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A device for nonsurgical transfer of bovine embryos and its effect on uterine contamination
THERIOGENOLOGY
A DEVICE FOR NONSURGICAL TRANSFER OF BOVINE EMBRYOS AND ITS EFFECT ON UTERINE CONTAMINATION A. Branda, M. Drostb, M.H. Aartsa and J.W. Gunninka aClinic for Veterinary Obstetrics, Gynecology and A.I. State University at Utrecht, The Netherlands b
Department of Reproduction, School of Veterinary Medicine University of California, Davis, California 95616 Received for publication:
August 2, 1976
ABSTRACT A simple, inexpensive, concentric cannula device is described for the transfer of embryos through the cervix in cattle. Its effectiveness in minimizing the introduction of contamination into the uterus was tested by bacteriological surveillance of the various components after use, and of the uterus of 12 cows slaughtered 2 - 3 days after sham transfer. The results of the bacteriological examination of the outer cannula, middle cannula and inner catheter were positive in lOD%, 66% and 50% respectively. Bacteria were always found in the samples obtained from the vulva and vagina, but in only 55% of those taken from the external OS of the cervix. Pregnancy resulted in 5/1D cows that were inseminated and subsequently subjected to a sham transfer to the horn contralateral to the corpus luteum. INTRODUCTION Uterine infection and ejection of the ova were believed to be major factors in the lack of success during early attempts at nonsurgical transfer of bovine embryos through the cervix. Several investigators (1,8,12) have subsequently succeeded in obtaining variable pregnancy rates in cattle using nonsurgical transfer techniques. The pregnancy rate after nonsurgical transfer via the cervix is generally lower than that reported after surgical transfer. In an effort to approximate the asepsis achieved during surgical transfer procedures, a nonsurgical transfer device was designed which would minimize the introduction of contamination into the uterus. The uterus is more susceptible to infection during the luteal phase than during the follicular phase (3,5,10). This paper describes a simple transfer device which is atraumatic, easy to sterilize and inexpensive. Results are presented of the bacteriological examination of the various components of the device after its use, as well as the uterus of 12 cows slaughtered 48-72 hr after nonsurgical sham embryo transfer.
NOVEMBER
1976
VOL. 6 NO. 5
509
THERIOGENOLOGY
MATERIALS AND METHODS The nonsurgicaltransfer device consists of two concentric, stainless steel cannulas, 6 and 3 mm in diameter, and an inner flexible urethral catheter (RUschelit ureteric catheter, Willy Rtisch,Stuttgart, West Germany), 1.65 or 1.98 mm in diameter, which are 35, 45 and 70 cm long, respectively). The outer cannula is covered at one end with a piece of nonabsorbent paper prior to autoclaving. An epidural injection of 4-6 ml 2% xylocaine is given to suspend defecation and prevent straining. The vulva of the recipient is cleaned with 70% alcohol. The labia are parted and the outer two sterile cannulas, one inside the other, are introduced into the vagina and 1 - 2 cm into the external OS of the cervix. The second cannula is then pushed through the protective paper cover and extended 3 - 4 cm into the horn ipsilateral to the corpus luteum per rectal guidance. Next, the flexible catheter is introduced, using sterile technique, into the middle cannula and advanced as far as possible into the uterine horn. Its passage is facilitated by gentle undulating movements of the horn as it is manipulated per rectum. The embryo contained in 0.5 ml medium is deposited in the middle of the uterine horn via the flexible catheter. After this, the catheter is flushed with an additional 0.5 ml of medium. The content of the catheter is 0.5 ml. Ten virgin heifers with normal estrous cycles were selected from an experimental herd for bacteriological examination of the vulva, vagina and external OS of the cervix. Ten lactating cows (3-6 yr old) were selected at the slaughterhouse for the same purpose. The vulva was sampled with a sterile cotton swab. After parting the lips of the vulva as far as possible, the vagina was sampled by introduction of a sterile cotton swab. A sterile tube speculum (diameter 4-5 cm) with a built-in light source was introduced into the vagina and kept in position just in front of the cervix to obtain a sample from the external OS of the cervix with a third swab. The samples were transported to the laboratory in sterile tubes. Nonsurgical sham embryo transfers were performed on a farm in 12 cycling, lactating cows (3-7 yr old) which were slaughtered 48-72 hr later, The uteri of animals which were slaughtered, were flamed locally with a Bunsen burner and incised. A sample was taken with a bacteriological loop from each uterine horn, the body of the uterus and the cervix. Cultures were prepared as soon as possible after collection by inoculation of horse blood agar, serum broth and liver broth. Aerobic and anaerobic incubation were used. A group of 10 lactating cows (3-8 yr old) were inseminated and a nonsurgical sham transfer was made to the horn contralateral to the corpus luteum, 6-10 days after estrus. Subsequently, the three components of the device were examined bacteriologically imnediately upon withdrawal from the genital tract.
