THERIOGENOLOGY
A SINGLE CANNLJL& TECHNIQUE FOR NONSURGICAL COLLECTION OF OVA FROM CATTLE=
R. F. Roweb , M. R. Del Campob, C. L. Eilts', L. R. Frenchd, R. P. Winch= and 0. J. Gintherb
Departments of Veterinary Science and Animal Science University of Wisconsin-Madison Madison, Wisconsin 53706
Received for publication:
July 6, 1976
a Research supported by College of Agricultural and Life Sciences, University of Wisconsin-Madison, CBR Ova Transfer, Inc., Fennimore, Wisconsin, and Rock-A-Bye Farms, Richland Center, Wisconsin. b Department of Veterinary Science, University of Wisconsin-Madison, Wisconsin. Dr. Rowe is a post-doctoral trainee supported by the Wisconsin Animal Health Laboratory. ' CBR Ova Transfer, Inc., Fennimore, Wisconsin. d
Department of Meat and Animal Science, University of Wisconsin-Madison.
ACKNOWLEDGMENTS The authors thank American Breeders Service, Deforest, Wisconsin for a grant of semen and Upjohn Company, Kalamazoo Michigan for the gift of prostaglandin Fza. Excellent technical assistance was contributed by Mrs. J. Meschievitz, Mr. Max Carter and Mr. Tom Oettiker. Special thanks to Donna Van Horn for illustrations and graphics.
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THERIOGENOLOGY
Abstract The uteri of 34 heifers were flushed for ova six to nine days following The technique estrus using a single cannula nonsurgical technique. involved the infusion of fluid by gravity and agitation within the U’CSKUS by to-and-fro action of a syringe followed by unassisted fluid collection. Each horn was flushed five times using 30-150 ml of flushing fluid per flush. Recovered fluid (flushing fluid plus uterine secretion) was an average of 95% of the volume of the fluid inserted. Ova were recovered from 12 of 19 nontreated, single ovulating heifers (63%) and from all of 15 super-ovulated heifers (mean and S.D. for number of ova, 6.3 ? 4.4/ superovulated heifer; range, 1 to 14 ova). Based on the number of corpora lutea, the ova recovery index was 54% as averaged over the 15 superovulated heifers. The technique has been used in 4 additional super-ovulated heifers with modification (increased number of flushes to 8) subsequent to the termination of the planned project. Recovery index for the first 5 flushes was 58%. However, some ova were recovered in the 6th, 7th, and 8th flushes resulting in an apparent improved recovery index of 69%. Introduction Past attempts for nonsurgical recovery of ova from cows (1,2,3,4,5) have utilized intricate techniques for intrauterine placement of multiple cannulae, but have met with limited succass. Graham (6) and Schultz (7) summarized the problems associated with previous attempts at nonsurgical ova collection as poor percent recovery of the fluid infused, low recovery percentages of available ova, handling and processing large amounts of flushing fluid and trauma to the uterus from passage of a catheter to the tip of the horn. Recovery rates have been far below that reported for surgical collections. The present project was undertaken to develop a simple, reliable method of nonsurgical ova collection. Materials and Methods Twenty-eight Holstein and 6 beef heifers were used (19 nontreated, single ovulating and 15 superovulated). Ten of the single ovulating heifers were bred and 9 were not. The superovulation regimen utilized a commercial pituitary 72 hours after the initial gonadotropin injection. preparation,e and PGF Heifers were insemina ga ed with two ampules of frozen semen at the beginning of standing estrus and every 12 hours until the termination of estrus. Most of the heifers were bred 3 times. Prior to collection, an epidural anesthetic was administered and the labia were washed and disinfected. No attempt was made to douch the vagina. Heifers were restrained in a squeeze chute to minimize movement. All collections were done 6 to 9 days after estrus. Numbers of corpora lutea in superovulated heifers were estimated both by rectal palpation and laparoscopy (8).
e Armour Std FSH-PR, Burns Bio-tech, Omaha, Neb.
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The collection device (Fig. 1) consisted of a Foley catheterf (French No. 12 to 20 with 30 ml cuff, Fig. Z), stainless steel stylet (Fig. Z), teflon coated surgical tubing, 3 tubing clamps, 2 glass T's, 2 settling bulbsg (Fig. 4) one of which was equipped with a 2-holed rubber cork and glass tubing, and a 50 ml syringe. Associated equipment included a small diameter uterine infusion catheter (Luchens uterine infusion catheter, Fig. 2), ova examination cups, and flushing medium. All glassware and tubing which had the potential for contact with ova were siliconized with a commercial producth in an attempt to reduce the occurrence of adhering of ova to the equipment. The equipment was siliconised and sterilized prior to each use. A range of sizes of Foley catheters was kept on hand and the largest size which could be passed through the cervix was used. One of the settling bulbs was used as a media reservoir and the other for collection and settling. Several settling bulbs were kept on hand for repeated flushing. The Luchens catheter was passed through the cervix first in an attempt to facilitate subsequent passage of the Foley catheter. The ova examination cups were prepared by a glass-maker from 250 ml round-bottom flasks as shown (Fig. 3). The cups were cut from the flasks on the bias so that flaws present on the bottom of the flask would not be on the bottom or viewing area of the examination cups. The flushing medium was tissue culture medium (TCM)199i to which had been added 100 IU potassium penicillin G,I 100 mcg streptomycin sulfate,k and 1 mg bovine serum albumin1 per ml. The medium and glassware were warmed to body temperature prior to use. Throughout the collection procedure the reproductive tract was manipulated and monitored by rectal palpation. The steps of the collection procedure were as follows (Fig. 1): 1) passage into the uterus and subsequent removal of the Luchens catheter, 2) passage and placement of the Foley catheter with stylet into the caudal portion of one horn, 3) inflation of the cuff with air and removal of the stylet, 4) attachment of the remainder of the collection device except the collection settling bulb, 5) filling of reservoir with flushing media with clamp A attached, 6) removal of air from exterior portion of the tubing by clamping exterior portion of the Foley catheter and removal of clamp A with clamp B in place, followed by attachment of clamp C after the air was expelled, 7) removal of clamp from Foley catheter and infusion of media by gravity flow, 8) attachment of collection settling bulb, 9) attachment
f
BardexR, C.R. Bard Inc., Murray Hill, N. J.
