Transcervical embryo collection in Boer goats
Suyadi, B. Sohnrey, W. Holtz
*Institute for Animal Husbandry and Genetics, University of Goettingen, Albrecht-Thaer-Weg 3, 37075 Goettingen, Germany
Abstract
Two experiments were conducted, aimed at improving the practicability of the method for transcervical embryo collection in Boer goats described by Pereira et al. [Pereira, R.J.T.A., Sohnrey, B., Holtz, W., 1998. J. Anim. Sci. 76, 360±363]. Invention of a hammock-like restraining device, use of a wider-bore ¯ushing catheter and a modi®ed ¯ushing mode contributed toward this end. The importance of a luteolytic prostaglandin F2a-treatment [Pereira et al., 1998] was con®rmed. In Experiment 1,
administration of PGF2a8 h before does are ¯ushed, increased the recovery rate from 43 to 79% (P<0.05). Advancing the PG
F2a-treatment to 24 h before ¯ushing was instrumental in further enhancing embryo recovery rate. The amount of time
required for ¯ushing was reduced by about 20 min (P<0.05) and the number of embryos recovered from the ®rst 10 out of 30 ¯ushes amounted to more than 80%, compared to 50% (P<0.05) when treating 8 h before ¯ushing. Administration of 1 IU of oxytocin at the onset of ¯ushing did not have any signi®cant effect. When applying the ®ndings of this investigation, the time required for ¯ushing may be reduced from about 4 h [Pereira et al., 1998] to less than 45 min per doe and the required number of person involved decreased from four to two persons.#2000 Elsevier Science B.V. All rights reserved.
Keywords:Goat; Embryo transfer; Embryo collection; Prostaglandin F2a
1. Introduction
Boer goats were initially introduced into Germany in 1980. Since 1985, repeated imports of frozen semen as well as frozen embryos in 1994, led to the establish-ment of a population of about 1500 registered goats, distributed over much of the Federal Republic of Germany. The nucleus herd is maintained at the Institute in GoÈttingen where rigorous selection for weight gain and conformation is practiced. Insemina-tion with frozen semen imported from South Africa
contributes to continuous herd improvement. Breed-ing stock and frozen embryos are sold to breeders in several European countries and abroad (Brazil, Republic of China etc.). The GoÈttingen herd is inten-sely utilized for research revolving primarily around reproductive functions and biotechnologies, as well as growth and carcass traits. Embryo transfer and embryo transfer-associated technologies have been a central activity at the Institute during the last decade. The areas covered in Boer goat research were superovula-tion (Nowshari et al., 1992, 1995), cryopreservasuperovula-tion (Yuswiati and Holtz, 1990; Puls-Kleingeld et al., 1992; Nowshari and Holtz, 1993, 1995), transfer technique (Besenfelder et al., 1994), in vitro-fertiliza-tion (Pereira et al., 1995) and micromanipulavitro-fertiliza-tion (Nowshari and Holtz, 1993).
*Corresponding author. Tel.:49-551-395605;
fax:49-551-395587.
E-mail address: [email protected] (W. Holtz)
The commonly applied surgical approach to embryo collection in goats has several disadvantages, such as the stress of anaesthesia and surgery, post-operative adhesions limiting the number of possible interventions and high expenses (Holtz, 1996). A laparoscopic approach puts less strain on the animals, but requires special instruments and skill. Transcervical embryo collection has repeatedly been attempted in goats and sometimes led to a degree of success (Bondurant et al., 1984; Nagashima et al., 1987; Bessoudo et al., 1988; Flores-Foxworth et al., 1992). In most cases the animals were sedated or anaesthetized and ¯ushed in ventral or dorsal recum-bency. A major break-through was achieved with the establishment of a practicable technique for non-sur-gical embryo collection (Pereira and Holtz, 1996; Pereira et al., 1998). The present investigation focuses on ways of improving the existing technique of trans-cervical embryo collection in Boer goats.