510
NOVEMBER
1976
VOL. 6 NO. 5
THERIOGENOLOGY
RESULTS Bacteriawere always found in the samples obtained from the vulva and vagina but In only 55% of those taken from the external OS of the
cervix of the IO virgin heifers and 10 lactating cows. Streptococci and Bacillus species were the most cornnonbacteria found In the tract, fol'lowed numbers by staphylococci, stre tococci E. coli and C while diphtheroids were seen ln only +----fa ew an maim greatest num er of isolates, as well as the largest number of colonies, were recovered from the vulva, and the smallest number of isolates and colonies from the cervix. The results of the bacteriological examination of the instruments used for nonsurgical sham embryo transfer and of the uteri of recipient animals slaughtered 48-72 hr after sham transfer, are shown in Table I. The results of the bacteriological examination of the outer cannula, middle cannula and inner catheter were positive in 12/12 (lOO%), 8/12 (66%) and 6/12 (50%) respectively. Five of 10 cows which were inseminated and in which a subsequent contralateral sham transfer was done became pregnant. Bacteria were recovered from the middle cannula and the inner flexible catheter of one of the cows which conceived and from the middle cannula only from a second cow which conceived. Bacteriological findings were negative for the middle and inner catheter used in the remaining 8 cows. DISCUSSION The bacteriological findings in the vulva, vagina and external OS of the cervix are comparable to those reported in the literature (2,6, 7,73) with a decrease in the number of isolates in moving from the vulva to the cervix. A similar decrease in number of isolates could be observed from the outer cannula to the inner catheter. Contamination of the third catheter occurred in only 50% of the cases and, when contamination did take place, fewer than 10 colonies were cultured. In contrast, more than 10 colonies were cultured from 60% of the isolates from the outer cannula. Thus, the use of a set of three concentric cannulas offers protection against contamination of the uterus by potential pathogenic bacteria from the vulva, vagina and cervix. Biopsies taken after inoculation showed that the bacteria and/or trauma caused only a slight reaction of the endometrium characterized by small accumulations of inflam matory cells (6). The endometrium returned to normal in 5-6 days. This emphasizes the possibility that bacteria, or trauma inflicted upon the endometrium, may alter the intrauterine environment and interfere with conception without necessarily producing clinically detectable endometritis. At slaughter, bacteria were found in the uterus of 2/12 cows to which a nonsurgical sham transfer had been made. These bacteria could have entered the uterus with vaginal fluids upon relaxation of the cervix after slaughter when the animals were suspended by the hindlegs (639).
NOVEMBER
1976
VOL.
