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Intrauterine transfer of early canine embryos
Theriogenology 66 (2006) 1703–1705 www.journals.elsevierhealth.com/periodicals/the
Intrauterine transfer of early canine embryos Toshihiko Tsutsui *, Tatsuya Hori, Shinobu Endo, Arata Hayama, Eiichi Kawakami Department of Reproduction, Nippon Veterinary and Animal Science University, 1-7-1 Kyonan-cho, Musashino-shi, Tokyo 180-8602, Japan
Abstract The success rate of in vitro fertilization (IVF) in dogs is low and only early embryos have been obtained. In the present study, we investigated the use of intrauterine transfer of early-stage canine embryos to obtain pups. Twenty-three female dogs, in which the date of ovulation (based on plasma progesterone concentrations) differed by 1 day, were used (10 donors and 13 recipients). The uterine tube was extirpated under general anesthesia 1–4 days after mating (5–7 days after ovulation), and descending perfusion was done to collect embryos. Embryos were examined and transferred into the uterine horn of a recipient, ipsilateral to the ovary with the most corpora lutea. Pregnancy was established in one of eight bitches that received early embryos (zygote to 4-cell embryos); she received two zygotes and one 2-cell embryo and delivered two puppies. Although intrauterine transfer of early embryos (zygote to 4-cell embryos) was difficult, pregnancy was achieved, suggesting that uterine tube transfer is appropriate for these early-stage embryos. # 2006 Elsevier Inc. All rights reserved. Keywords: Canine; Early embryo; Intrauterine transfer; Embryo transfer
1. Introduction Five studies have reported production of in vitroderived canine embryos from in vitro fertilization (IVF) of in vitro matured oocytes [1–5]. In those studies, development rates to early embryonic stages ranged from 8.2 to 33.3%. The embryos of dogs enter the uterine horn as morulae, 9 days after ovulation. To obtain newborn pups using early embryos, England et al. [1] transferred two 2-cell embryos obtained by IVF into the uterine horn (ET), and confirmed implantation. We reported that lower uterine tube transfer of early canine embryos achieved conception [6]. However, lower uterine tube transfer of embryos
* Corresponding author. Tel.: +81 422 39 7340; fax: +81 422 39 7340. E-mail address: [email protected] (T. Tsutsui). 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.01.009
from the fimbriate tubae of dogs using a small catheter requires considerable skill, since the anatomy of the uterine tube is complex. The objective of the present study was to investigate the use of intrauterine transfer of early-stage canine embryos to obtain pups. 2. Materials and methods 2.1. Animals and estimation of time of ovulation Twenty-three Beagle bitches, aged 1–7 years, with a 1 day difference in day of ovulation, were used (10 were donors and 13 were recipients). To determine the ovulation day in the dogs tested, blood was collected from the cephalic vein daily starting 6 days after the beginning of vulval bleeding of estrus and the ovulation day was estimated from the blood progesterone (P4) concentration. The initial day with a P4 concentration of 2 ng/mL or higher was defined as the ovulation day [7].
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T. Tsutsui et al. / Theriogenology 66 (2006) 1703–1705
Table 1 Results of embryos recovery in bitches Donor number (age)
346 (5) 9 (10) 314 (8)
Days until embryo recovery
Number of CL
Number of ova recovered
Rate of ova recovered (%)
Number of ova recovered
After ovulation
After mating
5
1
La Rb
6 6
5 6
91.7
5 6
5
2
L R
4 5
4 3
77.8
4 2
1
L R
4 5
4 5
100 4
4 1
5
2
One-cell
337 (6)
5
2
L R
2 7
2 7
100
2 7
316 (7)
6
1
L R
6 4
6 4
100
5 3
348 (3)
6
2
L R
2 3
1 3
80
L R
2 8
2 8
100 100
319 (8)
6
3
369 (2)
6
3
L R
0 6
0 6
320 (8)
6
4
L R
11 0
4 0
336 (6)
7
3
L R
4 8
4 8
a b
Left ovary. Right ovary.
