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Effects of embryo-freezing and thawing techniques on the survivability of Brucella abortus
THERIOGENOLOGY
EFFECTS OF EMBRYO-FREEZING AND THAWING TECHNIQUES ON THR SURVIVABILITYOF BRUCELLAABORTUS 4 D.A. Stringfellpw,'D.F. Wo fe,2 J.A. McGuire,3 4 L.H. Lauerman, B.W. Gray, and P.H. Sparling Departmentof Mi5robiology,'Departmentof Large Animal Megicine and Surgery, and Departmentof Anatomy and Histology, College of Veteriyry Medicine,Departmentof Research Data Analysis, Alabama AgriculturalExperiment Station,Auburn University,SAL36849 and USDA, APHIS, VS Received for publication: November 21, 1985 Accepted: September 12, 1986 ABSTRACT
A suspensionof a pathogenicstrain (2308) of Brucella abortus was aliquoted,centrifuged,resuspendedin 6 treatment media and quantitated. Ten l-ml samples of each treatmentwere subjected to a standardembryo-freezingtechnique. The treatments were selected to examine the effects of 1) freezing and thawing, 2) cryoprotectantsand 3) antibioticson the survivabilityof Brucella suspendedin embryo-support media. Five samples of each treatmentwere thawed and quantitatedafter a 2-wk storage period and five samples were thawed and quantitatedafter a 6-mo storage period. Means and percent reductionswere determinedfor each treatment. There was no statisticaldifferencebetween means at 2 wk and 6 mo within anv treatment. Freezingand thawing caused a 64% reductionin the number of viable B&ella. Th; addition of antibioticscaused a 99.9% reductionin viabilityof the organism. Glycerol protected the organismduring freezing and thawing-inthe absence of antibiotics but did not interferewith the high percent reduction seen when antibioticswere present. Dimethylsulfoxide(DMSO),however,not only protected the organism during freezing and thawingbut also appeared to negate the effects of the antibiotics. Key
words: Brucellaabortus,embryo freezing,antibiotics, cryprotectants
Acknowledgments Published as PublicationNo. 1790, Auburn University,College of VeterinaryMedicine and Alabama AgriculturalExperiment Station Journal Series No. 5-85924. This study was funded in part under CooperativeAgreementNo. 1216-93027-8sbetweenAuburn Universityand USDA, APHIS, VS.
NOVEMBER
1986 VOL. 26 NO. 5
553
THERIOGENOLOGY
INTRODUCTION Embryo transfer has been proposed as a means of salvaging genetic material from infected populations of livestock and for introduction of genetic material into closed populations without introducing infectious disease agents (1). Further, the development of safe transfer techniques would permit intranational or international movement of genetic material free of disease and with a minimum of restrictions. Several studies to assess the risk of transmitting Brucella abortus through embryo-transfer techniques have examined the exposure potential of embryos collected from superovulated infected cows (2-S). The results of these studies support the hypothesis that embryo exposure to -B. abortus in the uterus of superovulated infected cows is minimal if embryos are not collected shortly after abortion. The possibility for exposure in a small number of cases cannot be discounted (6). Two studies have examined the in vitro interaction of xona pellucida-intact (ZP-I) bovine embryos with B. abortus (7,8). These workers concluded that Brucella would -adhere if exposure of ZP-I embryos did occur. Embryo-transfer procedures provide the opportunity for washing ZP-I embryos to remove infectious agents. In addition, other components of the in vitro environment may serve to affect the viability of these agents. This study was designed to examine the effects of standard embryo-freezing and thawing techniques ou the viability of -B. abortus. MATERIALS AND METHODS A trypticase agar slant with 5% bovine serum was inoculated with g. abortus, biotype 1, strain 2308 and incubated in a humidified atmosphere of air and B% CO2 at 37'C for 3 days. Two mi li liters phosphate-buffered saline (PBS) with 10% heat-inactivated fetal bovine serum (PBS) were used to harvest the suspension. The harvested suspension was diluted in PBS-lo% FBS tg provide a Brucella stock suspension estimated to contain 10 cells/ml. Ten milliliters of the stock suspension were pipetted into each of seven 15-ml tubes and centrifuged at 1500 g for 15 min; the supernatant was discarded. Ten milliliters of treatment media zero through six were added to one of the seven tubes:
554
NOVEMBER 1986 VOL. 26 NO. 5
THERIOGENOLOGY
Treatment 0:
PBS-lo% FBS, before freezing and thawing
Treatment 1:
PBS-lo% FBS
Treatment 2:
PBS-lo% FBS, 1.4 M glycerol
Treatment 3:
PBS-lo% FBS, 1.5 M dimethylsulfoxide (DMSO)
Treatment 4:
PBS-lo% FBS, antibiotics [lo0 u penicillin (base), 100 mcg streptomycin (base), 0.25 mcg fungisone/ml treatment medium]
Treatment 5:
PBS-lo% FBS, antibiotic (as in Treatment A), 1.4 M glycerol
Treatment 6:
PBS-lo% FDS, antibiotics (as in Treatment I), 1.5 M DMSO
After the addition of treatment medium to each tube, the tubes were vortexed to suspend the cel!.sand ten l-ml aliquots from each of treatment tubes one through six were placed in cryotubes for freezing. A l-ml aliquot of Treatment 0 serially diluted and quantitated according to the method of Miles and Misra (9). Color-coded and labelled cryotubes were attached to metal canes and placed vertically in a room-temperature (27'~) methanol bath in a mechanically rate-controlled freezer. The specimens were cooled to -7'C at lo/minute and then each was seeded by touching with metal forceps that had been submerged in liquid nitrogen. Following seeding, the cryotubes were held at -7'C for 10 min and then cooled to -35'C at 0.3'/minute and then to -38'C at O.l'/minute. Following 10 min equilibration at -38'C, the cryotubes were plunged into liquid nitrogen for storage. Cryotubes were thawed quickly by removing them from the liquid nitrogen and placing them in a 37'C water bath for 30 seconds. Five of the cryotubes in each treatment group were thawed approximately 2 wk after freezing and five of the cryotubes were thawed approximately 6 mo after freezing. After thawing, the contents of each cryotube were washed into a 15-ml tube with approximately 10 ml of 0.85 M sodium chloride, centrifuged at 1500 g for 15 min and the supernatant was discarded. The residue was resuspended in 1 ml of 0.85 M sodium chloride, serially diluted and quantitated according to the method of Miles and Misra (9). Means and percent reductions of colony-forming units (CFU) vere determined for each treatment group at the 2-wk and 6-mo thawing times. Means were compared according to Duncan's Multiple Range test.
NOVEMBER 1986 VOL. 26 NO. 5
555
THERIOGENOLOGY
RESULTS
Table 1 shows a significant drop in numbers of CFU in all treatment groups (p
Table 1.
Treatment
Mean number of colony forming units x lo6 of Brucella abortus in 1 ml of treatment suspension after embryo freezing and thawing procedures. Thawed at 2 weeks
213.0a 69.8d 102.0c llO.Ob~C .0073e .lg48e 124.6 abcde t ,,
,
Thawed at 6 months
213.0a 83.0' 113.4b 117.0b .0096; .29%0 123.0
Total Thawed at 2 weeks and 6 months
213.0a 76.4d 107.7= 113.5c .0C85e .19 4e B 123.8
Means within a column with the same letter superscript are not significantly different (p
As shown in Table 2, freezing and thawing in the absence of antibiotics and cryoprotectants (Treatment 1) resulted in a 64% reduction in CFU. In the presence of antibiotics (Treatment 4) and antibiotics plus glycerol (Treatment 5), there was a 99.9% reduction. Freezing and thawing with the cryoprotectants glycerol (Treatment 2) and EMSO (Treatment 3) without antibiotics resulted in 49% and 47% CFU reductions, respectively. Antibiotics and DMSO together resulted in only a 42% reduction of CFU (Treatment 6).
556
NOVEMBER
1986 VOL.
26 NO. 5
THERIOGENOLOGY
Table 2.