6 NO. 5
511
THERIOGENOLOGY
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NOVEMBER
1976
VOL. 6 NO. 5
THERIOGENOLOGY Griffin et al., (4) using a similar set of concentrictubes for sampling the bane uterus, found that the sampling procedureyielded uncontaminatedsamples and that the technfquedid not lead to colonization of the uterus by exogenous bacteria, even when cows were sampled repeatedlyduring the luteal phase of the estrous cycle. Pregnancy resulted in 5/10 cows that were inseminated and subsequently subjected to a sham transfer to the horn contralateral to the corpus luteum. This is in agreement with the findings of Seidel et al., (11) who performed sham transfers to the horn ipsilateral to the corpus luteum using the same approach. Contamination of the uterus after nonsurgical embryo transfer using three concentric cannulas does not appear to be a major cause of the reduced pregnancy rates reported (8,lZ). It is possible that technical failures, such as lack of experience, are of greater importance. Trauma of the endometrium in combination with an induced leucocytosis, or a disturbance of the endocrinological and biochemical environmentof the uterus may also play a role. REFERENCES
1.
Brand, A., Taverne, M. A. M., Van der Weyden, G. C., Aarts, M. H., Dieleman, S. J., Fontijne, P., Drost, M., and de Bois, C. H. W. Non-surgical embryo transfer in cattle. I. Myometrial activity as a possible cause of embryo expulsion. Proc. Agric. Res. Seminar: Egg Transfer in Cattle, Commission European Communities, EUR 5491: 41-56 (1976).
2.
Gibbons, W. J., Attleberger, M. H., Kiesel, G. K., and Dacres, W. G. The bacteriology of the cervical mucus of cattle. Cornell Vet. 9: 255-264 (1958).
3.
Gunnink, 3. W. Een onderzoek naar het afweermechanisme van de uterus. Thesis, University of Utrecht (1973).
4.
Griffin, J. F. T., Murphy, J. A., Nunn, W. R., and Hartigan, P. J. Repetitive in vivo sampling of the bovine uterus under field conditions. Br. Vet. J. 130: 259-264 (1974).
5.
Hawk, H. W., Brinsfield, T. H., Turner, G. D., Whitmore, G. W., and Norcross, M. A. Effect of ovarian status on induced acute inflammatory responses in cattle uteri. Am. J. Vet. Res. 25: 362366 (1964).
6.
Kampelmacher, E. H. Een orienterend onderzoek omtrent de microbiologie en histologie van de uterus bij onvruchtbare runderen met behulp van een biopsieapparaat. Thesis, University of Utrecht (1954).
7.
Khalil, 0. La microflore ginitale chez la vache a l'etat normal et pathologique. Thesis, Lyon (1970).
a.
Lawson, R. A. S., Rowson, L. E. 4., Moor, R. Y., and Tervit, R. M. Experiments on egg transfer in the cow and ewe: Dependence of conception rate on the transfer procedure and stage of the oestrous cycle. J. Reprod. Fert. 45: 101-107 (1975).
NOVEMBER 1976 VOL. 6 NO. 5
513
THJZRIOGENOLOGY
9.
514
Nakamura, R.M., Miyahara, A.Y., Koshi, J.H., Olbrich, S.E., Change, E., Buntain, B., Kondo, R., and Wakatsuki J. W-112 Regional research project, Annual Report, Reproductive Performance in Beef Cattle and Sheep. University of Hawaii (1975).
10.
Rowson, L.E.A., Lamming, G.E., and Fry, R.M. The relationship between ovarian hormones and uterine infection. Vet. Rec. 65: 355 (1953).
11.
Seidel, G.E., Jr., Bowen, J.M., Homan, N.R., and Okun, N.R. Fertility of heifers with sham embryo transfer through the cervix. Vet. Rec. 97: 307-308 (1975).
12.
Sreenan, J.M. Successful nonsurgical transfer of fertilised cow eggs. Vet. Rec. 96: 490-491 (1975).
13.
Tdubrich, F. Die Bakteriinflora der Vagina des gesunden Rindes unter BerUcksichtigung des Geschlechtszyklus, der Trdcht igkeitphase, des Alters und des pH-Wertes. Zbl. Vet. med. 5(4 ): 373389 (1958).