g Corning GlassWorks, Corning N. Y. h SilicladR, Becton, Dickinson and Co., Parsippany, N. J. i Flow Laboratories, Rockville, Maryland ' GIBCO, Grand Island, N. Y. k Pfizer, Inc., New York, N. Y. 1 Sigma Chemical Co., St. Louis, Missouri.
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of clamp A and removal of clamp B, 10) agitation of the flushing fluid in the uterus by vigorous to-and-fro movement of syringe plunger, 11) immediate removal of clamp C and collection of fluid by gravity flow, 12) flushing of the tubing by removing clamp A, and 13) replacement of clamps A, B and C, refilling reservoir and repeating infusion collection steps. Single ovulating heifers were collected from the uterine horn ipsilateral to the CL and superovulated heifers were collected from both horns. The cuff of the Foley catheter was inflated with air until an obvious uterine distention occurred as determined by rectal palpation and the catheter seemed firmly anchored. Excessive insufflation, beyond that necessary for anchoring and preventing leakage of flushing medium, was avoided. Infusion of the medium was by gravity from a height of approximately 50 cm. This appeared to produce adequate distention of the horn. The degree of distention was monitored per rectum and the passage of fluid into the horn was readily detectable. Agitation of the fluid in the horn was done by the to-and-fro movement of the syringe plunger to favor dislodgement and movement of ova. Approximately 15-25% of the volume of fluid that had been infused was withdrawn and reinserted rapidly 3 times. Clamp C was removed immediately after the third emptying of the syringe. The flushing medium rapidly entered the settling bulb without manual evacuation of the horn. When indicated, evacuation of the fluid was assisted by gentle uterine massage. The fluid from each of the 5 flushes was collected into a separate settling bulb so that the effect of sequence of flushes could be evaluated. The flushing medium in each settling bulb was allowed to stand undisturbed at least 20 minutes. Fluid was then withdrawn from the valve at the bottom of the bulb, sequentially, in quantities of 30 to 40 ml per examination cup. For purposes of comparison of ova collection efficiency between nonsurgical and surgical techniques data accumulated in conjunction with surgical collection of ova at a professional ova transfer establishment were examined. The collections involved 29 heifers of the recently imported beef breeds (Maine-Anjou, Normandy, and Sirmnental). Collections were done by insertion and collection of flushing fluid from each horn through a midventral laparatomy as described by Rowson, et al (9). All heifers for which collections ware attempted over a period of time were included without selection bias. Results Two single ovulating heifers were replaced because the operator was unable to pass the catheter through the cervix and 1 superovulated heifer was eliminated due to metritis. Recovery data for ova and flushing fluids are shown for the nonsurgically collected single ovulating heifers (Table 1) and for the nonsurgically and surgically collected superovulated heifers (Table 2). ow recovery rates for the nonsurgical technique were 70% (bred, single ovulators), 56% (nonbred, single ovulators), and 54% (superovulated heifers). Recovery rate for the surgical technique in superovulated heifers was 58%. Fluid recovery rates for the nonsurgical technique were 96% (bred,
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single ovulators), 97% (nonbred, single ovulators), and 92% (superovulated heifers). In superovulated heifers the percent of recovered ova which were found in each of the 5 flushes were 31, 27, 17, 4 and 21, respectively. Discussion The recovery index used to evaluate the efficiency of ova recovery "as based on the number of corpora lutea (No. ova collected i No. corpora lutea x 100) and is therefore likely a censervative estimate. For example, the index does not take into account formation of corpora lutea from unovulated follicles, failure of pick-up of ova by the infundibulum, degeneration of ova beyond a recognizable stage by the time of flushing, and failure to find ova that were collected in the flushing fluid. Any of these events would lead to an underestimation of collection efficiency. Another source of error involves the estimation of number of corpora lutea. In the present series 11 of the superovulated heifers were examined both by rectal palpation and by laparoscopy. The overall means and standard deviations for the 2 techniques were quite close (rectal palpation, 13.0 i 4.1; laparoscopy 13.1 ? 4.3). HOWeVsl-, sometimes the estimate from one'method "as higher and sometimes lower than the other so that the average disagreement between methods "as 2.5 CL per CO". The correlation between methods "as +0.76. Failure to approach a correlation of 1 reflects the degree of disagreement between methods. On the assumption that laparoscopy "as more accurate it "as given preference in calculation of the recovery index (available in 11 of 15 superovulated cattle). It "as noted that even with close inspection during surgical collections there "as occasional disagreement between observers on CL numbers. Corpora lutea that were close together were not always clearly discernible. Selection bias "as eliminated by using a preplanned number of heifers for the non-surgical technique and by including all heifers which were submitted to surgery at a professional ova transfer establishment for the surgical technique. The recovery indices reported herein, therefore, are useful and reasonable and, if anything, are on the conservative side. It is emphasized that valid statistical comparisons between the nonsurgical and surgical techniques are not indicated, since the two techniques were done independently, at different locations, and by different operators. The comparisons must, therefore, be considered with some reservation. However, it should be noted that the difference in efficiency between techniques in superovulated cattle did not appear to be great (54% vs 58% for nonsurgical and surgical techniques, respectively). The single cannula technique "as used in 12 superovulated cattle under field conditions subsequent to the planned project. Flushes were combined and the number of flushes "as not carefully monitored, but "as estimated to be 4-6/born. The recovery index was 59% which is quite comparable to the 54% obtained in the planned project and the 58% for surgical recovery. The results for other nonsurgical techniques were reviewed by Foote and Onuma (10). It is impossible to make critical comparisonsbetween previously reported techniques and the one used herein as most of the data on efficiency of ova collection are incomplete. Often the data are reported as successful
