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A new alternative for embryo transfer and artificial insemination in mares: ultrasound-guided intrauterine injection
Veterinary Review A New Alternative for Embryo Transfer and Artificial Insemination in Mares: Ultrasound-Guided Intrauterine Injection L. A. Silva, DVM,a,c E. L. Gastal, DVM,a,c M. O. Gastal, DVM,b,c J. C. F. Jacob, DVM,b C. P. Reis, DVM,a and O. J. Ginther, VMD,c
SUMMARY
A transvaginal ultrasound-guided intrauterine injection (IUI) technique was developed for embryo transfer and for injection of small quantities of sperm in mares. The target area of a horn was positioned by transrectal manipulation against the wall of the vaginal fornix over the face of a transvaginal transducer. A needle with a catheter containing the embryo or semen was inserted through the needle guide of the transducer into the uterine lumen. The tips of the needle and catheter, the movement of the catheter in the uterine lumen, and the ejection of fluid was monitored on the ultrasound screen. Pregnancy rate 15 days after ovulation for the IUI embryo transfer technique (30/39, 77%) was similar to the pregnancy rate for transcervical (TC) embryo transfer (30/38, 79%). The pregnancy rate for IUI insemination of 20 × 106 progressively motile sperm into the tip of the uterine horn ipsilateral to ovulation was 5/10 (50%). Results indicated that the IUI approach is a viable alternative for embryo transfer. Results also supported the potential of IUI for insemination of low numbers of sperm, but more extensive studies with various doses of sperm are needed.
Part of these data were presented at the 8th International Symposium on Equine Reproduction, Fort Collins, Colorado, July 2002, and reported in an abstract of the proceedings. From the Departments of Veterinary Sciencea and Animal Science,b Federal University of Viçosa, Brazil; and the Eutheria Foundation,c Cross Plains, Wisconsin. E. L. and M. O. Gastal are on leave from the Departments of Veterinary Science and Animal Science, respectively, Federal University of Viçosa, Brazil. Reprint requests: O. J. Ginther, Eutheria Foundation, 4343 Garfoot Rd, Cross Plains, WI 53528, USA. 0737-0806/$ - see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2004.07.006
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Keywords: Ultrasound-guided; Artificial insemination; Mares
Embryo
transfer;
INTRODUCTION
Embryo transfer and artificial insemination are powerful techniques for increasing reproductive efficiency and genetic gain in horses. Currently, equine embryos are being transferred transcervically or surgically through a flank incision.1 Reported pregnancy rates for TC transfers have ranged from 26% to 83%.2-7 The large variability in pregnancy rates can be related1 to such factors as operator experience for depositing the embryo into the uterus, expulsion of the embryo from the uterus, introduction of contaminants through the cervix, and manipulation of the genital tract, thereby stimulating uterine contractions by the production of prostaglandin F2a (PGF2a) 8,9 and oxytocin.8,10 Pregnancy rates after surgical embryo transfer through a flank incision have been more consistent among reports, ranging from 65% to 80%.1,3-6,11,12 However, the surgical method is an invasive procedure requiring time, equipment, medicines, and facilities, all of which increase the cost and restrict its application. Alternative nonsurgical methods that bypass the cervix have been tried for embryo transfer in mares to avoid or minimize cervical manipulations. Resulting pregnancy rates have been low, ranging from 33% to 40%.5,13,14 These nonsurgical methods used the transvaginal route for IUI aided by a 3-channel apparatus previously used in cattle,13 a colpotomy,14 or a tubular speculum.5 The authors of the speculum approach expressed difficulty in confirming the entry of the needle and plastic tube into the uterine lumen. A transvaginal ultrasound-guided IUI technique has been developed in mares and is efficient (96%) for delivering solutions into the uterine lumen and allowing constant monitoring by ultrasound.15 The authors suggested
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that with refinement, this technique could be developed for embryo transfer in mares. The technique avoids cervical entry and therefore may reduce the variability in pregnancy rates. Subsequently, an in vitro study16 evaluated the efficiency of ultrasound-guided IUI, using genital tracts from a slaughterhouse. Good success rates (88%-90%) were found for insertion of the needle and plastic tube into the uterine lumen and for delivering vehicle into the uterus. A high certainty score of 4 was associated with more successful injections (90%) than a certainty score of 3 (68%). The result of this study demonstrated that scoring the procedure from the ultrasound images is useful for assessing efficiency of the IUI. Alternative artificial insemination techniques using low number of sperm are needed to increase reproductive efficiency per ejaculate and to favor the use of frozen or sexed semen. Intracornual, intraoviductal, and hysteroscopic insemination methods have been described using low sperm doses.17-19 Results of deep intracornual insemination have varied widely.17,20 Intraoviductal insemination17,18 is an invasive procedure that requires appropriate facilities and qualified people. It has been demonstrated that direct deposition of a low sperm dose into the oviduct is not necessary.17 Hysteroscopic insemination has been the most used and tested method for insemination with low sperm number.19,21,22 The sperm dose used in these studies ranged between 1 × 106 and 20 × 106 of progressive motile sperm, and pregnancy rates ranged between 13% and 67% using fresh, frozen, or sexed semen. The objectives of the current in vivo studies in mares, using the ultrasound-guided IUI technique, were a) to evaluate the technique for delivering fluid into the uterine lumen (Experiment 1); b) to assess a certainty score system for enhancing operator skills (Experiment 1); c) to test the technique as an alternative method for embryo transfer and compare its efficiency with the TC method (Experiment 2); and d) to preliminarily assess the use of the technique for artificial insemination with low number of sperm (Experiment 3). MATERIALS AND METHODS
Mares and Transrectal Ultrasonography Nonlactating, cycling, small draft-type, crossbred Breton mares between 3 and 15 years of age, weighing 350 to 600 kg, and in good body condition were used in 3 experiments during October to March (spring to early autumn) of 3 ovulatory seasons in the Southern Hemisphere. The mares were kept on pasture under natural light, had access to water and trace-mineralized salt, and were supplemented with grain as needed. Mares
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were selected at the beginning of each ovulatory season using transrectal palpation and ultrasound examinations. Mares were not used if they had indications of ovarian or uterine abnormalities.23 The ultrasound scanner was equipped with a 5-MHz linear-array transducer (Aloka SSD-500V, Aloka America, Wallingford, CT) for transrectal examinations. All mares ovulated before treatment as determined by ultrasound examinations for detection of ovulation (day 0) and formation of a corpus luteum. Uterine tone and endometrial echotexture were evaluated23 on the day of the procedures (Experiments 1 and 2). Pregnancy diagnoses were done by ultrasonography on day 15 (Experiments 2 and 3). Pregnancies were terminated at day 16 by locating the embryonic vesicle by transrectal ultrasonography and rupturing the vesicle by transrectal digital pressure.23 Administration of dCloprostenol (75 µg, intramuscular) (Prolise, Tecnopex, Brazil) was done to induce luteolysis, and mares were reused if they continued to meet the selection criteria.