NOVEMBER
1976 VOL. 6 NO. 5
A DEVICE FOR NONSURGICAL TRANSFER OF BOVINE EMBRYOS AND ITS EFFECT ON UTERINE CONTAMINATION A. Branda, M. Drostb, M.H. Aartsa and J.W. Gunninka aClinic for Veterinary Obstetrics, Gynecology and A.I. State University at Utrecht, The Netherlands b
Department of Reproduction, School of Veterinary Medicine University of California, Davis, California 95616 Received for publication:
August 2, 1976
ABSTRACT A simple, inexpensive, concentric cannula device is described for the transfer of embryos through the cervix in cattle. Its effectiveness in minimizing the introduction of contamination into the uterus was tested by bacteriological surveillance of the various components after use, and of the uterus of 12 cows slaughtered 2 - 3 days after sham transfer. The results of the bacteriological examination of the outer cannula, middle cannula and inner catheter were positive in lOD%, 66% and 50% respectively. Bacteria were always found in the samples obtained from the vulva and vagina, but in only 55% of those taken from the external OS of the cervix. Pregnancy resulted in 5/1D cows that were inseminated and subsequently subjected to a sham transfer to the horn contralateral to the corpus luteum. INTRODUCTION Uterine infection and ejection of the ova were believed to be major factors in the lack of success during early attempts at nonsurgical transfer of bovine embryos through the cervix. Several investigators (1,8,12) have subsequently succeeded in obtaining variable pregnancy rates in cattle using nonsurgical transfer techniques. The pregnancy rate after nonsurgical transfer via the cervix is generally lower than that reported after surgical transfer. In an effort to approximate the asepsis achieved during surgical transfer procedures, a nonsurgical transfer device was designed which would minimize the introduction of contamination into the uterus. The uterus is more susceptible to infection during the luteal phase than during the follicular phase (3,5,10). This paper describes a simple transfer device which is atraumatic, easy to sterilize and inexpensive. Results are presented of the bacteriological examination of the various components of the device after its use, as well as the uterus of 12 cows slaughtered 48-72 hr after nonsurgical sham embryo transfer.
NOVEMBER
1976
VOL. 6 NO. 5
509
THERIOGENOLOGY
MATERIALS AND METHODS The nonsurgicaltransfer device consists of two concentric, stainless steel cannulas, 6 and 3 mm in diameter, and an inner flexible urethral catheter (RUschelit ureteric catheter, Willy Rtisch,Stuttgart, West Germany), 1.65 or 1.98 mm in diameter, which are 35, 45 and 70 cm long, respectively). The outer cannula is covered at one end with a piece of nonabsorbent paper prior to autoclaving. An epidural injection of 4-6 ml 2% xylocaine is given to suspend defecation and prevent straining. The vulva of the recipient is cleaned with 70% alcohol. The labia are parted and the outer two sterile cannulas, one inside the other, are introduced into the vagina and 1 - 2 cm into the external OS of the cervix. The second cannula is then pushed through the protective paper cover and extended 3 - 4 cm into the horn ipsilateral to the corpus luteum per rectal guidance. Next, the flexible catheter is introduced, using sterile technique, into the middle cannula and advanced as far as possible into the uterine horn. Its passage is facilitated by gentle undulating movements of the horn as it is manipulated per rectum. The embryo contained in 0.5 ml medium is deposited in the middle of the uterine horn via the flexible catheter. After this, the catheter is flushed with an additional 0.5 ml of medium. The content of the catheter is 0.5 ml. Ten virgin heifers with normal estrous cycles were selected from an experimental herd for bacteriological examination of the vulva, vagina and external OS of the cervix. Ten lactating cows (3-6 yr old) were selected at the slaughterhouse for the same purpose. The vulva was sampled with a sterile cotton swab. After parting the lips of the vulva as far as possible, the vagina was sampled by introduction of a sterile cotton swab. A sterile tube speculum (diameter 4-5 cm) with a built-in light source was introduced into the vagina and kept in position just in front of the cervix to obtain a sample from the external OS of the cervix with a third swab. The samples were transported to the laboratory in sterile tubes. Nonsurgical sham embryo transfers were performed on a farm in 12 cycling, lactating cows (3-7 yr old) which were slaughtered 48-72 hr later, The uteri of animals which were slaughtered, were flamed locally with a Bunsen burner and incised. A sample was taken with a bacteriological loop from each uterine horn, the body of the uterus and the cervix. Cultures were prepared as soon as possible after collection by inoculation of horse blood agar, serum broth and liver broth. Aerobic and anaerobic incubation were used. A group of 10 lactating cows (3-8 yr old) were inseminated and a nonsurgical sham transfer was made to the horn contralateral to the corpus luteum, 6-10 days after estrus. Subsequently, the three components of the device were examined bacteriologically imnediately upon withdrawal from the genital tract.