Treatment
Mean percent reduction in colony forming units of Brucella abortus in treatment suspensions after embryo-freezing and thawing procedures. Thawed at 2 weeks
67.2b 52.1' 48.4C'd 99.ga 99.ga 41.5d
a,b,c,d
Thawed at 6 months
61.0b 46.8' 45.1C 99.ga 99.ga 42.3'
Total thawed at 2 weeks and 6 months
64.1b 49.4c 46JCsd 99.ga 99.ga 41.9d
Means within a column with the same letter superscr,ipt are not significantly different (~(0.05). Treat 1: PBS-lo% FBS. Treat 2: PBS-lo% FBS, 1.4M glycerol. Treat 3: PBS-lo% FBS, 1.5M DMSO. Treat 4: PBS-LOX FBS, antibiotics. Treat 5: PBS-lo% FBS, antibiotics, 1.4M glycerol. Treat 6: PBS-lo% FBS, antibiotics, 1.5M DMSO.
DISCUSSION The maintenance of embryos in an in vitro environment for a variable period of time is a necessary part of embryo transfer procedures. In standard culture media when temperature, osmolarity and pH are controlled, embryos can be kept at O-38'C for 20 to 30 h with only a slight deterioration of pregnancy rates (10). Freezing and thawing of cattle embryos is now a common component of the in vitro environment. The effects of the embryo's in vitro environment on bovine pathogen survivability are important in determining the risk of transferring pathogens in embryo-transfer procedures. Factors of importance in assessing the survivability of bacteria in the in vitro environment will include temperature, antibiotics, cryoprotectaats and time. The antibiotic combination selected is commonly used in transfer media and embryo collection and culture. The procedures for freezing and thawing were selected because they were in use at this institution at the time of the trials. The two cryoprotectaats selected have been the most commonly used commercially, although glycerol is presently considered the most desirable (11).
NOVEMBER
1986 VOL. 26 NO. 5
557
THERIOGENOLOGY
Treatments 1 to 4 were designed to separately test the effects of the components of standard embryo-freezing and thawing techniques on the survivability of-Brucella.- Freezing and thawing resulted in a 64% reduction (Table 2) in the viability of the Brucella , but the 36% which.might survive when embryos were not washed properly could be an infective dose for a recipient cow. Loss of viability was reduced 15% in the presence of 1.4 M glycerol and 18% in the presence of 1.5 M DMSO (Tables l-2). This could be important if antibiotics were not used or were lacking in potency. Both cryoprotectants provided some protection to the organisms in suspension during the freeze-thaw process. The combined effects of freezing and thawing and the use of antibiotics resulted in a 99.9% reduction in viability of the organism. We should not feel overly confident that the prophylactic use of antibiotics will b3 100% effective since some organisms survived (less than 2.0 X 10 CFU/m&). However, our initial concentration of organisms (2.13 X 10 CFU/ml) was much higher than would be found in media containing embryos which had been properly washed. Treatments 5 and 6 represent media which are actually used for freezing of embryos. The results from these treatments represent what might be expected if embryos were collected from infected covs, contaminated via recovery medium and not washed sufficiently to remove all Brucella. The percent reduction in CFU of Brucella suspended in PBS-LO% FBS, antibiotics and glycerol was identical to that obtained in Treatment 4 that tested the effect of antibiotics in the absence of cryoprotectants (99.9%). The use of DE.0 in the presence of the antibiotics resulted in only a 42% reduction in viability, indicating that the presence of DMSO has some detrimental effect on the efficiency of the antibiotic. The results of this experiment should be placed in perspective. Those using embryo transfer techniaues to nrevent the spread of Brucella should be aware that freeiing and-thawing of embryos in the medium used in Treatment 5 can be expected to destroy 99.9% of any Brucella which might be present after the dilution occurring in normal embryo recovery and washing procedures. The use of SrsO as the cryoprotectant is _ contraindicated since its presence reduced the percent reduction of viable organisms by almost 60%. The use of glycerol did not have this effect. Therefore, the medium of choice for controlling Brucella where transferred embryos are to be frozen and thawed is that used in Treatment 5.
558
NOVEMBER 1986 VOL. 26 NO. 5
THERIOGENOLOGY
REFERENCES 1.
Hare, W.C.D. Embryo transfer and disease transmission (an overview). Proceedings, 10th International Congress on Animal Reproduction and Artificial Insemination. Vol IV: IX 1-1X 8 (1984).
2.
Stringfellow, D.A., Bowell, V.L. and Schnurrenberger, P.R. Investigations into the potential for embryo transfer from Brucella abortus infected cows without transmission of infection. Theriogenology -18:733-743 (1982).
3.