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attempts (defined as collection of at least one ovum from an individual) and do not indicate whether single ovulating or superovulated animals were used. Dziuk, et al. (4) estimated the number of potential collectable ova by rectal palpation of corpora lutea in 34 superovulated cattle. An average of 0.9 ova/cow was recovered with a recovery index of 10% (calculated by the present authors). From 50 to 1000 ml of fluid was forced into the uterus and withdrawn by negative pressure and 77% of the fluid was recovered. Sugie (5), using a device for continuous flushing (double cannulae), reported recovery of ova from 81% of 32 superovulated cattle and an average of 5.1 ova recovered per animal. However, it is not clear whether the mean number of recovered ova includes cattle from which no ova were obtained. Corresponding results for the present technique in superovulated cattle were 100% and 6.3 ova, respectively, but information needed for further comparisons are not available. Although not critically studied, there has been no suggestion based on limited numbers of transfers to recipients that the present nonsurgical technique was detrimental to the collected ova. The percent of recovered ova from superovulated cattle that appeared to be fertilized was 65% for the nonsurgical technique and 64% for the surgical technique. One of the initial concerns in development of techniques for ova recovery was the possibility that the flushing medium and, therefore, the ova would be lost through the oviducts. In the present project, there was no indication that the tubouterine junction opened. More than 95% of the total volume of infused medium was recovered. Occasional leakage occurred around the cuff due to faulty inflation or partial deflation of the balloon, however, this was not a serious problem. Conversely, over distention of the cuff was noted to cause occlusion of the lumen of the catheter resulting in reduced flow rate. No attempt was made to critically evaluate the importance of various steps in the procedure and it is likely that modifications can be made to adjust to various field conditions. Preliminary trials were done using 4 technique modifications: 1) agitation with gravity collection as described herein, 2) gravity collection without agitation, 3) agitation with collection by aspiration of air from the collecting bulb, and 4) collection by aspiration without agitation. Gravity collection with agitation seemed the most effective of the modifications, but ova were obtained with all 4 procedures. The number of animals was too small for critical evaluation. The usefulness of the Luchens catheter to facilitate subsequent passage of the Foley catheter was not tested. As experience with the technique progressed, the step involving the Luchens catheter was omitted and is currently being used only when difficulty occurs in passing the Foley catheter. It should be noted that previous workers have reported that the cervix of diestrus cattle (day 5) may be dilated with relaxin (11). Perhaps, therefore, relaxin should be tested as an aid in cattle in which the catheter cannot be passed. Experience is an important factor in passage of the catheter and in manipulating and placing the end of the catheter into each of the uterine horns. In this regard, the effectiveness of the epidural anesthesia seemed important. Inadequate anesthesia may result in rectal and abdominal contractions which hamper manipulative efforts and may prevent adequate filling or distention of the uterine horns. The authors believe that -in utero agitation of the fluid is an important aspect of the
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procedure but acknowledge that this has not been tested. While the fluid is agitated with the syringe, the surging and receding force of the medium can be felt, per rectum, throughout the horn. Agitation of the flushing medium within the horns could be done by manually induced contractions of the horn per rectum. In the authors' opinion, however, the use of the syringe provides better control and may reduce the possibilities of injury to the endometrium. The time course of the entire collection procedure in superovulated cattle was approximately 35 minutes from insertion of the Foley catheter into the first horn to withdrawal of the Foley catheter from the second horn. From the time clamp A was removed to begin the infusion of one flush until the clamp was again removed for the next flush was approximately 3 minutes. Use of settling bulbs (Fig. 4) appeared to be an especially useful innovation for rapid detection of ova in the flushing fluid. Examination of the literature indicates Dracy and Peterson (3) used a French separatory funnel for processing large quantities of fluid, but no data on the effectiveness of the funnel were given. In the present study, all ova except two that were degenerated were found in the 30 to 40 ml of fluid which was first drawn through the valve at the bottom of the bulb (contents of first examination cup). The settling bulbs can be also be used to process fluids used to rinse the glassware for possible adhering ova or to reprocess a series of cups that have already been examined. It is recommended that the use of the single cannula ova collection technique on a professional basis be preceded by practice. No attempt has been made by us or others to critically evaluate the possibilities for deleterious effects on the donor animal of nonsurgical ova collection techniques. Noticeable ruptures or loss of fluid into the uterine wall or broad ligament did not occur in the present study. However, during preliminary developmental trials, 2 ruptures occurred. Enlargement of the uterus occurred during infusion but the fluid could not be evacuated and was believed to have entered the uterine wall. Subsequent to the planned project complete penetration of the uterine wall occurred in one cow and flushing fluid was lost into the abdominal cavity. Rupture or trauma to the uterus is most likely to occur during placement of the catheter into the horn and during insufflation of the cuff. In approximately 10% of the collections, red blood cells appeared in the medium indicating uterine trauma. Since the red cells remained suspended in the flushing fluid, detection of ova was quite difficult. The presence of epithelial cells, mucus, and debris of unknown origin indicated that thorough agitation occurred. In many instances, ova were found in flushings which had the most debris. A time-saving step that can be used in the field is to combine more than one flush into each settling bulb. In the present work flushes were kept separate to evaluate the effectiveness of repeated flushing. The percent of recovered ova which were found in each of the 5 flushes was 31, 27, 17, 4 and 21, respective!lY Thus, the last flush produced a large number of ova. ( It is likely, therefore, that a greater number of flushes would have increased the recovery index. Subsequent to the completion of the present project, the single cannula technique has been used on 4 superovulated cattle with the modification that 8 instead of 5 flushes were used for each horn. The ova recovery index for the first 5 flushes was 58% which corresponds to 54% for the primary project. However, the index for all 8 flushes was 69%. Six, 6 and 3% of the recovered ova were in the 6th, 7th and 8th flushes, respectively. Thus, it appears that the recovery index of 54% obtained in the primary project can be increased, by increasing the number of flushes.