Ultrasound-Guided IUI The ultrasound-guided IUI technique used in these studies was adapted from a study of IUI of fluid.15 The ultrasound scanner was equipped with a 5-MHz convexarray transducer with a hard plastic extension for transvaginal procedures. Needles and human epidural catheters were used as described in each experiment. Immediately before the procedure, the mares were sedated using detomidine hydrochloride (1 mg per mare, intravenous [IV]) (Dormosedam, Orion Pharma, Finland). A tail bandage was applied, and the perineal area was prepared aseptically. An assistant was used for manipulation of the needle, catheter, and syringe during the IUIs. During the transfer, the operator wore a sterile surgical glove over a shoulder-length plastic sleeve. The transducer extension was covered with a nonlubricated condom (Blowtex, São Paulo, Brazil). An opening that was equivalent to about 30% of the surface area of the transducer face was made at the tip of the condom. The edge of the opening was stretched over the transducer face so that only the face was exposed. The assembled device was covered with an outer sterile plastic sheath. The protected transducer was inserted into the vagina, and the outer sheath was then removed. The middle to caudal portion of the right uterine horn (Experiments 1 and 2) or the tip of the uterine horn ipsilateral to the preovulatory follicle (Experiment 3) was positioned transrectally against the wall of the vaginal fornix over the face of the transducer (Fig 1). The target segment of the uterus was viewed initially in cross section, and then the transducer was rotated 90° to allow adequate monitoring of the uterine lumen. The lumen was identified
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as an echoic or white line23 when the uterus was viewed in a longitudinal section (Fig 2). The assistant inserted the needle with a catheter previously connected to a disposable syringe into the needleguide channel of the transducer extension after the transducer was inserted into the vagina. After the target segment was located, the needle was pushed through the protective condom and the vaginal and uterine walls. The position of the needle and catheter in the uterus was verified on the ultrasound screen by identification of 2 echogenic spots (tips of needle and catheter; Figs 2, 3) and also by at least 3 to-and-fro movements of the tip of the catheter against the opposite wall of the endometrium. When the image of the tip of the needle and catheter was in the uterine lumen as verified by the toand-fro movements, the catheter was advanced cranially approximately 1 to 2 cm (Experiments 1 and 2) or 3 to 5 cm (Experiment 3) into the lumen (Figs 2, 3). The tip of the catheter was blunt, and there was no known instances of puncture of the opposite endometrial wall or the uterine folds during advancement in the lumen. The medium with the embryo or the semen was deposited immediately after confirming the appropriate location of the catheter in the uterine lumen. Observation of fluid flow during injection (Experiments 1 and 2) or a small saculation at the tip of the horn (Experiment 3) during or after injection confirmed the correct placement of the tip of the catheter in the uterine lumen.
Experiment 1: Ultrasound-Guided IUI Technique for Delivering Fluid Into the Uterus
Figure 1. Schematic drawing of the ultrasound-guided intrauterine injection of an embryo into middle-caudal portion of a horn and semen into the tip. The operator’s arm manipulates the target area of the uterine horn transrectally over the face of the transvaginal probe. Note (dorsal view) the probe is rotated into the vaginal fornix at the side of the cervix during the final preinjection manipulation.
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Fifteen mares were used between days 7 and 10 after ovulation. The mares were reused 2 or 3 times if no uterine fluid was detected within 48 hours after injection, resulting in a total of 33 injections. A 16-gauge needle (56 cm), a radiopaque polyamide epidural catheter containing a tungsten strip (1.05 × 0.6 × 1000 mm) (Perifix, B. Braun, Germany), and 20 mL of saline in a disposable syringe were used for the injections. The echogenic markings allowed imaging of the catheter in the lumen. The images of ultrasonographic endpoints (tip of needle, tip of catheter, to-and-fro movements against the endometrium, and fluid flow during the injection) were scored as good, fair, or absent for each of the 4 endpoints. The overall degree of certainty of the injection into the lumen was estimated using a scoring system of 1 to 4 (low to high).16 The number of attempts per injection, the time (s) spent for each injection, and the uterine tone and echotexture were also recorded. The efficiency of the technique was evaluated immediately after injection by transrectal ultrasonography using a 5-MHz linear trans-
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Figure 3. Ultrasonograms taken during intrauterine insemination, showing (top to bottom) cranial end of uterine horn, needle tip (NT), and catheter (C) in the uterine lumen.
ducer to identify the injected fluid as an anechoic image in the uterine lumen.
Experiment 2: Ultrasound-Guided IUI Technique for Embryo Transfer Figure 2. Ultrasonograms taken during intrauterine embryo transfer, showing (top to bottom) cross section of uterine horn, longitudinal section of horn with an echoic line representing the uterine lumen (UL) and the needle tip (NT), catheter tip (CT), and catheter (C) in the uterine lumen.
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Donor and recipient mares were synchronized with d-Cloprostenol and 2500 IU of human chorionic gonadotropin. Criteria for the recipient mares at the time of transfer were 1) ovulation 1 day before to 2 days after the donor; and 2) uterine tone and endometrial echotexture characteristic of diestrus on the day of transfer.23
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Donors and embryo recovery. Donors were inseminated every other day from the day of detection of a *35mm follicle until ovulation. Inseminations were performed with fresh semen from fertile stallions containing 500 × 106 progressively motile sperm in 20 mL of skim milk extender.24 The embryos were recovered on day 7 by nonsurgical uterine flushing, using 3 L of ringer-lactate solution at 37°C.25 After identification under stereomicroscopy, the embryos were graded,26 and only embryos with a grade of 1 or 2 were used. The embryos were washed 9 times27 in Zwitterionic medium plus 0.5% bovine serum albumin (BSA) (Cultilab, Campinas, Brazil). Embryo transfers. During season 1 (2001-2002), the mares were randomly assigned to 2 groups of embryo transfer techniques: IUI (n = 23) and TC injection (n = 23). In season 2 (2002-2003), the mares were randomized into 3 groups: IUI (n = 16), TC (n = 15), and control (n = 16; inseminated mares). The same operator performed the embryo transfer techniques in both seasons. For the ultrasound-guided IUI technique, the embryo was drawn into an epidural catheter (1.05 × 0.6 × 1000 mm) between columns of air and medium (0.5 mL), using a 1-mL disposable syringe. The loaded catheter was inserted into a 16-gauge needle (56 cm), and the embryo transfer was performed as previously described. A score system, similar to the system used in Experiment 1, was used to evaluate the efficiency of the IUI procedure. For TC transfers, the embryo was loaded into a straw (0.25 mL) between columns of air and medium to minimize movement and to favor expulsion of the embryo. The straw and a stainless-steel gun were inserted into a disposable French-style sheath, and the entire device was placed into a plastic sterile sheath to minimize contamination. Before the TC embryo transfers, the recipient mares were prepared and sedated as described for the IUI technique. The transfer gun was placed into the cervix, the sterile sheath was penetrated, and the embryo was deposited deeply into the uterine body. Immediately after the transfers, the catheter within the needle (IUI groups) or the straw within the sheath (TC group) was rinsed with embryo medium into a Petri dish and searched as an aid in determining that the embryo had been expelled into the uterus.