Voelkel, S:A., Stucky, K.W., Looney, C-R., Enright, F.H., An attempt to isolate Brucella abortus from uterine flushings of brucellosis reactor donor cattle. Theriogenology g:355-366 (1983).
Fumes, P.E. and Godke, R.A.
4.
Stringfellow, D.A., Scanlan, C.M., Hannan, S-S., Panangala, V.S., Gray, B.W. and Galik, P.A. Culture of uterine flushings, cervical mucus, and udder secretions collected postabortion from heifers artificially exposed to Brucella abortus. Theriogenology -20~77-83 (1983).
5.
Stringfellow, D.A., Gray, B.W., Sparling, P-H., Panangala, v-s., Galik, P.A., and Young-White, R.R. The effects of superovulation on Brucella abortus infection in the bovine uterus. Theriogenology 23:701-710 (1985).
6.
Hawk, H.W., Kiddy, C.A., Wilson, J-A., Esposito, M. and Winter, A.J. Bacteriological studies of uteri of clinically normal cows of low fertility. J. Dairy Sci. g:120-128 (1958).
7.
Stringfellow, D.A., Scanlan, C.M., Brown, R.R., Meadows, G.B., Gray, B.W. and Young-White, R.R. Culture of bovine embryos after in vitro exposure to Brucella abortus. Theriogenology $_:1005-1012 (1984).
8.
Mallek, Z., Guerin, B. Nibart, M., Parer, M. and Thibier, M. Conseauences de la contamination in vitro des embryos de souris et de vaches par Brucella abortus. Built. Acad. Vet. de France x:479-490 (1984).
9.
Laboratory Alton, G.G., Jones, L.M. and Pietz, DE. Techniques in Brucellosis. World Health Organization Monograph Series No. 55, Geneva, 1975, p. 60.
10.
Seidel, G.E., Jr. Applications of embryo transfer and related technologies to Cattle. J. Dairy Sci. g:2786-2796 (1983).
11.
Lehn-Jensen, H. Deep freezing of cattle embryos. Proceedings of the 10th International Congress on Animal Reproduction and Artificial Insemination VI: II 1 - II 12 (1984).
NOVEMBER 1986 VOL. 26 NO. 5
559
EFFECTS OF EMBRYO-FREEZING AND THAWING TECHNIQUES ON THR SURVIVABILITYOF BRUCELLAABORTUS 4 D.A. Stringfellpw,'D.F. Wo fe,2 J.A. McGuire,3 4 L.H. Lauerman, B.W. Gray, and P.H. Sparling Departmentof Mi5robiology,'Departmentof Large Animal Megicine and Surgery, and Departmentof Anatomy and Histology, College of Veteriyry Medicine,Departmentof Research Data Analysis, Alabama AgriculturalExperiment Station,Auburn University,SAL36849 and USDA, APHIS, VS Received for publication: November 21, 1985 Accepted: September 12, 1986 ABSTRACT
A suspensionof a pathogenicstrain (2308) of Brucella abortus was aliquoted,centrifuged,resuspendedin 6 treatment media and quantitated. Ten l-ml samples of each treatmentwere subjected to a standardembryo-freezingtechnique. The treatments were selected to examine the effects of 1) freezing and thawing, 2) cryoprotectantsand 3) antibioticson the survivabilityof Brucella suspendedin embryo-support media. Five samples of each treatmentwere thawed and quantitatedafter a 2-wk storage period and five samples were thawed and quantitatedafter a 6-mo storage period. Means and percent reductionswere determinedfor each treatment. There was no statisticaldifferencebetween means at 2 wk and 6 mo within anv treatment. Freezingand thawing caused a 64% reductionin the number of viable B&ella. Th; addition of antibioticscaused a 99.9% reductionin viabilityof the organism. Glycerol protected the organismduring freezing and thawing-inthe absence of antibiotics but did not interferewith the high percent reduction seen when antibioticswere present. Dimethylsulfoxide(DMSO),however,not only protected the organism during freezing and thawingbut also appeared to negate the effects of the antibiotics. Key
words: Brucellaabortus,embryo freezing,antibiotics, cryprotectants
Acknowledgments Published as PublicationNo. 1790, Auburn University,College of VeterinaryMedicine and Alabama AgriculturalExperiment Station Journal Series No. 5-85924. This study was funded in part under CooperativeAgreementNo. 1216-93027-8sbetweenAuburn Universityand USDA, APHIS, VS.