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References 1. Rowson, L. E. A. and D. F. Dowling. of fertilized eggs from the living cow.
An apparatus for the extraction Vet. Record 61: 191 (1949)
2. Dowling, D. F. Problems of the transplantation of fertilized ova. J. Agr. Sci. (England) x: 374 (1949) 3. Dracy, A. E. and W. E. Peterson. Isolation of ova from the living bovine. J. Dairy Sci. 2: 797 (1950) 4. Dziuk, P. J., J. D. Donker, J. R. Nichols and W. E. Peterson. Problems associated with the transfer of ova between cattle. Minn. Agr. Exp. Sta. Tech. Bull. 222 (1958) 5. Sugie, T. Non surgical ova collection in cattle (Jap.). Ann. Report Nat. Inst. An. Ind., Chiba-shi, Japan 1968. 8: 55 (1970) 6. Graham, E. F. Ova Transfer. Proceedings Fifth Tech. Conf. on Art. Insem. and Reprod. Nat. Ass. of An. Breeders (1975) 7. Schultz, R. H. Techniques and problems associated with collection and transfer of bovine ova. Annual Meeting of Am. Vet. Sot. for Study of Breeding Soundness. Columbia, Missouri, Sept. 6-7, 1974
a. Wishart, D. F. and J. B. Snowball. of the ovary in situ.
Vet:Rec.
Endoscopy in cattle: Observation 92: 139-143 (1973)
9. Rowson, L. E. A., R. M. Moor, and R. A. S. Lawson. Fertility following egg tranfer in the cow; effect of method, medium and synchronization of estrus. J. Reprod. Fert. 18: 517 (1969) 10. Foote, R. H. and H. Onuma. Superovulation, ovum collection, culture and transfer. A review. J. Dairy Sci. 53: 1681 (1970) 11. Graham, E. F. and A. E. Dracy. The effect of relaxin and mechanical dilation on the bovine cervix. J. Dairy Sci., 36: 772 (1953)
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9
10
Bred
Non Bred
No. heifers
Group
480
478
56
k172
+156
96
97
265-705
250-700 k3
+3
90-100
90-100
40
43
Fluid collected/heifer (%I 1st Mean SD Range
Recovery Rates for Ova and Flushing Fluid From Single Ovulating Heifers
Fluid infused/ heifer (ml) Range Mean SD
70
% from which ovum was recovered
Table 1.
20
43
40
0
0
14
0
0
% of recovered ova in each flush 2nd 5th 3rd 4th
1
en
8
o\
r
6
-&
z T;:
E
54
933
92
Index of recovery of ova/ heifer (%)c
Fluid inserted/heifer (ml)
Fluid recovered/heifer (W)
C
b
4 21
17
of heifers in parenthesis.
flush flush flush flush flush
k31 +20 +23 +8 f22
k6
?280
k29
24.9
t4.4
S.D.
9.9
l-22
o-22 O-67
O-75
O-100 O-60
78-99
543-1390
58
6.2
1-14
13-100
Mean
Range
229
k6.3
k5.5
S.D.
No. ova recovered G estimated no. corpora lutea x 100.
O-100
l-29
o-21
Range
Surgical recovery (29)
No. of corpora lutea estimated by laparoscopy in 11 heifers and by rectal palpation in 4 heifers for nonsurgical recovery and by visual inspection for surgical recovery.
a No.
1st 2nd 3rd 4th 5th
31 27
12.2
No. corpora lutea/heiferb
% of recovered ova/heifer which were in:
6.3
Mean
Nonsurgical recovery (15)a
Recovery Rates for Ova and Flushing Fluid from Superovulated Heifers
No. ova recovered/heifer
Item
Table 2.
a
!G
$!
a
!
THERIOGENOLOGY
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1976
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THERIOGENOLOGY
Luchens
Fig. 2.
Fig. 3.
4.82
cat hetsr
Foley catheter and companion instruments
Method for preparation of ova examination cup from a round bottom flask.
NOVEMBER
1976 VOL. 6 NO. 5
THERIOGENOLOGY
Fig.
NOVEMBER1976
4.
Collection and settling bulb.