Experiment 3: Ultrasound-Guided IUI Technique for Artificial Insemination Ten mares were used during 1 reproductive season (2003-2004). The mares were scanned by transrectal ultrasonography every other day until detection of a *30-mm follicle. Thereafter, scanning was done daily until ovulation. After detection of a *35-mm follicle, ovulation was induced with 2500 IU of human chorionic gonadotropin.
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The artificial insemination was performed 30 hours after human chorionic gonadotropin injection, regardless of uterine tone and echotexture scores. Fresh semen (*70% progressive motility) from a fertile stallion was used. Mares were inseminated once with 20 × 106 progressively motile sperm diluted in 1 mL of skim milk extender.24 The semen was aspirated into an epidural catheter (0.85 × 0.45 × 1000 mm) with a 1-mL disposable syringe, which was inserted into a 17-gauge needle (56 cm). Immediately before insemination, the mares were prepared and sedated as described for the IUI technique. Insertion of the inseminating catheter was done by the ultrasound-guided IUI technique as previously described. After the tip of the catheter reached the tip of the horn and/or resistance was felt, the semen was deposited. The uterine horn was pushed down and held by the operator for 3 minutes to favor contact of semen with the oviductal papilla.
Statistical Analyses The efficiency of the ultrasound-guided IUI technique with 1 versus 2 uterine punctures, embryo recovery rate, embryo quality, and the pregnancy rate were analyzed by r2 tests. Qualitative data for certainty score for the IUI (Experiment 1) were examined by a MannWhitney test. RESULTS
Experiment 1: Ultrasound-Guided IUI for Delivering Fluid Into the Uterus The ultrasound-guided IUI was 97% (32/33) efficient for delivering saline into the uterine lumen. After insertion of the apparatus into the vagina, a mean of 48.0 ± 1.3 seconds was required to complete the procedure. The image of the tip of the needle, tip of the catheter, to-and-fro movements of the catheter, and fluid flow during the injection were seen in 97% to 100% of the injections. The overall certainty score of the ultrasound-guided IUI was 3.8 ± 0.0 for the successes and 3.0 for the 1 failure. The majority (88%, 29/33) of the ultrasound-guided IUIs were done with 1 uterine puncture. The efficiency for delivering saline into the uterus did not differ (P > .05) between procedures with 1 or 2 punctures. The mean scores for uterine tone (1.9 ± 0.1 and 1.9 ± 0.2), uterine echotexture (3.1 ± 0.1 and 2.8 ± 0.4), and certainty (3.7 ± 0.1 and 3.8 ± 0.1) were similar between procedures that required 1 and 2 punctures, respectively. In addition, when the mares were subgrouped into classes of uterine echotexture (scores: 1 to 2, 2.1 to 3, 3.1 to 3.5, and 3.5 to
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4) and uterine tone (scores: 1.5, 2, and 2.5), the certainty scores did not differ (P > .05).
Table
Experiment 2: Embryo Transfer Embryo recovery and quality. The embryo recovery rate and the quality of the embryos did not differ (P > .05) between seasons. The overall embryo recovery rate was 72% and 88% for embryos graded 1 or 2 in each season. The time spent to perform the uterine flushing for the embryo recovery was 18 ± 0.5 minutes, and the percentage of fluid recovered was 98%. Embryo transfers. The pregnancy rates during seasons 1 and 2 for the ultrasound-guided IUI and TC methods and the control group are shown (Table). The pregnancy rate was similar among groups and between seasons. One puncture versus 2 or more punctures with the ultrasound-guided IUI technique did not affect the pregnancy rates (76%, 19/25; and 79%, 11/14, respectively). Pregnancy rates were not different for recipient mares used once (80%; n = 37) and more than once
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Pregnancy rates obtained by IUI or TC embryo transfer techniques in 2 reproductive seasons: Experiment 2 Pregnancy rates
Season 2001-2002 2002-2003
Combined
Technique IUI TC IUI TC Control IUI TC
n (%) 18/23 19/23 12/16 11/15 11/16 30/39 30/38
(78.3) (82.6) (75.0) (73.3) (68.8) (76.9) (78.9)
No differences (P > .05) between embryo transfer techniques or between seasons within techniques or between the embryo transfer techniques and control group (inseminated mares) in the 2002-2003 season. IUI, intrauterine injection; TC, transcervical.
(76%; n = 15). The score for certainty of the IUI procedure for embryo transfers was not different between pregnant (3.9 ± 0.0) or nonpregnant (3.8 ± 0.1) mares.
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After rinsing the catheter or the tip of the straw within the sheath immediately after the embryo transfers, 1 embryo was found for each technique. The embryos were reevaluated under stereomicroscopy and graded 2 and were transferred again to the same recipient mares. One reinserted embryo (found in rinse of the IUI catheter) resulted in pregnancy.
Experiment 3: Ultrasound-Guided IUI for Artificial Insemination All 10 mares ovulated after 30 and before 48 hours after human chorionic gonadotropin administration or within 18 hours after insemination. The pregnancy rate on day 15 after IUI artificial insemination with 20 × 106 progressive motile sperm was 50% (5/10). DISCUSSION
The efficiency (97%) of the in vivo ultrasoundguided IUI of saline (Experiment 1) seemed numerically superior to reported in vitro results for delivering vehicle into the uterus (88%)16 and was similar to an in vivo study using treatment solutions (96%).15 The apparent reduced success for the in vitro studies may have been a result of using softer thawed genital tracts. The ultrasonographic endpoints (tips of needle and catheter, to-and-fro movements of catheter, fluid ejection) were imaged successfully during almost all IUI procedures (Experiment 1). The tip of the needle on the needle-guide line and toand-fro movements in the lumen were always imaged, and the tip of the catheter was imaged in 97% of the injections. Although fluid injection was imaged in 97% of the procedures, it seemed more difficult to discern. The injection of fluid was done only after imaging the to-andfro movemens of the catheter in the uterine lumen. A second puncture was performed when the ultrasonographic end points were not clearly imaged during the first attempt. The number of puncture attempts did not affect the certainty scores and efficiency of the IUIs. Uterine tone and echotexture scores before the procedure did not affect the certainty scores. However, high uterine echotexture scores (3 and 4) or low uterine tone score (<1.5) seemed associated with difficulty during the IUIs in Experiment 1, presumably because of the larger number of endometrial folds with edema or uterine softness. However, even under these conditions, the IUIs were successful. During development of the IUI technique for embryo transfer, an attempt was made to use 0.10 to 0.15 mL of medium. This resulted in no pregnancies in 7 consecutive attempts, and the volume was changed to 0.50 mL. Some uterine infections occurred during the developmental
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stage preceding Experiment 2 and were corrected by using the protective condom and outer sheath; antibiotics were not used in these experiments. Five embryos were lost during the IUI in Experiment 2 because of unsuitable behavior of the mare or handling accidents during loading of the catheter or inserting the needle. These failures are not included in the table of pregnancy results. The 16gauge needle used for IUI embryo transfers did not accommodate a catheter with an outer diameter >1.05 mm. The inner diameter (0.6 mm) of the catheter accommodated about 95% of the embryos. The remaining embryos were too large for this set of needles and catheters. The operator therefore should be prepared to use a set with a larger needle and catheter for IUI or to use the TC route when needed. The overall pregnancy rates (Experiment 2) did not differ between the ultrasound-guided IUI (77%) and the TC (79%) embryo transfer techniques and were similar to the highest pregnancy rates found in the literature for TC embryo transfers2,7 or surgical transfers.1,11,12 The pregnancy rates of the 2 embryo transfer techniques were similar between seasons and during the second reproductive season did not differ (P > .05) when compared to the control group (inseminated mares). The technician’s ability, evaluation of the embryo, and the rigorous selection of recipients may have contributed to the high pregnancy rates on day 15. The overall pregnancy rate with the ultrasound-guided IUI technique (Experiment 2) was numerically lower than the efficiency rate obtained with IUIs of saline into the uterus (Experiment 1). However, efficiency in Experiment 1 was evaluated immediately after injection, whereas pregnancy diagnoses were not done until day 15. No uterine inflammation or rectal irritation was observed in any of the animals after the adequate development of the ultrasound-guided IUI technique for embryo transfer. It has been suggested that entry and manipulation of the cervix during TC embryo transfer may stimulate the release of PGF2a8,9,28 and oxytocin8,10 and that these hormones may lead to reduced and inconsistent pregnancy rates after TC embryo transfer. In addition, invasion and dilation of the cervix may increase the occurrence of uterine infection and reflux of transferred embryos.6 However, in the current studies no difference in pregnancy rate occurred between the IUI and TC techniques. In addition, the release of PGF2a and oxytocin during the IUI procedure has not been studied. The pregnancy rates were not altered by 1 versus more than 1 uterine puncture during IUI. The pregnancy rates for mares used more than once were similar to the rate in mares used once and to the rates in control mares.