NOVEMBER
1986 VOL. 26 NO. 5
553
THERIOGENOLOGY
INTRODUCTION Embryo transfer has been proposed as a means of salvaging genetic material from infected populations of livestock and for introduction of genetic material into closed populations without introducing infectious disease agents (1). Further, the development of safe transfer techniques would permit intranational or international movement of genetic material free of disease and with a minimum of restrictions. Several studies to assess the risk of transmitting Brucella abortus through embryo-transfer techniques have examined the exposure potential of embryos collected from superovulated infected cows (2-S). The results of these studies support the hypothesis that embryo exposure to -B. abortus in the uterus of superovulated infected cows is minimal if embryos are not collected shortly after abortion. The possibility for exposure in a small number of cases cannot be discounted (6). Two studies have examined the in vitro interaction of xona pellucida-intact (ZP-I) bovine embryos with B. abortus (7,8). These workers concluded that Brucella would -adhere if exposure of ZP-I embryos did occur. Embryo-transfer procedures provide the opportunity for washing ZP-I embryos to remove infectious agents. In addition, other components of the in vitro environment may serve to affect the viability of these agents. This study was designed to examine the effects of standard embryo-freezing and thawing techniques ou the viability of -B. abortus. MATERIALS AND METHODS A trypticase agar slant with 5% bovine serum was inoculated with g. abortus, biotype 1, strain 2308 and incubated in a humidified atmosphere of air and B% CO2 at 37'C for 3 days. Two mi li liters phosphate-buffered saline (PBS) with 10% heat-inactivated fetal bovine serum (PBS) were used to harvest the suspension. The harvested suspension was diluted in PBS-lo% FBS tg provide a Brucella stock suspension estimated to contain 10 cells/ml. Ten milliliters of the stock suspension were pipetted into each of seven 15-ml tubes and centrifuged at 1500 g for 15 min; the supernatant was discarded. Ten milliliters of treatment media zero through six were added to one of the seven tubes:
554
NOVEMBER 1986 VOL. 26 NO. 5
THERIOGENOLOGY
Treatment 0:
PBS-lo% FBS, before freezing and thawing
Treatment 1:
PBS-lo% FBS
Treatment 2:
PBS-lo% FBS, 1.4 M glycerol
Treatment 3:
PBS-lo% FBS, 1.5 M dimethylsulfoxide (DMSO)
Treatment 4:
PBS-lo% FBS, antibiotics [lo0 u penicillin (base), 100 mcg streptomycin (base), 0.25 mcg fungisone/ml treatment medium]
Treatment 5:
PBS-lo% FBS, antibiotic (as in Treatment A), 1.4 M glycerol
Treatment 6:
PBS-lo% FDS, antibiotics (as in Treatment I), 1.5 M DMSO
After the addition of treatment medium to each tube, the tubes were vortexed to suspend the cel!.sand ten l-ml aliquots from each of treatment tubes one through six were placed in cryotubes for freezing. A l-ml aliquot of Treatment 0 serially diluted and quantitated according to the method of Miles and Misra (9). Color-coded and labelled cryotubes were attached to metal canes and placed vertically in a room-temperature (27'~) methanol bath in a mechanically rate-controlled freezer. The specimens were cooled to -7'C at lo/minute and then each was seeded by touching with metal forceps that had been submerged in liquid nitrogen. Following seeding, the cryotubes were held at -7'C for 10 min and then cooled to -35'C at 0.3'/minute and then to -38'C at O.l'/minute. Following 10 min equilibration at -38'C, the cryotubes were plunged into liquid nitrogen for storage. Cryotubes were thawed quickly by removing them from the liquid nitrogen and placing them in a 37'C water bath for 30 seconds. Five of the cryotubes in each treatment group were thawed approximately 2 wk after freezing and five of the cryotubes were thawed approximately 6 mo after freezing. After thawing, the contents of each cryotube were washed into a 15-ml tube with approximately 10 ml of 0.85 M sodium chloride, centrifuged at 1500 g for 15 min and the supernatant was discarded. The residue was resuspended in 1 ml of 0.85 M sodium chloride, serially diluted and quantitated according to the method of Miles and Misra (9). Means and percent reductions of colony-forming units (CFU) vere determined for each treatment group at the 2-wk and 6-mo thawing times. Means were compared according to Duncan's Multiple Range test.