Table 2 Results of intrauterine transplantation of early canine embryos
Two-cell
Four-cell
Eight-cell
De-generate
1 1 1 3 1 2 6
36.4
2
1 6 0
2
100
4 8
T. Tsutsui et al. / Theriogenology 66 (2006) 1703–1705
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Donor bitches were mated once, 3–5 days after ovulation. The left and right uterine tubes from 10 bitches were excised under general anesthesia 1–4 days after mating (5–7 days after ovulation). The perfusate used to collect the embryos was 20% canine serumsupplemented Ringer’s solution, as described by Tsutsui et al. [9]. Embryos were examined and then transferred into the uterine horn of a recipient, ipsilateral to the ovary with the most corpora lutea [9]. In the recipient bitches, pregnancy diagnosis was done 25–30 days after ovulation, using an ultrasonographic imaging diagnosis system (Echovision SSD500EV; Aloka Co., Tokyo, Japan).
achieved conception [6,9]. Eight-cell embryos from dogs correspond to 7 days after ovulation; therefore, pregnancy can be established when these embryos are transferred from the uterine tube to the uterine horn 2 days earlier [9]. Pregnancy was established in one of the eight dogs that underwent intrauterine transfer of early embryos (zygote to 4-cell embryos). In this dog, two zygotes and one 2-cell embryo were transferred, and the bitch delivered two puppies. England et al. [1] transferred 2cell embryos into the uterine horn and subsequently used ultrasonography to detect a fetal sac. However, in the present study, we did not detect any fetal sacs in the seven recipients that received only zygotes or 4-cellstage embryos, and none gave birth to any puppies. In conclusion, although intrauterine transfer of early embryos (zygote to 4-cell embryos) was difficult, pregnancy was achieved, suggesting that uterine tube transfer is appropriate for early-stage canine embryos.
3. Results
References
The mean (S.E.M.) number of embryos collected was 8.2 0.9 (range, 4–12; Table 1). The collection rate was 88.6 6.8% (range, 36.4–100%). Furthermore, 88.6% of these embryos were normal, whereas the remaining embryos were degenerated. With respect to the stage of development, zygote to 2-cell embryos were collected 5 days after ovulation (1–2 days after mating), and 4-cell embryos or 4-and 8-cell embryos 6 days after ovulation (1–4 days after mating). Pregnancy was established in one of two dogs that received both a zygote and a 2-cell embryo, whereas in all four dogs that received only zygotes, pregnancy was not established (Table 2). One of three dogs that received one 4-cell and up to three 8-cell embryos became pregnant, but aborted. Two dogs that each received four 8-cell embryos delivered one and two pups, respectively.
[1] England GCW, Verstegen JP, Hewitt DA. Pregnancy following in vitro fertilization of canine oocytes. Vet Rec 2001;148:20–2. [2] Hori T, Tsutsui T. In vitro fertilization of mature canine ova. Vet Rec 2003;152:688–90. [3] Otoi T, Murakami M, Fujii M, Tanaka M, Ooka A, Une S, et al. Development of canine oocytes matured and fertilised in vitro. Vet Rec 2000;146:52–3. [4] Rodrigues Bde A, dos Santos LC, Rodrigues JL. Embryonic development of in vitro matured and in vitro fertilized dog oocytes. Mol Reprod Dev 2004;67:215–23. [5] Yamada S, Shimazu Y, Kawaji H, Nakazawa M, Naito K, Toyoda Y. Maturation, fertilization, and development of dog oocytes in vitro. Biol Reprod 1992;46:853–8. [6] Tsutsui T, Hori T, Kawakami E. Intratubal transplantation of early canine embryos. J Reprod Fertil Suppl 2001;57:309–14. [7] Hase M, Hori T, Kawakami E, Tsutsui T. Plasma LH and progesterone levels before and after ovulation and observation of ovarian follicles by ultrasonographic diagnosis system in dogs. J Vet Med Sci 2000;62:243–8. [8] Munro C, Stabenfeldt GT. Development of a microtiter plate enzyme immunoassay for the determination of the progesterone. J Endocrinol 1983;101:41–9. [9] Tsutsui T, Hori T, Okazaki H, Tanaka A, Shiono M, Yokosuka M, et al. Transfer of canine embryos at various developmental stages recovered by hysterectomy or surgical uterine flushing. J Vet Med Sci 2001;63:401–5.