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These results indicate that the pregnancy rates were not affected detrimentally by the previous embryo transfer techniques. Experiment 2 demonstrated an advantage to rinsing the catheter or straw immediately after the embryo transfer procedure to confirm that the embryo was expelled into the uterus. An embryo found in the catheter after the IUI originated a pregnancy after being transferred again by the IUI technique. The preliminary study using the ultrasound-guided IUI technique for artificial insemination in the mare indicated that this technique has potential as an alternative for insemination with low sperm number; 5 of 10 mares became pregnant when inseminated with 20 × 106 progressively motile sperm. This result was similar to the pregnancy rates obtained by others,17-19 using intracornual, intraoviductal, or hysteroscopic insemination, with low number of sperm. The IUI inseminator did not notice any difficulties associated with injecting the semen at the tip of the uterine horn during the estrous phase. Rectal irritation or uterine infections were not observed in any ani-
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mal, even though the manipulation of the uterine horn was more intense than for embryo transfer. When compared with the other techniques available for artificial insemination with low sperm dose, such as the intraoviductal and hysteroscopic approaches, the IUI technique may be less invasive and faster. Recent initial trials in our laboratory suggested that IUI insemination may be used with more reduced number of sperm; 3 of 5 and 2 of 4 mares became pregnant with 5 × 106 and 10 × 106 progressively motile sperm, respectively. Although results of Experiment 3 offer encouragement for the potential use of IUI for insemination with low numbers of sperm, more extensive studies are needed, especially with various doses of sperm. CONCLUSIONS
The ultrasound-guided IUI of saline solution in vivo was efficient and showed highly repeatable results. Observation of the 4 ultrasonographic endpoints (tip of the needle, tip of the catheter, to-and-fro movements of
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the catheter in the uterine lumen, and ejection of fluid) offered guidance during the IUI technique. The ultrasound-guided IUI embryo transfer technique produced pregnancy rates comparable to the TC procedure and the pregnancy rate of inseminated mares. The technique is therefore an alternative for embryo transfer in mares. The IUI technique could represent a valuable option for mares with poor history of cervical penetration or poor pregnancy rates after TC embryo transfers. In addition, this technique may be an alternative to maximize the use of embryo transfer in other species when cervical penetration is difficult. The ultrasound-guided IUI technique seems to be a notable alternative for future use in artificial insemination with low number of sperm in horses and possibly other species. Acknowledgments The authors thank Alexandre Nascimento Rocha Filho, Daniela Chiquetto, and Fernando Antonio de Freitas for care and handling of the animals and Tecnopec for a gift of drugs used in these studies.
11.
12.
13. 14.
15. 16. 17. 18. 19.
REFERENCES 1.
Squires EL, McCue PM, Vanderwall D. The current status of equine embryo transfer. Theriogenology 1999;51:91-104. 2. Fleury JJ. Transferencia não cirúrgica de embriões eqüinos colhidos no oitavo dia pós-ovulação. Arq Fac Vet, UFRGS 1998;26 (Suppl 1):266. 3. Imel KJ, Squires EL, Elsden RP, Shideler RK. Collection and transfer of equine embryos. J Am Vet Med Assoc 1981;179:987-91. 4. Iuliano MS, Squires EL, Cook VM. Effect of age of equine embryos and method of transfer on pregnancy rate. J Anim Sci 1985;60:258-63. 5. Muller Z, Cunat L. Special surgical transfers of horse embryos: embryo transfer III. Equine Vet J 1993;Suppl 15:113-5. 6. Squires EL, Imel KJ, Iuliano MF, Shideler RK. Factors affecting reproductive efficiency in equine embryo transfer programme. J. Reprod Fertil 1982;Suppl 32:409-14. 7. Squires EL, Carnevale EM, McCue PM, Bruemmer JE. Embryo technologies in the horse. Theriogenology 2003;59:151-70. 8. Handler J, Königshofer M, Kindahl H, Schams D, Aurich C. Secretion patterns of oxytocin and PGF2a-metabolite in response to cervical dilatation in cyclic mares. Theriogenology 2003;59:1381-91. 9. Sirois J, Betteridge KJ, Goff AK. PGF2a release, progesterone secretion and conceptus growth associated with successful and unsuccessful transcervical embryo transfer and reinsertion in the mare. J Reprod Fertil 1987;Suppl 35:419-27. 10. Nikolakopoulos E, Kindahl H, Gilbert CL, Goode J, Watson ED. Release of oxytocin and prostaglandin F2a around teasing, natural
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service and associated events in the mare. Anim Rep Sci 2000;63:89-99. Carnevale EM, Ramirez RJ, Squires EL, Alvarenga MA, Vanderwall DK, McCue PM. Factors affecting pregnancy rates and early embryonic death after equine embryo transfer. Theriogenology 2000;54:965-79. Carney NJ, Squires EL, Cook VM, Seidel GR Jr, Jasko DJ. Comparison of pregnancy rates from transfer of fresh versus cooled, transported equine embryos. Theriogenology 1991;36: 23-32. Oguri N, Tsutsumi Y. Non-surgical egg transfer in mares. J Reprod Fertil 1974;41:313-20. Palmer E, Duchamp G, Lagneaux D. A new technique for equine embryo transfer by colpotomy and blind puncture of the uterine horn. Third International Symposium on Equine Embryo Transfer. Buenos Aires, Argentina; 1992. p. 43. Gastal MO, Gastal EL, Torres CA.A., Ginther OJ. Effect of PGE2 on uterine contractility and tone in mares. Theriogenology 1998;50: 989-99. Gastal MO, Gastal EL, Rivera AP, Silva LA. Evaluation of the transvaginal ultrasound-guided intrauterine injection technique for embryo transfer in mares. Theriogenology 2002;58:725-8. Buchanan BR, Seidel GE Jr, McCue PM, Schenk JL, Herickhoff LA, Squires EL. Insemination of mares with low numbers of either unsexed or sexed spermatozoa. Theriogenology 2000;53:1333-44. McCue PM, Fleury JJ, Denniston DJ, Graham JK, Squires EL. Oviductal insemination in the mare. J Reprod Fertil 2000;Suppl 56:499-502. Morris LHA, Hunter RHF, Allen WR. Hysteroscopic insemination of small numbers of spermatozoa at the uterotubal junction of preovulatory mares. J Reprod Fertil 2000;118:95-100. Woods J, Rigby S, Brinsko S, Stephens R, Varner D, Blanchard T. Effect of intrauterine treatment with prostaglandin E2 prior to insemination of mares in the uterine horn or body. Theriogenology 2000;53:1827-36. Brinsko SP, Rigby SL, Lindsey AC, Blanchard TL, Love CC, Varner DD. Pregnancy rates in mares following hysteroscopic or transrectally guided insemination with low sperm numbers at the utero-tubal papilla. Theriogenology 2003;59:1001-9. Lindsey AC, Schenk JL, Graham JF, Bruemmer JE, Squires EL. Hysteroscopic insemination of low numbers of flow sorted fresh and frozen/thawed stallion spermatozoa. Equine Vet J 2002;34:121-7. Ginther OJ. Ultrasonic Imaging and Animal Reproduction: Horses. Book 2. Cross Plains, WI: Equiservices; 1995. p. 394. Varner DD. Collection and preservation of stallion spermatozoa. Theriogenology 1986;AQv13-33. Alvarenga MA, Alvarenga FCL, Meira C. Some modifications in the technique used for recovery equine embryos. Equine Vet J 1993;15:111-2. McKinnon AO, Squires EL. Morphologic assessment of the equine embryo. J Am Vet Med Assoc 1988;192:401-6. Stringfellow DA. Recommendations for the sanitary handling of in vivo–derived embryos. In: D. A. Stringfellow and S. M. Seidel, editors. Manual of the International Embryo Transfer Society. Savoy, IL; Savoy. 1998. pp. 79-84. Kask K, Odensvik K, Kindahl H. Prostaglandin F2a release associated with an embryo transfer procedure in the mare. Equine Vet J 1997;29:286-9.