VOL.6
NO.5
483
A SINGLE CANNLJL& TECHNIQUE FOR NONSURGICAL COLLECTION OF OVA FROM CATTLE=
R. F. Roweb , M. R. Del Campob, C. L. Eilts', L. R. Frenchd, R. P. Winch= and 0. J. Gintherb
Departments of Veterinary Science and Animal Science University of Wisconsin-Madison Madison, Wisconsin 53706
Received for publication:
July 6, 1976
a Research supported by College of Agricultural and Life Sciences, University of Wisconsin-Madison, CBR Ova Transfer, Inc., Fennimore, Wisconsin, and Rock-A-Bye Farms, Richland Center, Wisconsin. b Department of Veterinary Science, University of Wisconsin-Madison, Wisconsin. Dr. Rowe is a post-doctoral trainee supported by the Wisconsin Animal Health Laboratory. ' CBR Ova Transfer, Inc., Fennimore, Wisconsin. d
Department of Meat and Animal Science, University of Wisconsin-Madison.
ACKNOWLEDGMENTS The authors thank American Breeders Service, Deforest, Wisconsin for a grant of semen and Upjohn Company, Kalamazoo Michigan for the gift of prostaglandin Fza. Excellent technical assistance was contributed by Mrs. J. Meschievitz, Mr. Max Carter and Mr. Tom Oettiker. Special thanks to Donna Van Horn for illustrations and graphics.
NOVEMBER
1976
VOL. 6 NO. 5
471
THERIOGENOLOGY
Abstract The uteri of 34 heifers were flushed for ova six to nine days following The technique estrus using a single cannula nonsurgical technique. involved the infusion of fluid by gravity and agitation within the U’CSKUS by to-and-fro action of a syringe followed by unassisted fluid collection. Each horn was flushed five times using 30-150 ml of flushing fluid per flush. Recovered fluid (flushing fluid plus uterine secretion) was an average of 95% of the volume of the fluid inserted. Ova were recovered from 12 of 19 nontreated, single ovulating heifers (63%) and from all of 15 super-ovulated heifers (mean and S.D. for number of ova, 6.3 ? 4.4/ superovulated heifer; range, 1 to 14 ova). Based on the number of corpora lutea, the ova recovery index was 54% as averaged over the 15 superovulated heifers. The technique has been used in 4 additional super-ovulated heifers with modification (increased number of flushes to 8) subsequent to the termination of the planned project. Recovery index for the first 5 flushes was 58%. However, some ova were recovered in the 6th, 7th, and 8th flushes resulting in an apparent improved recovery index of 69%. Introduction Past attempts for nonsurgical recovery of ova from cows (1,2,3,4,5) have utilized intricate techniques for intrauterine placement of multiple cannulae, but have met with limited succass. Graham (6) and Schultz (7) summarized the problems associated with previous attempts at nonsurgical ova collection as poor percent recovery of the fluid infused, low recovery percentages of available ova, handling and processing large amounts of flushing fluid and trauma to the uterus from passage of a catheter to the tip of the horn. Recovery rates have been far below that reported for surgical collections. The present project was undertaken to develop a simple, reliable method of nonsurgical ova collection. Materials and Methods Twenty-eight Holstein and 6 beef heifers were used (19 nontreated, single ovulating and 15 superovulated). Ten of the single ovulating heifers were bred and 9 were not. The superovulation regimen utilized a commercial pituitary 72 hours after the initial gonadotropin injection. preparation,e and PGF Heifers were insemina ga ed with two ampules of frozen semen at the beginning of standing estrus and every 12 hours until the termination of estrus. Most of the heifers were bred 3 times. Prior to collection, an epidural anesthetic was administered and the labia were washed and disinfected. No attempt was made to douch the vagina. Heifers were restrained in a squeeze chute to minimize movement. All collections were done 6 to 9 days after estrus. Numbers of corpora lutea in superovulated heifers were estimated both by rectal palpation and laparoscopy (8).
e Armour Std FSH-PR, Burns Bio-tech, Omaha, Neb.
472
NOVEMBER
1976
VOL. 6 NO. 5
THERIOGENOLOGY
The collection device (Fig. 1) consisted of a Foley catheterf (French No. 12 to 20 with 30 ml cuff, Fig. Z), stainless steel stylet (Fig. Z), teflon coated surgical tubing, 3 tubing clamps, 2 glass T's, 2 settling bulbsg (Fig. 4) one of which was equipped with a 2-holed rubber cork and glass tubing, and a 50 ml syringe. Associated equipment included a small diameter uterine infusion catheter (Luchens uterine infusion catheter, Fig. 2), ova examination cups, and flushing medium. All glassware and tubing which had the potential for contact with ova were siliconized with a commercial producth in an attempt to reduce the occurrence of adhering of ova to the equipment. The equipment was siliconised and sterilized prior to each use. A range of sizes of Foley catheters was kept on hand and the largest size which could be passed through the cervix was used. One of the settling bulbs was used as a media reservoir and the other for collection and settling. Several settling bulbs were kept on hand for repeated flushing. The Luchens catheter was passed through the cervix first in an attempt to facilitate subsequent passage of the Foley catheter. The ova examination cups were prepared by a glass-maker from 250 ml round-bottom flasks as shown (Fig. 3). The cups were cut from the flasks on the bias so that flaws present on the bottom of the flask would not be on the bottom or viewing area of the examination cups. The flushing medium was tissue culture medium (TCM)199i to which had been added 100 IU potassium penicillin G,I 100 mcg streptomycin sulfate,k and 1 mg bovine serum albumin1 per ml. The medium and glassware were warmed to body temperature prior to use. Throughout the collection procedure the reproductive tract was manipulated and monitored by rectal palpation. The steps of the collection procedure were as follows (Fig. 1): 1) passage into the uterus and subsequent removal of the Luchens catheter, 2) passage and placement of the Foley catheter with stylet into the caudal portion of one horn, 3) inflation of the cuff with air and removal of the stylet, 4) attachment of the remainder of the collection device except the collection settling bulb, 5) filling of reservoir with flushing media with clamp A attached, 6) removal of air from exterior portion of the tubing by clamping exterior portion of the Foley catheter and removal of clamp A with clamp B in place, followed by attachment of clamp C after the air was expelled, 7) removal of clamp from Foley catheter and infusion of media by gravity flow, 8) attachment of collection settling bulb, 9) attachment
f
BardexR, C.R. Bard Inc., Murray Hill, N. J.