NOVEMBER 1986 VOL. 26 NO. 5
555
THERIOGENOLOGY
RESULTS
Table 1 shows a significant drop in numbers of CFU in all treatment groups (p
Table 1.
Treatment
Mean number of colony forming units x lo6 of Brucella abortus in 1 ml of treatment suspension after embryo freezing and thawing procedures. Thawed at 2 weeks
213.0a 69.8d 102.0c llO.Ob~C .0073e .lg48e 124.6 abcde t ,,
,
Thawed at 6 months
213.0a 83.0' 113.4b 117.0b .0096; .29%0 123.0
Total Thawed at 2 weeks and 6 months
213.0a 76.4d 107.7= 113.5c .0C85e .19 4e B 123.8
Means within a column with the same letter superscript are not significantly different (p
As shown in Table 2, freezing and thawing in the absence of antibiotics and cryoprotectants (Treatment 1) resulted in a 64% reduction in CFU. In the presence of antibiotics (Treatment 4) and antibiotics plus glycerol (Treatment 5), there was a 99.9% reduction. Freezing and thawing with the cryoprotectants glycerol (Treatment 2) and EMSO (Treatment 3) without antibiotics resulted in 49% and 47% CFU reductions, respectively. Antibiotics and DMSO together resulted in only a 42% reduction of CFU (Treatment 6).
556
NOVEMBER
1986 VOL.
26 NO. 5
THERIOGENOLOGY
Table 2.
Treatment
Mean percent reduction in colony forming units of Brucella abortus in treatment suspensions after embryo-freezing and thawing procedures. Thawed at 2 weeks
67.2b 52.1' 48.4C'd 99.ga 99.ga 41.5d
a,b,c,d
Thawed at 6 months
61.0b 46.8' 45.1C 99.ga 99.ga 42.3'
Total thawed at 2 weeks and 6 months
64.1b 49.4c 46JCsd 99.ga 99.ga 41.9d
Means within a column with the same letter superscr,ipt are not significantly different (~(0.05). Treat 1: PBS-lo% FBS. Treat 2: PBS-lo% FBS, 1.4M glycerol. Treat 3: PBS-lo% FBS, 1.5M DMSO. Treat 4: PBS-LOX FBS, antibiotics. Treat 5: PBS-lo% FBS, antibiotics, 1.4M glycerol. Treat 6: PBS-lo% FBS, antibiotics, 1.5M DMSO.
DISCUSSION The maintenance of embryos in an in vitro environment for a variable period of time is a necessary part of embryo transfer procedures. In standard culture media when temperature, osmolarity and pH are controlled, embryos can be kept at O-38'C for 20 to 30 h with only a slight deterioration of pregnancy rates (10). Freezing and thawing of cattle embryos is now a common component of the in vitro environment. The effects of the embryo's in vitro environment on bovine pathogen survivability are important in determining the risk of transferring pathogens in embryo-transfer procedures. Factors of importance in assessing the survivability of bacteria in the in vitro environment will include temperature, antibiotics, cryoprotectaats and time. The antibiotic combination selected is commonly used in transfer media and embryo collection and culture. The procedures for freezing and thawing were selected because they were in use at this institution at the time of the trials. The two cryoprotectaats selected have been the most commonly used commercially, although glycerol is presently considered the most desirable (11).