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SUMMARY
A transvaginal ultrasound-guided intrauterine injection (IUI) technique was developed for embryo transfer and for injection of small quantities of sperm in mares. The target area of a horn was positioned by transrectal manipulation against the wall of the vaginal fornix over the face of a transvaginal transducer. A needle with a catheter containing the embryo or semen was inserted through the needle guide of the transducer into the uterine lumen. The tips of the needle and catheter, the movement of the catheter in the uterine lumen, and the ejection of fluid was monitored on the ultrasound screen. Pregnancy rate 15 days after ovulation for the IUI embryo transfer technique (30/39, 77%) was similar to the pregnancy rate for transcervical (TC) embryo transfer (30/38, 79%). The pregnancy rate for IUI insemination of 20 × 106 progressively motile sperm into the tip of the uterine horn ipsilateral to ovulation was 5/10 (50%). Results indicated that the IUI approach is a viable alternative for embryo transfer. Results also supported the potential of IUI for insemination of low numbers of sperm, but more extensive studies with various doses of sperm are needed.
Part of these data were presented at the 8th International Symposium on Equine Reproduction, Fort Collins, Colorado, July 2002, and reported in an abstract of the proceedings. From the Departments of Veterinary Sciencea and Animal Science,b Federal University of Viçosa, Brazil; and the Eutheria Foundation,c Cross Plains, Wisconsin. E. L. and M. O. Gastal are on leave from the Departments of Veterinary Science and Animal Science, respectively, Federal University of Viçosa, Brazil. Reprint requests: O. J. Ginther, Eutheria Foundation, 4343 Garfoot Rd, Cross Plains, WI 53528, USA. 0737-0806/$ - see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2004.07.006
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Keywords: Ultrasound-guided; Artificial insemination; Mares
Embryo
transfer;
INTRODUCTION
Embryo transfer and artificial insemination are powerful techniques for increasing reproductive efficiency and genetic gain in horses. Currently, equine embryos are being transferred transcervically or surgically through a flank incision.1 Reported pregnancy rates for TC transfers have ranged from 26% to 83%.2-7 The large variability in pregnancy rates can be related1 to such factors as operator experience for depositing the embryo into the uterus, expulsion of the embryo from the uterus, introduction of contaminants through the cervix, and manipulation of the genital tract, thereby stimulating uterine contractions by the production of prostaglandin F2a (PGF2a) 8,9 and oxytocin.8,10 Pregnancy rates after surgical embryo transfer through a flank incision have been more consistent among reports, ranging from 65% to 80%.1,3-6,11,12 However, the surgical method is an invasive procedure requiring time, equipment, medicines, and facilities, all of which increase the cost and restrict its application. Alternative nonsurgical methods that bypass the cervix have been tried for embryo transfer in mares to avoid or minimize cervical manipulations. Resulting pregnancy rates have been low, ranging from 33% to 40%.5,13,14 These nonsurgical methods used the transvaginal route for IUI aided by a 3-channel apparatus previously used in cattle,13 a colpotomy,14 or a tubular speculum.5 The authors of the speculum approach expressed difficulty in confirming the entry of the needle and plastic tube into the uterine lumen. A transvaginal ultrasound-guided IUI technique has been developed in mares and is efficient (96%) for delivering solutions into the uterine lumen and allowing constant monitoring by ultrasound.15 The authors suggested
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that with refinement, this technique could be developed for embryo transfer in mares. The technique avoids cervical entry and therefore may reduce the variability in pregnancy rates. Subsequently, an in vitro study16 evaluated the efficiency of ultrasound-guided IUI, using genital tracts from a slaughterhouse. Good success rates (88%-90%) were found for insertion of the needle and plastic tube into the uterine lumen and for delivering vehicle into the uterus. A high certainty score of 4 was associated with more successful injections (90%) than a certainty score of 3 (68%). The result of this study demonstrated that scoring the procedure from the ultrasound images is useful for assessing efficiency of the IUI. Alternative artificial insemination techniques using low number of sperm are needed to increase reproductive efficiency per ejaculate and to favor the use of frozen or sexed semen. Intracornual, intraoviductal, and hysteroscopic insemination methods have been described using low sperm doses.17-19 Results of deep intracornual insemination have varied widely.17,20 Intraoviductal insemination17,18 is an invasive procedure that requires appropriate facilities and qualified people. It has been demonstrated that direct deposition of a low sperm dose into the oviduct is not necessary.17 Hysteroscopic insemination has been the most used and tested method for insemination with low sperm number.19,21,22 The sperm dose used in these studies ranged between 1 × 106 and 20 × 106 of progressive motile sperm, and pregnancy rates ranged between 13% and 67% using fresh, frozen, or sexed semen. The objectives of the current in vivo studies in mares, using the ultrasound-guided IUI technique, were a) to evaluate the technique for delivering fluid into the uterine lumen (Experiment 1); b) to assess a certainty score system for enhancing operator skills (Experiment 1); c) to test the technique as an alternative method for embryo transfer and compare its efficiency with the TC method (Experiment 2); and d) to preliminarily assess the use of the technique for artificial insemination with low number of sperm (Experiment 3). MATERIALS AND METHODS
