sper- matozoa frozen-thawed in EYT (3). Gentamicin appeared to reduce ferti l i ty of bull semen in a field trial (P<.05) but the NR were generally comparable to other ferti l i ty trials presented in this report (Table 2).

Experiment 2. Clindamycin

Percent MS was depressed (P<.05) in all extenders containing 250/ag/ml of clindamycin. Clindamyin is effective against mycoplasma strains of bovine origin in vitro (10) but it was spermicidal in semen inoculated with my- coplasma (15). Because of this toxici ty to sperm, clindamycin does not appear to be a useful antibiotic to add to bull semen, and it was not used for insemination.

Experiment 3: Amikacin

Increasing concentrations of amikacin to 500 /ag/ml depressed percent MS in WM (Table 1), but again this minor effect may have been due to chance. The concentration of 500/ag/ml of amikacin was used previously (2, 5) without affecting percent MS. Amikacin, in WM-pro- cessed semen, did not affect fertility of sper- matozoa (Table 2), which is consistent with previous ferti l i ty studies with amikacin (2).

Experiment 4: Minocin

Depressing effects (P<.05) of minocin were observed in EYe and EYT on post thaw percent MS (Table 1). However, post thaw percent MS in WM was not affected by increasing minocin from 100 to 500 /~g/ml. Also, minocin in WM-processed semen had no effect on ferti l i ty of bull sperm (Table 2). These results are consistent with an earlier study (3) and with those reported by Truscott and Abreo (17) and Abreo et al. (1) indicating toxici ty of minocin in egg yolk extenders. In view of these results, minocin should only be considered for use with

WM-processed semen for controlling possible contamination with mycoplasma.

Experiment 5: Tylosin

Postthaw percent MS averaged over 18 ejaculates treated with tylosin was not affected except for a small decrease (P<.05) in EYe containing 500 /~g/ml of tylosin (Table 1). Toxici ty of tylosin (500/ag/ml) in semen frozen in EYe was previously demonstrated (7). However, it was not detrimental to spermato- zoa at 250 lag/ml in nonglycerol extender, which is consistent with present findings, Fert i l i ty with tylosin-treated bull sperm was comparable with ferti l i ty of control semen (Table 2), which agrees with an earlier study with tylosin (7). As tylosin is effective against mycoplasma and ureaplasma in bovine semen (17), addition of this antibiotic to processed bull semen may be useful to inhibit these potential semen contaminants.

Experiment 6: Linco-Spectin ®

Postthaw percent MS in extenders con- taining 300/600 /Jg/ml of Linco-Spectin® was not different from control (Table 1). This is consistent with a previous report when similar concentrations of Linco-Spectin® were added to raw semen (9). There was a tendency for a progressive decline in percent MS in WM containing increasing Linco-Spectin® (Table 1). Although percent MS in this extender was significantly affected by 600/1200 /~g/ml of Linco-Spectin® (P<.05), similar concentrations in WM-processed semen did not affect ferti l i ty (Table 2). Comparable fertility rates have been reported previously with Linco-Spectin® in semen processed in WM (16), and in EYe (7). Thus it appears that appropriate concentrations of Linco-Spectin® can be used in bull semen as an antimycoplasma agent without affecting sperm moti l i ty or fertility.

Conclusions

Only clindamycin decreased the MS in all extenders. Minocin was toxic to sperm pre- served in EYe and EYT. However, fertility of sperm treated with experimental antibiotics was comparable with ferti l i ty of sperm of controls at concentrations of antibiotics reported by others to be effective against microorganisms.

Journal of Dairy Science Vol. 70, No. 11, 1987

OUR INDUSTRY TODAY 2443

In conclusion, m o s t o f these ant ibiot ics can be safely used in bull semen to con t ro l specific con tamina t ing organisms, and the combina t i on o f gentamicin , ty losin , and Linco-Spect in® is being a d o p t e d by the artificial breeding in- dust ry (12).

A C K N O W L E D G M E N T S

Amikacin was kindly suppl ied by D. Gin- gerich, Bristol Laborator ies . We apprecia te the technical assistance of S. Allen and typ ing of the manuscr ip t by D. Bevins.