510
NOVEMBER
1976
VOL. 6 NO. 5
THERIOGENOLOGY
RESULTS Bacteriawere always found in the samples obtained from the vulva and vagina but In only 55% of those taken from the external OS of the
cervix of the IO virgin heifers and 10 lactating cows. Streptococci and Bacillus species were the most cornnonbacteria found In the tract, fol'lowed numbers by staphylococci, stre tococci E. coli and C while diphtheroids were seen ln only +----fa ew an maim greatest num er of isolates, as well as the largest number of colonies, were recovered from the vulva, and the smallest number of isolates and colonies from the cervix. The results of the bacteriological examination of the instruments used for nonsurgical sham embryo transfer and of the uteri of recipient animals slaughtered 48-72 hr after sham transfer, are shown in Table I. The results of the bacteriological examination of the outer cannula, middle cannula and inner catheter were positive in 12/12 (lOO%), 8/12 (66%) and 6/12 (50%) respectively. Five of 10 cows which were inseminated and in which a subsequent contralateral sham transfer was done became pregnant. Bacteria were recovered from the middle cannula and the inner flexible catheter of one of the cows which conceived and from the middle cannula only from a second cow which conceived. Bacteriological findings were negative for the middle and inner catheter used in the remaining 8 cows. DISCUSSION The bacteriological findings in the vulva, vagina and external OS of the cervix are comparable to those reported in the literature (2,6, 7,73) with a decrease in the number of isolates in moving from the vulva to the cervix. A similar decrease in number of isolates could be observed from the outer cannula to the inner catheter. Contamination of the third catheter occurred in only 50% of the cases and, when contamination did take place, fewer than 10 colonies were cultured. In contrast, more than 10 colonies were cultured from 60% of the isolates from the outer cannula. Thus, the use of a set of three concentric cannulas offers protection against contamination of the uterus by potential pathogenic bacteria from the vulva, vagina and cervix. Biopsies taken after inoculation showed that the bacteria and/or trauma caused only a slight reaction of the endometrium characterized by small accumulations of inflam matory cells (6). The endometrium returned to normal in 5-6 days. This emphasizes the possibility that bacteria, or trauma inflicted upon the endometrium, may alter the intrauterine environment and interfere with conception without necessarily producing clinically detectable endometritis. At slaughter, bacteria were found in the uterus of 2/12 cows to which a nonsurgical sham transfer had been made. These bacteria could have entered the uterus with vaginal fluids upon relaxation of the cervix after slaughter when the animals were suspended by the hindlegs (639).
NOVEMBER
1976
VOL.