Mares and Transrectal Ultrasonography Nonlactating, cycling, small draft-type, crossbred Breton mares between 3 and 15 years of age, weighing 350 to 600 kg, and in good body condition were used in 3 experiments during October to March (spring to early autumn) of 3 ovulatory seasons in the Southern Hemisphere. The mares were kept on pasture under natural light, had access to water and trace-mineralized salt, and were supplemented with grain as needed. Mares
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were selected at the beginning of each ovulatory season using transrectal palpation and ultrasound examinations. Mares were not used if they had indications of ovarian or uterine abnormalities.23 The ultrasound scanner was equipped with a 5-MHz linear-array transducer (Aloka SSD-500V, Aloka America, Wallingford, CT) for transrectal examinations. All mares ovulated before treatment as determined by ultrasound examinations for detection of ovulation (day 0) and formation of a corpus luteum. Uterine tone and endometrial echotexture were evaluated23 on the day of the procedures (Experiments 1 and 2). Pregnancy diagnoses were done by ultrasonography on day 15 (Experiments 2 and 3). Pregnancies were terminated at day 16 by locating the embryonic vesicle by transrectal ultrasonography and rupturing the vesicle by transrectal digital pressure.23 Administration of dCloprostenol (75 µg, intramuscular) (Prolise, Tecnopex, Brazil) was done to induce luteolysis, and mares were reused if they continued to meet the selection criteria.
Ultrasound-Guided IUI The ultrasound-guided IUI technique used in these studies was adapted from a study of IUI of fluid.15 The ultrasound scanner was equipped with a 5-MHz convexarray transducer with a hard plastic extension for transvaginal procedures. Needles and human epidural catheters were used as described in each experiment. Immediately before the procedure, the mares were sedated using detomidine hydrochloride (1 mg per mare, intravenous [IV]) (Dormosedam, Orion Pharma, Finland). A tail bandage was applied, and the perineal area was prepared aseptically. An assistant was used for manipulation of the needle, catheter, and syringe during the IUIs. During the transfer, the operator wore a sterile surgical glove over a shoulder-length plastic sleeve. The transducer extension was covered with a nonlubricated condom (Blowtex, São Paulo, Brazil). An opening that was equivalent to about 30% of the surface area of the transducer face was made at the tip of the condom. The edge of the opening was stretched over the transducer face so that only the face was exposed. The assembled device was covered with an outer sterile plastic sheath. The protected transducer was inserted into the vagina, and the outer sheath was then removed. The middle to caudal portion of the right uterine horn (Experiments 1 and 2) or the tip of the uterine horn ipsilateral to the preovulatory follicle (Experiment 3) was positioned transrectally against the wall of the vaginal fornix over the face of the transducer (Fig 1). The target segment of the uterus was viewed initially in cross section, and then the transducer was rotated 90° to allow adequate monitoring of the uterine lumen. The lumen was identified
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as an echoic or white line23 when the uterus was viewed in a longitudinal section (Fig 2). The assistant inserted the needle with a catheter previously connected to a disposable syringe into the needleguide channel of the transducer extension after the transducer was inserted into the vagina. After the target segment was located, the needle was pushed through the protective condom and the vaginal and uterine walls. The position of the needle and catheter in the uterus was verified on the ultrasound screen by identification of 2 echogenic spots (tips of needle and catheter; Figs 2, 3) and also by at least 3 to-and-fro movements of the tip of the catheter against the opposite wall of the endometrium. When the image of the tip of the needle and catheter was in the uterine lumen as verified by the toand-fro movements, the catheter was advanced cranially approximately 1 to 2 cm (Experiments 1 and 2) or 3 to 5 cm (Experiment 3) into the lumen (Figs 2, 3). The tip of the catheter was blunt, and there was no known instances of puncture of the opposite endometrial wall or the uterine folds during advancement in the lumen. The medium with the embryo or the semen was deposited immediately after confirming the appropriate location of the catheter in the uterine lumen. Observation of fluid flow during injection (Experiments 1 and 2) or a small saculation at the tip of the horn (Experiment 3) during or after injection confirmed the correct placement of the tip of the catheter in the uterine lumen.
Experiment 1: Ultrasound-Guided IUI Technique for Delivering Fluid Into the Uterus
Figure 1. Schematic drawing of the ultrasound-guided intrauterine injection of an embryo into middle-caudal portion of a horn and semen into the tip. The operator’s arm manipulates the target area of the uterine horn transrectally over the face of the transvaginal probe. Note (dorsal view) the probe is rotated into the vaginal fornix at the side of the cervix during the final preinjection manipulation.
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Fifteen mares were used between days 7 and 10 after ovulation. The mares were reused 2 or 3 times if no uterine fluid was detected within 48 hours after injection, resulting in a total of 33 injections. A 16-gauge needle (56 cm), a radiopaque polyamide epidural catheter containing a tungsten strip (1.05 × 0.6 × 1000 mm) (Perifix, B. Braun, Germany), and 20 mL of saline in a disposable syringe were used for the injections. The echogenic markings allowed imaging of the catheter in the lumen. The images of ultrasonographic endpoints (tip of needle, tip of catheter, to-and-fro movements against the endometrium, and fluid flow during the injection) were scored as good, fair, or absent for each of the 4 endpoints. The overall degree of certainty of the injection into the lumen was estimated using a scoring system of 1 to 4 (low to high).16 The number of attempts per injection, the time (s) spent for each injection, and the uterine tone and echotexture were also recorded. The efficiency of the technique was evaluated immediately after injection by transrectal ultrasonography using a 5-MHz linear trans-
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Figure 3. Ultrasonograms taken during intrauterine insemination, showing (top to bottom) cranial end of uterine horn, needle tip (NT), and catheter (C) in the uterine lumen.
ducer to identify the injected fluid as an anechoic image in the uterine lumen.
Experiment 2: Ultrasound-Guided IUI Technique for Embryo Transfer Figure 2. Ultrasonograms taken during intrauterine embryo transfer, showing (top to bottom) cross section of uterine horn, longitudinal section of horn with an echoic line representing the uterine lumen (UL) and the needle tip (NT), catheter tip (CT), and catheter (C) in the uterine lumen.