g Corning GlassWorks, Corning N. Y. h SilicladR, Becton, Dickinson and Co., Parsippany, N. J. i Flow Laboratories, Rockville, Maryland ' GIBCO, Grand Island, N. Y. k Pfizer, Inc., New York, N. Y. 1 Sigma Chemical Co., St. Louis, Missouri.
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of clamp A and removal of clamp B, 10) agitation of the flushing fluid in the uterus by vigorous to-and-fro movement of syringe plunger, 11) immediate removal of clamp C and collection of fluid by gravity flow, 12) flushing of the tubing by removing clamp A, and 13) replacement of clamps A, B and C, refilling reservoir and repeating infusion collection steps. Single ovulating heifers were collected from the uterine horn ipsilateral to the CL and superovulated heifers were collected from both horns. The cuff of the Foley catheter was inflated with air until an obvious uterine distention occurred as determined by rectal palpation and the catheter seemed firmly anchored. Excessive insufflation, beyond that necessary for anchoring and preventing leakage of flushing medium, was avoided. Infusion of the medium was by gravity from a height of approximately 50 cm. This appeared to produce adequate distention of the horn. The degree of distention was monitored per rectum and the passage of fluid into the horn was readily detectable. Agitation of the fluid in the horn was done by the to-and-fro movement of the syringe plunger to favor dislodgement and movement of ova. Approximately 15-25% of the volume of fluid that had been infused was withdrawn and reinserted rapidly 3 times. Clamp C was removed immediately after the third emptying of the syringe. The flushing medium rapidly entered the settling bulb without manual evacuation of the horn. When indicated, evacuation of the fluid was assisted by gentle uterine massage. The fluid from each of the 5 flushes was collected into a separate settling bulb so that the effect of sequence of flushes could be evaluated. The flushing medium in each settling bulb was allowed to stand undisturbed at least 20 minutes. Fluid was then withdrawn from the valve at the bottom of the bulb, sequentially, in quantities of 30 to 40 ml per examination cup. For purposes of comparison of ova collection efficiency between nonsurgical and surgical techniques data accumulated in conjunction with surgical collection of ova at a professional ova transfer establishment were examined. The collections involved 29 heifers of the recently imported beef breeds (Maine-Anjou, Normandy, and Sirmnental). Collections were done by insertion and collection of flushing fluid from each horn through a midventral laparatomy as described by Rowson, et al (9). All heifers for which collections ware attempted over a period of time were included without selection bias. Results Two single ovulating heifers were replaced because the operator was unable to pass the catheter through the cervix and 1 superovulated heifer was eliminated due to metritis. Recovery data for ova and flushing fluids are shown for the nonsurgically collected single ovulating heifers (Table 1) and for the nonsurgically and surgically collected superovulated heifers (Table 2). ow recovery rates for the nonsurgical technique were 70% (bred, single ovulators), 56% (nonbred, single ovulators), and 54% (superovulated heifers). Recovery rate for the surgical technique in superovulated heifers was 58%. Fluid recovery rates for the nonsurgical technique were 96% (bred,
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single ovulators), 97% (nonbred, single ovulators), and 92% (superovulated heifers). In superovulated heifers the percent of recovered ova which were found in each of the 5 flushes were 31, 27, 17, 4 and 21, respectively. Discussion The recovery index used to evaluate the efficiency of ova recovery "as based on the number of corpora lutea (No. ova collected i No. corpora lutea x 100) and is therefore likely a censervative estimate. For example, the index does not take into account formation of corpora lutea from unovulated follicles, failure of pick-up of ova by the infundibulum, degeneration of ova beyond a recognizable stage by the time of flushing, and failure to find ova that were collected in the flushing fluid. Any of these events would lead to an underestimation of collection efficiency. Another source of error involves the estimation of number of corpora lutea. In the present series 11 of the superovulated heifers were examined both by rectal palpation and by laparoscopy. The overall means and standard deviations for the 2 techniques were quite close (rectal palpation, 13.0 i 4.1; laparoscopy 13.1 ? 4.3). HOWeVsl-, sometimes the estimate from one'method "as higher and sometimes lower than the other so that the average disagreement between methods "as 2.5 CL per CO". The correlation between methods "as +0.76. Failure to approach a correlation of 1 reflects the degree of disagreement between methods. On the assumption that laparoscopy "as more accurate it "as given preference in calculation of the recovery index (available in 11 of 15 superovulated cattle). It "as noted that even with close inspection during surgical collections there "as occasional disagreement between observers on CL numbers. Corpora lutea that were close together were not always clearly discernible. Selection bias "as eliminated by using a preplanned number of heifers for the non-surgical technique and by including all heifers which were submitted to surgery at a professional ova transfer establishment for the surgical technique. The recovery indices reported herein, therefore, are useful and reasonable and, if anything, are on the conservative side. It is emphasized that valid statistical comparisons between the nonsurgical and surgical techniques are not indicated, since the two techniques were done independently, at different locations, and by different operators. The comparisons must, therefore, be considered with some reservation. However, it should be noted that the difference in efficiency between techniques in superovulated cattle did not appear to be great (54% vs 58% for nonsurgical and surgical techniques, respectively). The single cannula technique "as used in 12 superovulated cattle under field conditions subsequent to the planned project. Flushes were combined and the number of flushes "as not carefully monitored, but "as estimated to be 4-6/born. The recovery index was 59% which is quite comparable to the 54% obtained in the planned project and the 58% for surgical recovery. The results for other nonsurgical techniques were reviewed by Foote and Onuma (10). It is impossible to make critical comparisonsbetween previously reported techniques and the one used herein as most of the data on efficiency of ova collection are incomplete. Often the data are reported as successful