NOVEMBER
1986 VOL. 26 NO. 5
557
THERIOGENOLOGY
Treatments 1 to 4 were designed to separately test the effects of the components of standard embryo-freezing and thawing techniques on the survivability of-Brucella.- Freezing and thawing resulted in a 64% reduction (Table 2) in the viability of the Brucella , but the 36% which.might survive when embryos were not washed properly could be an infective dose for a recipient cow. Loss of viability was reduced 15% in the presence of 1.4 M glycerol and 18% in the presence of 1.5 M DMSO (Tables l-2). This could be important if antibiotics were not used or were lacking in potency. Both cryoprotectants provided some protection to the organisms in suspension during the freeze-thaw process. The combined effects of freezing and thawing and the use of antibiotics resulted in a 99.9% reduction in viability of the organism. We should not feel overly confident that the prophylactic use of antibiotics will b3 100% effective since some organisms survived (less than 2.0 X 10 CFU/m&). However, our initial concentration of organisms (2.13 X 10 CFU/ml) was much higher than would be found in media containing embryos which had been properly washed. Treatments 5 and 6 represent media which are actually used for freezing of embryos. The results from these treatments represent what might be expected if embryos were collected from infected covs, contaminated via recovery medium and not washed sufficiently to remove all Brucella. The percent reduction in CFU of Brucella suspended in PBS-LO% FBS, antibiotics and glycerol was identical to that obtained in Treatment 4 that tested the effect of antibiotics in the absence of cryoprotectants (99.9%). The use of DE.0 in the presence of the antibiotics resulted in only a 42% reduction in viability, indicating that the presence of DMSO has some detrimental effect on the efficiency of the antibiotic. The results of this experiment should be placed in perspective. Those using embryo transfer techniaues to nrevent the spread of Brucella should be aware that freeiing and-thawing of embryos in the medium used in Treatment 5 can be expected to destroy 99.9% of any Brucella which might be present after the dilution occurring in normal embryo recovery and washing procedures. The use of SrsO as the cryoprotectant is _ contraindicated since its presence reduced the percent reduction of viable organisms by almost 60%. The use of glycerol did not have this effect. Therefore, the medium of choice for controlling Brucella where transferred embryos are to be frozen and thawed is that used in Treatment 5.
558
NOVEMBER 1986 VOL. 26 NO. 5
THERIOGENOLOGY
REFERENCES 1.
Hare, W.C.D. Embryo transfer and disease transmission (an overview). Proceedings, 10th International Congress on Animal Reproduction and Artificial Insemination. Vol IV: IX 1-1X 8 (1984).
2.
Stringfellow, D.A., Bowell, V.L. and Schnurrenberger, P.R. Investigations into the potential for embryo transfer from Brucella abortus infected cows without transmission of infection. Theriogenology -18:733-743 (1982).
3.
Voelkel, S:A., Stucky, K.W., Looney, C-R., Enright, F.H., An attempt to isolate Brucella abortus from uterine flushings of brucellosis reactor donor cattle. Theriogenology g:355-366 (1983).
Fumes, P.E. and Godke, R.A.
4.
Stringfellow, D.A., Scanlan, C.M., Hannan, S-S., Panangala, V.S., Gray, B.W. and Galik, P.A. Culture of uterine flushings, cervical mucus, and udder secretions collected postabortion from heifers artificially exposed to Brucella abortus. Theriogenology -20~77-83 (1983).
5.
Stringfellow, D.A., Gray, B.W., Sparling, P-H., Panangala, v-s., Galik, P.A., and Young-White, R.R. The effects of superovulation on Brucella abortus infection in the bovine uterus. Theriogenology 23:701-710 (1985).
6.
Hawk, H.W., Kiddy, C.A., Wilson, J-A., Esposito, M. and Winter, A.J. Bacteriological studies of uteri of clinically normal cows of low fertility. J. Dairy Sci. g:120-128 (1958).
7.
Stringfellow, D.A., Scanlan, C.M., Brown, R.R., Meadows, G.B., Gray, B.W. and Young-White, R.R. Culture of bovine embryos after in vitro exposure to Brucella abortus. Theriogenology $_:1005-1012 (1984).
8.
Mallek, Z., Guerin, B. Nibart, M., Parer, M. and Thibier, M. Conseauences de la contamination in vitro des embryos de souris et de vaches par Brucella abortus. Built. Acad. Vet. de France x:479-490 (1984).
9.
Laboratory Alton, G.G., Jones, L.M. and Pietz, DE. Techniques in Brucellosis. World Health Organization Monograph Series No. 55, Geneva, 1975, p. 60.
10.
Seidel, G.E., Jr. Applications of embryo transfer and related technologies to Cattle. J. Dairy Sci. g:2786-2796 (1983).
11.
Lehn-Jensen, H. Deep freezing of cattle embryos. Proceedings of the 10th International Congress on Animal Reproduction and Artificial Insemination VI: II 1 - II 12 (1984).
NOVEMBER 1986 VOL. 26 NO. 5
559