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Donor and recipient mares were synchronized with d-Cloprostenol and 2500 IU of human chorionic gonadotropin. Criteria for the recipient mares at the time of transfer were 1) ovulation 1 day before to 2 days after the donor; and 2) uterine tone and endometrial echotexture characteristic of diestrus on the day of transfer.23
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Donors and embryo recovery. Donors were inseminated every other day from the day of detection of a *35mm follicle until ovulation. Inseminations were performed with fresh semen from fertile stallions containing 500 × 106 progressively motile sperm in 20 mL of skim milk extender.24 The embryos were recovered on day 7 by nonsurgical uterine flushing, using 3 L of ringer-lactate solution at 37°C.25 After identification under stereomicroscopy, the embryos were graded,26 and only embryos with a grade of 1 or 2 were used. The embryos were washed 9 times27 in Zwitterionic medium plus 0.5% bovine serum albumin (BSA) (Cultilab, Campinas, Brazil). Embryo transfers. During season 1 (2001-2002), the mares were randomly assigned to 2 groups of embryo transfer techniques: IUI (n = 23) and TC injection (n = 23). In season 2 (2002-2003), the mares were randomized into 3 groups: IUI (n = 16), TC (n = 15), and control (n = 16; inseminated mares). The same operator performed the embryo transfer techniques in both seasons. For the ultrasound-guided IUI technique, the embryo was drawn into an epidural catheter (1.05 × 0.6 × 1000 mm) between columns of air and medium (0.5 mL), using a 1-mL disposable syringe. The loaded catheter was inserted into a 16-gauge needle (56 cm), and the embryo transfer was performed as previously described. A score system, similar to the system used in Experiment 1, was used to evaluate the efficiency of the IUI procedure. For TC transfers, the embryo was loaded into a straw (0.25 mL) between columns of air and medium to minimize movement and to favor expulsion of the embryo. The straw and a stainless-steel gun were inserted into a disposable French-style sheath, and the entire device was placed into a plastic sterile sheath to minimize contamination. Before the TC embryo transfers, the recipient mares were prepared and sedated as described for the IUI technique. The transfer gun was placed into the cervix, the sterile sheath was penetrated, and the embryo was deposited deeply into the uterine body. Immediately after the transfers, the catheter within the needle (IUI groups) or the straw within the sheath (TC group) was rinsed with embryo medium into a Petri dish and searched as an aid in determining that the embryo had been expelled into the uterus.
Experiment 3: Ultrasound-Guided IUI Technique for Artificial Insemination Ten mares were used during 1 reproductive season (2003-2004). The mares were scanned by transrectal ultrasonography every other day until detection of a *30-mm follicle. Thereafter, scanning was done daily until ovulation. After detection of a *35-mm follicle, ovulation was induced with 2500 IU of human chorionic gonadotropin.
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The artificial insemination was performed 30 hours after human chorionic gonadotropin injection, regardless of uterine tone and echotexture scores. Fresh semen (*70% progressive motility) from a fertile stallion was used. Mares were inseminated once with 20 × 106 progressively motile sperm diluted in 1 mL of skim milk extender.24 The semen was aspirated into an epidural catheter (0.85 × 0.45 × 1000 mm) with a 1-mL disposable syringe, which was inserted into a 17-gauge needle (56 cm). Immediately before insemination, the mares were prepared and sedated as described for the IUI technique. Insertion of the inseminating catheter was done by the ultrasound-guided IUI technique as previously described. After the tip of the catheter reached the tip of the horn and/or resistance was felt, the semen was deposited. The uterine horn was pushed down and held by the operator for 3 minutes to favor contact of semen with the oviductal papilla.
Statistical Analyses The efficiency of the ultrasound-guided IUI technique with 1 versus 2 uterine punctures, embryo recovery rate, embryo quality, and the pregnancy rate were analyzed by r2 tests. Qualitative data for certainty score for the IUI (Experiment 1) were examined by a MannWhitney test. RESULTS
Experiment 1: Ultrasound-Guided IUI for Delivering Fluid Into the Uterus The ultrasound-guided IUI was 97% (32/33) efficient for delivering saline into the uterine lumen. After insertion of the apparatus into the vagina, a mean of 48.0 ± 1.3 seconds was required to complete the procedure. The image of the tip of the needle, tip of the catheter, to-and-fro movements of the catheter, and fluid flow during the injection were seen in 97% to 100% of the injections. The overall certainty score of the ultrasound-guided IUI was 3.8 ± 0.0 for the successes and 3.0 for the 1 failure. The majority (88%, 29/33) of the ultrasound-guided IUIs were done with 1 uterine puncture. The efficiency for delivering saline into the uterus did not differ (P > .05) between procedures with 1 or 2 punctures. The mean scores for uterine tone (1.9 ± 0.1 and 1.9 ± 0.2), uterine echotexture (3.1 ± 0.1 and 2.8 ± 0.4), and certainty (3.7 ± 0.1 and 3.8 ± 0.1) were similar between procedures that required 1 and 2 punctures, respectively. In addition, when the mares were subgrouped into classes of uterine echotexture (scores: 1 to 2, 2.1 to 3, 3.1 to 3.5, and 3.5 to
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4) and uterine tone (scores: 1.5, 2, and 2.5), the certainty scores did not differ (P > .05).
Table
Experiment 2: Embryo Transfer Embryo recovery and quality. The embryo recovery rate and the quality of the embryos did not differ (P > .05) between seasons. The overall embryo recovery rate was 72% and 88% for embryos graded 1 or 2 in each season. The time spent to perform the uterine flushing for the embryo recovery was 18 ± 0.5 minutes, and the percentage of fluid recovered was 98%. Embryo transfers. The pregnancy rates during seasons 1 and 2 for the ultrasound-guided IUI and TC methods and the control group are shown (Table). The pregnancy rate was similar among groups and between seasons. One puncture versus 2 or more punctures with the ultrasound-guided IUI technique did not affect the pregnancy rates (76%, 19/25; and 79%, 11/14, respectively). Pregnancy rates were not different for recipient mares used once (80%; n = 37) and more than once
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Pregnancy rates obtained by IUI or TC embryo transfer techniques in 2 reproductive seasons: Experiment 2 Pregnancy rates
Season 2001-2002 2002-2003
Combined
Technique IUI TC IUI TC Control IUI TC
n (%) 18/23 19/23 12/16 11/15 11/16 30/39 30/38
(78.3) (82.6) (75.0) (73.3) (68.8) (76.9) (78.9)
No differences (P > .05) between embryo transfer techniques or between seasons within techniques or between the embryo transfer techniques and control group (inseminated mares) in the 2002-2003 season. IUI, intrauterine injection; TC, transcervical.
(76%; n = 15). The score for certainty of the IUI procedure for embryo transfers was not different between pregnant (3.9 ± 0.0) or nonpregnant (3.8 ± 0.1) mares.