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attempts (defined as collection of at least one ovum from an individual) and do not indicate whether single ovulating or superovulated animals were used. Dziuk, et al. (4) estimated the number of potential collectable ova by rectal palpation of corpora lutea in 34 superovulated cattle. An average of 0.9 ova/cow was recovered with a recovery index of 10% (calculated by the present authors). From 50 to 1000 ml of fluid was forced into the uterus and withdrawn by negative pressure and 77% of the fluid was recovered. Sugie (5), using a device for continuous flushing (double cannulae), reported recovery of ova from 81% of 32 superovulated cattle and an average of 5.1 ova recovered per animal. However, it is not clear whether the mean number of recovered ova includes cattle from which no ova were obtained. Corresponding results for the present technique in superovulated cattle were 100% and 6.3 ova, respectively, but information needed for further comparisons are not available. Although not critically studied, there has been no suggestion based on limited numbers of transfers to recipients that the present nonsurgical technique was detrimental to the collected ova. The percent of recovered ova from superovulated cattle that appeared to be fertilized was 65% for the nonsurgical technique and 64% for the surgical technique. One of the initial concerns in development of techniques for ova recovery was the possibility that the flushing medium and, therefore, the ova would be lost through the oviducts. In the present project, there was no indication that the tubouterine junction opened. More than 95% of the total volume of infused medium was recovered. Occasional leakage occurred around the cuff due to faulty inflation or partial deflation of the balloon, however, this was not a serious problem. Conversely, over distention of the cuff was noted to cause occlusion of the lumen of the catheter resulting in reduced flow rate. No attempt was made to critically evaluate the importance of various steps in the procedure and it is likely that modifications can be made to adjust to various field conditions. Preliminary trials were done using 4 technique modifications: 1) agitation with gravity collection as described herein, 2) gravity collection without agitation, 3) agitation with collection by aspiration of air from the collecting bulb, and 4) collection by aspiration without agitation. Gravity collection with agitation seemed the most effective of the modifications, but ova were obtained with all 4 procedures. The number of animals was too small for critical evaluation. The usefulness of the Luchens catheter to facilitate subsequent passage of the Foley catheter was not tested. As experience with the technique progressed, the step involving the Luchens catheter was omitted and is currently being used only when difficulty occurs in passing the Foley catheter. It should be noted that previous workers have reported that the cervix of diestrus cattle (day 5) may be dilated with relaxin (11). Perhaps, therefore, relaxin should be tested as an aid in cattle in which the catheter cannot be passed. Experience is an important factor in passage of the catheter and in manipulating and placing the end of the catheter into each of the uterine horns. In this regard, the effectiveness of the epidural anesthesia seemed important. Inadequate anesthesia may result in rectal and abdominal contractions which hamper manipulative efforts and may prevent adequate filling or distention of the uterine horns. The authors believe that -in utero agitation of the fluid is an important aspect of the
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procedure but acknowledge that this has not been tested. While the fluid is agitated with the syringe, the surging and receding force of the medium can be felt, per rectum, throughout the horn. Agitation of the flushing medium within the horns could be done by manually induced contractions of the horn per rectum. In the authors' opinion, however, the use of the syringe provides better control and may reduce the possibilities of injury to the endometrium. The time course of the entire collection procedure in superovulated cattle was approximately 35 minutes from insertion of the Foley catheter into the first horn to withdrawal of the Foley catheter from the second horn. From the time clamp A was removed to begin the infusion of one flush until the clamp was again removed for the next flush was approximately 3 minutes. Use of settling bulbs (Fig. 4) appeared to be an especially useful innovation for rapid detection of ova in the flushing fluid. Examination of the literature indicates Dracy and Peterson (3) used a French separatory funnel for processing large quantities of fluid, but no data on the effectiveness of the funnel were given. In the present study, all ova except two that were degenerated were found in the 30 to 40 ml of fluid which was first drawn through the valve at the bottom of the bulb (contents of first examination cup). The settling bulbs can be also be used to process fluids used to rinse the glassware for possible adhering ova or to reprocess a series of cups that have already been examined. It is recommended that the use of the single cannula ova collection technique on a professional basis be preceded by practice. No attempt has been made by us or others to critically evaluate the possibilities for deleterious effects on the donor animal of nonsurgical ova collection techniques. Noticeable ruptures or loss of fluid into the uterine wall or broad ligament did not occur in the present study. However, during preliminary developmental trials, 2 ruptures occurred. Enlargement of the uterus occurred during infusion but the fluid could not be evacuated and was believed to have entered the uterine wall. Subsequent to the planned project complete penetration of the uterine wall occurred in one cow and flushing fluid was lost into the abdominal cavity. Rupture or trauma to the uterus is most likely to occur during placement of the catheter into the horn and during insufflation of the cuff. In approximately 10% of the collections, red blood cells appeared in the medium indicating uterine trauma. Since the red cells remained suspended in the flushing fluid, detection of ova was quite difficult. The presence of epithelial cells, mucus, and debris of unknown origin indicated that thorough agitation occurred. In many instances, ova were found in flushings which had the most debris. A time-saving step that can be used in the field is to combine more than one flush into each settling bulb. In the present work flushes were kept separate to evaluate the effectiveness of repeated flushing. The percent of recovered ova which were found in each of the 5 flushes was 31, 27, 17, 4 and 21, respective!lY Thus, the last flush produced a large number of ova. ( It is likely, therefore, that a greater number of flushes would have increased the recovery index. Subsequent to the completion of the present project, the single cannula technique has been used on 4 superovulated cattle with the modification that 8 instead of 5 flushes were used for each horn. The ova recovery index for the first 5 flushes was 58% which corresponds to 54% for the primary project. However, the index for all 8 flushes was 69%. Six, 6 and 3% of the recovered ova were in the 6th, 7th and 8th flushes, respectively. Thus, it appears that the recovery index of 54% obtained in the primary project can be increased, by increasing the number of flushes.