6 NO. 5
511
THERIOGENOLOGY
/L
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-
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-
-
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ID
u)
-
2
L
al
.,E f
-
512
27
-
z
m
(D
-
-
NOVEMBER
1976
VOL. 6 NO. 5
THERIOGENOLOGY Griffin et al., (4) using a similar set of concentrictubes for sampling the bane uterus, found that the sampling procedureyielded uncontaminatedsamples and that the technfquedid not lead to colonization of the uterus by exogenous bacteria, even when cows were sampled repeatedlyduring the luteal phase of the estrous cycle. Pregnancy resulted in 5/10 cows that were inseminated and subsequently subjected to a sham transfer to the horn contralateral to the corpus luteum. This is in agreement with the findings of Seidel et al., (11) who performed sham transfers to the horn ipsilateral to the corpus luteum using the same approach. Contamination of the uterus after nonsurgical embryo transfer using three concentric cannulas does not appear to be a major cause of the reduced pregnancy rates reported (8,lZ). It is possible that technical failures, such as lack of experience, are of greater importance. Trauma of the endometrium in combination with an induced leucocytosis, or a disturbance of the endocrinological and biochemical environmentof the uterus may also play a role. REFERENCES
1.
Brand, A., Taverne, M. A. M., Van der Weyden, G. C., Aarts, M. H., Dieleman, S. J., Fontijne, P., Drost, M., and de Bois, C. H. W. Non-surgical embryo transfer in cattle. I. Myometrial activity as a possible cause of embryo expulsion. Proc. Agric. Res. Seminar: Egg Transfer in Cattle, Commission European Communities, EUR 5491: 41-56 (1976).
2.
Gibbons, W. J., Attleberger, M. H., Kiesel, G. K., and Dacres, W. G. The bacteriology of the cervical mucus of cattle. Cornell Vet. 9: 255-264 (1958).
3.
Gunnink, 3. W. Een onderzoek naar het afweermechanisme van de uterus. Thesis, University of Utrecht (1973).
4.
Griffin, J. F. T., Murphy, J. A., Nunn, W. R., and Hartigan, P. J. Repetitive in vivo sampling of the bovine uterus under field conditions. Br. Vet. J. 130: 259-264 (1974).
5.
Hawk, H. W., Brinsfield, T. H., Turner, G. D., Whitmore, G. W., and Norcross, M. A. Effect of ovarian status on induced acute inflammatory responses in cattle uteri. Am. J. Vet. Res. 25: 362366 (1964).
6.
Kampelmacher, E. H. Een orienterend onderzoek omtrent de microbiologie en histologie van de uterus bij onvruchtbare runderen met behulp van een biopsieapparaat. Thesis, University of Utrecht (1954).
7.
Khalil, 0. La microflore ginitale chez la vache a l'etat normal et pathologique. Thesis, Lyon (1970).
a.
Lawson, R. A. S., Rowson, L. E. 4., Moor, R. Y., and Tervit, R. M. Experiments on egg transfer in the cow and ewe: Dependence of conception rate on the transfer procedure and stage of the oestrous cycle. J. Reprod. Fert. 45: 101-107 (1975).
NOVEMBER 1976 VOL. 6 NO. 5
513
THJZRIOGENOLOGY
9.
514
Nakamura, R.M., Miyahara, A.Y., Koshi, J.H., Olbrich, S.E., Change, E., Buntain, B., Kondo, R., and Wakatsuki J. W-112 Regional research project, Annual Report, Reproductive Performance in Beef Cattle and Sheep. University of Hawaii (1975).
10.
Rowson, L.E.A., Lamming, G.E., and Fry, R.M. The relationship between ovarian hormones and uterine infection. Vet. Rec. 65: 355 (1953).
11.
Seidel, G.E., Jr., Bowen, J.M., Homan, N.R., and Okun, N.R. Fertility of heifers with sham embryo transfer through the cervix. Vet. Rec. 97: 307-308 (1975).
12.
Sreenan, J.M. Successful nonsurgical transfer of fertilised cow eggs. Vet. Rec. 96: 490-491 (1975).
13.
Tdubrich, F. Die Bakteriinflora der Vagina des gesunden Rindes unter BerUcksichtigung des Geschlechtszyklus, der Trdcht igkeitphase, des Alters und des pH-Wertes. Zbl. Vet. med. 5(4 ): 373389 (1958).
NOVEMBER
1976 VOL. 6 NO. 5