The P4 level was measured by enzyme immunoassay [8]. 2.2. Recovery of early embryos and embryo transfer
4. Discussion We previously reported that lower uterine tube or upper uterine horn transfer of 8-cell embryos from dogs
Intrauterine transfer of early canine embryos Toshihiko Tsutsui *, Tatsuya Hori, Shinobu Endo, Arata Hayama, Eiichi Kawakami Department of Reproduction, Nippon Veterinary and Animal Science University, 1-7-1 Kyonan-cho, Musashino-shi, Tokyo 180-8602, Japan
Abstract The success rate of in vitro fertilization (IVF) in dogs is low and only early embryos have been obtained. In the present study, we investigated the use of intrauterine transfer of early-stage canine embryos to obtain pups. Twenty-three female dogs, in which the date of ovulation (based on plasma progesterone concentrations) differed by 1 day, were used (10 donors and 13 recipients). The uterine tube was extirpated under general anesthesia 1–4 days after mating (5–7 days after ovulation), and descending perfusion was done to collect embryos. Embryos were examined and transferred into the uterine horn of a recipient, ipsilateral to the ovary with the most corpora lutea. Pregnancy was established in one of eight bitches that received early embryos (zygote to 4-cell embryos); she received two zygotes and one 2-cell embryo and delivered two puppies. Although intrauterine transfer of early embryos (zygote to 4-cell embryos) was difficult, pregnancy was achieved, suggesting that uterine tube transfer is appropriate for these early-stage embryos. # 2006 Elsevier Inc. All rights reserved. Keywords: Canine; Early embryo; Intrauterine transfer; Embryo transfer
1. Introduction Five studies have reported production of in vitroderived canine embryos from in vitro fertilization (IVF) of in vitro matured oocytes [1–5]. In those studies, development rates to early embryonic stages ranged from 8.2 to 33.3%. The embryos of dogs enter the uterine horn as morulae, 9 days after ovulation. To obtain newborn pups using early embryos, England et al. [1] transferred two 2-cell embryos obtained by IVF into the uterine horn (ET), and confirmed implantation. We reported that lower uterine tube transfer of early canine embryos achieved conception [6]. However, lower uterine tube transfer of embryos
* Corresponding author. Tel.: +81 422 39 7340; fax: +81 422 39 7340. E-mail address: [email protected] (T. Tsutsui). 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.01.009
from the fimbriate tubae of dogs using a small catheter requires considerable skill, since the anatomy of the uterine tube is complex. The objective of the present study was to investigate the use of intrauterine transfer of early-stage canine embryos to obtain pups. 2. Materials and methods 2.1. Animals and estimation of time of ovulation Twenty-three Beagle bitches, aged 1–7 years, with a 1 day difference in day of ovulation, were used (10 were donors and 13 were recipients). To determine the ovulation day in the dogs tested, blood was collected from the cephalic vein daily starting 6 days after the beginning of vulval bleeding of estrus and the ovulation day was estimated from the blood progesterone (P4) concentration. The initial day with a P4 concentration of 2 ng/mL or higher was defined as the ovulation day [7].
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T. Tsutsui et al. / Theriogenology 66 (2006) 1703–1705
Table 1 Results of embryos recovery in bitches Donor number (age)
346 (5) 9 (10) 314 (8)
Days until embryo recovery
Number of CL
Number of ova recovered
Rate of ova recovered (%)
Number of ova recovered
After ovulation
After mating
5
1
La Rb
6 6
5 6
91.7
5 6
5
2
L R
4 5
4 3
77.8
4 2
1
L R
4 5
4 5
100 4
4 1
5
2
One-cell
337 (6)
5
2
L R
2 7
2 7
100
2 7
316 (7)
6
1
L R
6 4
6 4
100
5 3
348 (3)
6
2
L R
2 3
1 3
80
L R
2 8
2 8
100 100
319 (8)
6
3
369 (2)
6
3
L R
0 6
0 6
320 (8)
6
4
L R
11 0
4 0
336 (6)
7
3
L R
4 8
4 8
a b
Left ovary. Right ovary.