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References 1. Rowson, L. E. A. and D. F. Dowling. of fertilized eggs from the living cow.
An apparatus for the extraction Vet. Record 61: 191 (1949)
2. Dowling, D. F. Problems of the transplantation of fertilized ova. J. Agr. Sci. (England) x: 374 (1949) 3. Dracy, A. E. and W. E. Peterson. Isolation of ova from the living bovine. J. Dairy Sci. 2: 797 (1950) 4. Dziuk, P. J., J. D. Donker, J. R. Nichols and W. E. Peterson. Problems associated with the transfer of ova between cattle. Minn. Agr. Exp. Sta. Tech. Bull. 222 (1958) 5. Sugie, T. Non surgical ova collection in cattle (Jap.). Ann. Report Nat. Inst. An. Ind., Chiba-shi, Japan 1968. 8: 55 (1970) 6. Graham, E. F. Ova Transfer. Proceedings Fifth Tech. Conf. on Art. Insem. and Reprod. Nat. Ass. of An. Breeders (1975) 7. Schultz, R. H. Techniques and problems associated with collection and transfer of bovine ova. Annual Meeting of Am. Vet. Sot. for Study of Breeding Soundness. Columbia, Missouri, Sept. 6-7, 1974
a. Wishart, D. F. and J. B. Snowball. of the ovary in situ.
Vet:Rec.
Endoscopy in cattle: Observation 92: 139-143 (1973)
9. Rowson, L. E. A., R. M. Moor, and R. A. S. Lawson. Fertility following egg tranfer in the cow; effect of method, medium and synchronization of estrus. J. Reprod. Fert. 18: 517 (1969) 10. Foote, R. H. and H. Onuma. Superovulation, ovum collection, culture and transfer. A review. J. Dairy Sci. 53: 1681 (1970) 11. Graham, E. F. and A. E. Dracy. The effect of relaxin and mechanical dilation on the bovine cervix. J. Dairy Sci., 36: 772 (1953)
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9
10
Bred
Non Bred
No. heifers
Group
480
478
56
k172
+156
96
97
265-705
250-700 k3
+3
90-100
90-100
40
43
Fluid collected/heifer (%I 1st Mean SD Range
Recovery Rates for Ova and Flushing Fluid From Single Ovulating Heifers
Fluid infused/ heifer (ml) Range Mean SD
70
% from which ovum was recovered
Table 1.
20
43
40
0
0
14
0
0
% of recovered ova in each flush 2nd 5th 3rd 4th
1
en
8
o\
r
6
-&
z T;:
E
54
933
92
Index of recovery of ova/ heifer (%)c
Fluid inserted/heifer (ml)
Fluid recovered/heifer (W)
C
b
4 21
17
of heifers in parenthesis.
flush flush flush flush flush
k31 +20 +23 +8 f22
k6
?280
k29
24.9
t4.4
S.D.
9.9
l-22
o-22 O-67
O-75
O-100 O-60
78-99
543-1390
58
6.2
1-14
13-100
Mean
Range
229
k6.3
k5.5
S.D.
No. ova recovered G estimated no. corpora lutea x 100.
O-100
l-29
o-21
Range
Surgical recovery (29)
No. of corpora lutea estimated by laparoscopy in 11 heifers and by rectal palpation in 4 heifers for nonsurgical recovery and by visual inspection for surgical recovery.
a No.
1st 2nd 3rd 4th 5th
31 27
12.2
No. corpora lutea/heiferb
% of recovered ova/heifer which were in:
6.3
Mean
Nonsurgical recovery (15)a
Recovery Rates for Ova and Flushing Fluid from Superovulated Heifers
No. ova recovered/heifer
Item
Table 2.
a
!G
$!
a
!
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Luchens
Fig. 2.
Fig. 3.
4.82
cat hetsr
Foley catheter and companion instruments
Method for preparation of ova examination cup from a round bottom flask.
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Fig.
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4.
Collection and settling bulb.
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