REFERENCES

1 Abreo, C. B., G. J. King, I. McMillan, and C. R. Reeds. 1979. Effects of increasing concentrations of minocin on the motility of bovine spermatozoa processed in various extenders. Can. Vet. J. 20: 233.

2 Abroad, K., and R. H. Foote. 1985. Motility and fertility of frozen bull spermatozoa in Tris-yolk and milk extenders containing amikacin sulfate. J. Dairy Sci. 68:2083.

3 Ahmad, K., and R. H. Foote. 1986. Postthaw survival and fertility of frozen bull spermatozoa treated with antibiotics and detergent. J. Dairy Sci. 69:535.

4 Almquist, J. O. 1951. A comparison of penicillin, streptomycin, and sulfanilamide for improving the fertility of semen from bulls of low fertility. J. Dairy Sci. 34:819.

5 Arriola, J., and R. H. Foote. 1982. Effects of amikacin sulfate on the motility of stallion and bull spermatozoa at different temperatures and intervals of storage. J. Anita. Sci. 54:1105.

6 Fish, N. A., S. Rosendal, and R. B. Miller. 1985. The distribution of mycoplasmas and ureaplasmas in the genital tract of normal artificial insemination bulls. Can. Vet. J. 26:13.

7 Foote, R. H., and W. E. Berndtson. 1976. An- tibacterial agents for bull semen; do they help? Page 23 in Proc. 6th Tech. Conf. Artif. Insem.

Reprod. Natl. Assoc. Anlm. Breeders, Columbia, MO.

8 Foote, R. H., and R. W. Bratton. 1950. The fertility of bovine semen in extenders containing sulfanilamide, penicillin, streptomycin, and poly- myxin. J. Dairy Sci. 33:544.

9 Hamdy, A. H. 1972. Linco-spectin as a bovine semen additive. Page 54 in Proc. 4th Teeh. Conf. Artif. Insem. Repro& Natl. Assoc. Anita. Breeders, Columbia, MO.

10 Hamdy, A. H., and C. C. Miller. 1971. Antibiotics for bovine mycoplasmas. J. Dairy Sci. 54:1541.

11 Jasper, D. E., J. M. Al-Aubaidi, and J. Fabrieant. 1973. Isolation of myeoplasma from preputial washings of bulls. Cornell Vet. 64:296.

12 Lorton, S. P. 1986. Laboratory and fertility studies using a combination of gentamicin, tylosin and lineospeetin for the control of microorganisms in bovine semen. Page 39 in Proc. 11th Teeh. Conf. Artif. Insem. Reprod. Natl. Assoc. Anim. Breeders, Columbia, MO.

13 Onoviran, O., R. B. Truscott, N. A. Fish, C.A.V. Barker, and H. L. Ruhnke. 1975. The recovery of mycoplasma from the genital tracts of bulls in artificial breeding units in Ontario. Can. J. Comp. Med. 39:474.

14 Statistical Analysis System Institute, Inc. 1982. SAS User's guide. Cary, NC.

15 Shin, S. J., D. H. Lein, and V. Patten. 1986. The control of mycoplasmas, ureaplasmas, Carnpyl- obacter fetus, and Hemopbilus somnus in frozen bovine semen. Page 33 in Proc. l l t h Tech. Conf. Artif. lnsem. Reprod. Natl. Assoc. Anim. Breeders, Columbia, MO.

16 Shin, S., M. Kaproth, D. Lein, H. Arlitseh, and B. Howe. 1985. Whole milk extender with antibiotics to eliminate Campylobacter fetus from frozen bovine semen J. Dairy Sci. 68:1280.

17 Truscott, R. B., and C. Abreo. 1978. Antibiotics for elimination of mycoplasma and ureaplasma from bovine semen. J. Dairy Sci. 60:954.

18 Truscott, R. B., O. Onoviran, H. L. Ruhnke, N. A. Fish, and C.A.V. Barker. 1975. In vitro anti- microbial sensitivity of mycoplasma isolated from the bovine genital tract. Can. J. Comp. Med. 39:416.

Journal of Dairy Science Vol. 70, No. 11, 1987