Table 2 Results of intrauterine transplantation of early canine embryos
Two-cell
Four-cell
Eight-cell
De-generate
1 1 1 3 1 2 6
36.4
2
1 6 0
2
100
4 8
T. Tsutsui et al. / Theriogenology 66 (2006) 1703–1705
1705
Donor bitches were mated once, 3–5 days after ovulation. The left and right uterine tubes from 10 bitches were excised under general anesthesia 1–4 days after mating (5–7 days after ovulation). The perfusate used to collect the embryos was 20% canine serumsupplemented Ringer’s solution, as described by Tsutsui et al. [9]. Embryos were examined and then transferred into the uterine horn of a recipient, ipsilateral to the ovary with the most corpora lutea [9]. In the recipient bitches, pregnancy diagnosis was done 25–30 days after ovulation, using an ultrasonographic imaging diagnosis system (Echovision SSD500EV; Aloka Co., Tokyo, Japan).
achieved conception [6,9]. Eight-cell embryos from dogs correspond to 7 days after ovulation; therefore, pregnancy can be established when these embryos are transferred from the uterine tube to the uterine horn 2 days earlier [9]. Pregnancy was established in one of the eight dogs that underwent intrauterine transfer of early embryos (zygote to 4-cell embryos). In this dog, two zygotes and one 2-cell embryo were transferred, and the bitch delivered two puppies. England et al. [1] transferred 2cell embryos into the uterine horn and subsequently used ultrasonography to detect a fetal sac. However, in the present study, we did not detect any fetal sacs in the seven recipients that received only zygotes or 4-cellstage embryos, and none gave birth to any puppies. In conclusion, although intrauterine transfer of early embryos (zygote to 4-cell embryos) was difficult, pregnancy was achieved, suggesting that uterine tube transfer is appropriate for early-stage canine embryos.
3. Results
References
The mean (S.E.M.) number of embryos collected was 8.2 0.9 (range, 4–12; Table 1). The collection rate was 88.6 6.8% (range, 36.4–100%). Furthermore, 88.6% of these embryos were normal, whereas the remaining embryos were degenerated. With respect to the stage of development, zygote to 2-cell embryos were collected 5 days after ovulation (1–2 days after mating), and 4-cell embryos or 4-and 8-cell embryos 6 days after ovulation (1–4 days after mating). Pregnancy was established in one of two dogs that received both a zygote and a 2-cell embryo, whereas in all four dogs that received only zygotes, pregnancy was not established (Table 2). One of three dogs that received one 4-cell and up to three 8-cell embryos became pregnant, but aborted. Two dogs that each received four 8-cell embryos delivered one and two pups, respectively.
[1] England GCW, Verstegen JP, Hewitt DA. Pregnancy following in vitro fertilization of canine oocytes. Vet Rec 2001;148:20–2. [2] Hori T, Tsutsui T. In vitro fertilization of mature canine ova. Vet Rec 2003;152:688–90. [3] Otoi T, Murakami M, Fujii M, Tanaka M, Ooka A, Une S, et al. Development of canine oocytes matured and fertilised in vitro. Vet Rec 2000;146:52–3. [4] Rodrigues Bde A, dos Santos LC, Rodrigues JL. Embryonic development of in vitro matured and in vitro fertilized dog oocytes. Mol Reprod Dev 2004;67:215–23. [5] Yamada S, Shimazu Y, Kawaji H, Nakazawa M, Naito K, Toyoda Y. Maturation, fertilization, and development of dog oocytes in vitro. Biol Reprod 1992;46:853–8. [6] Tsutsui T, Hori T, Kawakami E. Intratubal transplantation of early canine embryos. J Reprod Fertil Suppl 2001;57:309–14. [7] Hase M, Hori T, Kawakami E, Tsutsui T. Plasma LH and progesterone levels before and after ovulation and observation of ovarian follicles by ultrasonographic diagnosis system in dogs. J Vet Med Sci 2000;62:243–8. [8] Munro C, Stabenfeldt GT. Development of a microtiter plate enzyme immunoassay for the determination of the progesterone. J Endocrinol 1983;101:41–9. [9] Tsutsui T, Hori T, Okazaki H, Tanaka A, Shiono M, Yokosuka M, et al. Transfer of canine embryos at various developmental stages recovered by hysterectomy or surgical uterine flushing. J Vet Med Sci 2001;63:401–5.
The P4 level was measured by enzyme immunoassay [8]. 2.2. Recovery of early embryos and embryo transfer
4. Discussion We previously reported that lower uterine tube or upper uterine horn transfer of 8-cell embryos from dogs