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After rinsing the catheter or the tip of the straw within the sheath immediately after the embryo transfers, 1 embryo was found for each technique. The embryos were reevaluated under stereomicroscopy and graded 2 and were transferred again to the same recipient mares. One reinserted embryo (found in rinse of the IUI catheter) resulted in pregnancy.
Experiment 3: Ultrasound-Guided IUI for Artificial Insemination All 10 mares ovulated after 30 and before 48 hours after human chorionic gonadotropin administration or within 18 hours after insemination. The pregnancy rate on day 15 after IUI artificial insemination with 20 × 106 progressive motile sperm was 50% (5/10). DISCUSSION
The efficiency (97%) of the in vivo ultrasoundguided IUI of saline (Experiment 1) seemed numerically superior to reported in vitro results for delivering vehicle into the uterus (88%)16 and was similar to an in vivo study using treatment solutions (96%).15 The apparent reduced success for the in vitro studies may have been a result of using softer thawed genital tracts. The ultrasonographic endpoints (tips of needle and catheter, to-and-fro movements of catheter, fluid ejection) were imaged successfully during almost all IUI procedures (Experiment 1). The tip of the needle on the needle-guide line and toand-fro movements in the lumen were always imaged, and the tip of the catheter was imaged in 97% of the injections. Although fluid injection was imaged in 97% of the procedures, it seemed more difficult to discern. The injection of fluid was done only after imaging the to-andfro movemens of the catheter in the uterine lumen. A second puncture was performed when the ultrasonographic end points were not clearly imaged during the first attempt. The number of puncture attempts did not affect the certainty scores and efficiency of the IUIs. Uterine tone and echotexture scores before the procedure did not affect the certainty scores. However, high uterine echotexture scores (3 and 4) or low uterine tone score (<1.5) seemed associated with difficulty during the IUIs in Experiment 1, presumably because of the larger number of endometrial folds with edema or uterine softness. However, even under these conditions, the IUIs were successful. During development of the IUI technique for embryo transfer, an attempt was made to use 0.10 to 0.15 mL of medium. This resulted in no pregnancies in 7 consecutive attempts, and the volume was changed to 0.50 mL. Some uterine infections occurred during the developmental
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stage preceding Experiment 2 and were corrected by using the protective condom and outer sheath; antibiotics were not used in these experiments. Five embryos were lost during the IUI in Experiment 2 because of unsuitable behavior of the mare or handling accidents during loading of the catheter or inserting the needle. These failures are not included in the table of pregnancy results. The 16gauge needle used for IUI embryo transfers did not accommodate a catheter with an outer diameter >1.05 mm. The inner diameter (0.6 mm) of the catheter accommodated about 95% of the embryos. The remaining embryos were too large for this set of needles and catheters. The operator therefore should be prepared to use a set with a larger needle and catheter for IUI or to use the TC route when needed. The overall pregnancy rates (Experiment 2) did not differ between the ultrasound-guided IUI (77%) and the TC (79%) embryo transfer techniques and were similar to the highest pregnancy rates found in the literature for TC embryo transfers2,7 or surgical transfers.1,11,12 The pregnancy rates of the 2 embryo transfer techniques were similar between seasons and during the second reproductive season did not differ (P > .05) when compared to the control group (inseminated mares). The technician’s ability, evaluation of the embryo, and the rigorous selection of recipients may have contributed to the high pregnancy rates on day 15. The overall pregnancy rate with the ultrasound-guided IUI technique (Experiment 2) was numerically lower than the efficiency rate obtained with IUIs of saline into the uterus (Experiment 1). However, efficiency in Experiment 1 was evaluated immediately after injection, whereas pregnancy diagnoses were not done until day 15. No uterine inflammation or rectal irritation was observed in any of the animals after the adequate development of the ultrasound-guided IUI technique for embryo transfer. It has been suggested that entry and manipulation of the cervix during TC embryo transfer may stimulate the release of PGF2a8,9,28 and oxytocin8,10 and that these hormones may lead to reduced and inconsistent pregnancy rates after TC embryo transfer. In addition, invasion and dilation of the cervix may increase the occurrence of uterine infection and reflux of transferred embryos.6 However, in the current studies no difference in pregnancy rate occurred between the IUI and TC techniques. In addition, the release of PGF2a and oxytocin during the IUI procedure has not been studied. The pregnancy rates were not altered by 1 versus more than 1 uterine puncture during IUI. The pregnancy rates for mares used more than once were similar to the rate in mares used once and to the rates in control mares.
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These results indicate that the pregnancy rates were not affected detrimentally by the previous embryo transfer techniques. Experiment 2 demonstrated an advantage to rinsing the catheter or straw immediately after the embryo transfer procedure to confirm that the embryo was expelled into the uterus. An embryo found in the catheter after the IUI originated a pregnancy after being transferred again by the IUI technique. The preliminary study using the ultrasound-guided IUI technique for artificial insemination in the mare indicated that this technique has potential as an alternative for insemination with low sperm number; 5 of 10 mares became pregnant when inseminated with 20 × 106 progressively motile sperm. This result was similar to the pregnancy rates obtained by others,17-19 using intracornual, intraoviductal, or hysteroscopic insemination, with low number of sperm. The IUI inseminator did not notice any difficulties associated with injecting the semen at the tip of the uterine horn during the estrous phase. Rectal irritation or uterine infections were not observed in any ani-
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mal, even though the manipulation of the uterine horn was more intense than for embryo transfer. When compared with the other techniques available for artificial insemination with low sperm dose, such as the intraoviductal and hysteroscopic approaches, the IUI technique may be less invasive and faster. Recent initial trials in our laboratory suggested that IUI insemination may be used with more reduced number of sperm; 3 of 5 and 2 of 4 mares became pregnant with 5 × 106 and 10 × 106 progressively motile sperm, respectively. Although results of Experiment 3 offer encouragement for the potential use of IUI for insemination with low numbers of sperm, more extensive studies are needed, especially with various doses of sperm. CONCLUSIONS
The ultrasound-guided IUI of saline solution in vivo was efficient and showed highly repeatable results. Observation of the 4 ultrasonographic endpoints (tip of the needle, tip of the catheter, to-and-fro movements of
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the catheter in the uterine lumen, and ejection of fluid) offered guidance during the IUI technique. The ultrasound-guided IUI embryo transfer technique produced pregnancy rates comparable to the TC procedure and the pregnancy rate of inseminated mares. The technique is therefore an alternative for embryo transfer in mares. The IUI technique could represent a valuable option for mares with poor history of cervical penetration or poor pregnancy rates after TC embryo transfers. In addition, this technique may be an alternative to maximize the use of embryo transfer in other species when cervical penetration is difficult. The ultrasound-guided IUI technique seems to be a notable alternative for future use in artificial insemination with low number of sperm in horses and possibly other species. Acknowledgments The authors thank Alexandre Nascimento Rocha Filho, Daniela Chiquetto, and Fernando Antonio de Freitas for care and handling of the animals and Tecnopec for a gift of drugs used in these studies.
11.
12.
13. 14.
15. 16. 17. 18. 19.
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