2. Materials and methods
Altogether 74 parous female Boer goats aged 35.112.2 (17±66) mo and weighing 56.229.7 (35±77) kg were synchronized by treatment with a progestin ear implant containing 1.5 mg Norgestomet (Crestar1
, Intervet, Boxmeer, Netherlands) for 11 days (Holtz and Sohnrey, 1992) and two i.m. injec-tions of 5 mg PGF2a (Dinolytic
1
, Pharmacia and Upjohn, Erlangen, Germany) administered at 12 h intervals on the day of implant removal. Animals were superovulated with 4, 4, 2, 2, 2 and 2 Armour Units of pFSH at 12 h intervals as described by Nowshari et al. (1995) and were naturally mated once or twice on each day of standing estrus. The number of corpora lutea were determined laparoscopically on the day prior to embryo collection, which took place 6 days after the last day of standing estrus.
In Experiment 1, 10 superovulated Boer goat does were treated i.m. with a luteolytic dose of 5 mg PGF2a(Dinolytic1) 8 h before embryo collection.
Nine does served as controls, receiving no PGF2a.
In Experiment 2, all 55 superovulated does received the luteolytic PGF2a-treatment. In three of the 55 does,
passage of the catheter through the cervical canal could not be accomplished. Of the remaining 52 does, 12 were treated with PGF2a8 h before ¯ushing (ÿ8 h
PG). The remaining 40 does were treated 24 h before ¯ushing. Of these 40 does, 20 were administered 1 IU oxytocin (Oxytocin-S1
, Intervet, Toenisvorst, Ger-many) i.v. after the ¯ushing catheter had been passed through the cervix (ÿ24 h PGOXT). The other 20 does received no oxytocin (ÿ24 h PG). The procedure of transcervical embryo collection was based on the technique described by Pereira et al. (1998), with the following modi®cations: (a) a restraining device was used, consisting of a hammock with holes for the front legs that prevented the animals from moving about or squatting during the ¯ushing procedure (Fig. 1). (b) The ¯ushing catheter (Ch 12, Ruesch, Kernen, Ger-many, Catalogue No. 220500) was thinner, had no in¯atable cuff and a wider bore than the one used by Pereira et al. (1998). This made for copious drainage of the infused media. (c) In Experiment 1, the volume of medium infused per ¯ush was increased from 20 (used during the earlier study) to 40 ml, where the ¯ushing mode was altered from 24 ¯ushes including a 2 h interruption to 30 uninterrupted ¯ushes. In Experi-ment 2, the original volume of 20 ml of ¯ushing medium per ¯ush was re-established. After introdu-cing the catheter into one uterine horn, 10 ¯ushes were conducted. The catheter was then changed to the other uterine horn for another 10 ¯ushes. Thereafter, each uterine horn was ¯ushed another ®ve times. The recovered ef¯uent was inspected under a stereo micro-scope (WILD M8, Switzerland) at a magni®cation of
20±50, to identify and evaluate embryos or ova.
Embryos were classi®ed based on morphological characters according to the method of Lindner and Wright (1983). Morphologically intact blastocysts classi®ed as `very good' or `good' were cryopreserved to be transferred at a later stage. Morulae are known to be unsuitable for cryopreservation (Nowshari and Holtz, 1995) and were thus cultured in vitro for 24 h. Those that continued to develop into blastocysts of very good or good quality, were considered trans-ferable and were cryopreserved.
Data for the number of embryos or ova recovered, recovery rate, duration of ¯ushing and number of transferable embryos were analyzed with a general linear model, followed by the Sheffe test (SAS Version 6.0). The proportion of embryos or ova recovered and transferable embryos identi®ed were analyzed by the w2-test (Steel and Torrie, 1960).
3. Results
The use of the restraining device proved to be very effective, replacing two helpers who would have had to restrain the animal and prevent it from squatting. The catheter was successfully passed through the cervical canal in each of the 19 animals in Experiment 1. In each of the two groups one doe yielded no embryos. As shown in Table 1, the injection of PGF2a
8 h before the onset of embryo collection increased the average number of embryos or ova recovered, com-pared to that of the controls (8.4 versus 4.4;P<0.05). The recovery rate was improved from 43 to 79% (P<0.05). In theÿ8 h PG-group 55% of the embryos or ova were in the blastocyst stage, 35% were morulae, 6% degenerate embryos and 3% uncleaved ova. The corresponding values for the control group are 30, 30,
30 and 9%, respectively. The duration of the ¯ushing process was similar for both groups (80 versus 77 min, respectively).
In Experiment 2, the catheter was successfully passed through the cervical canal of 52 out of the 55 does (95%). In the remaining three does, it was not possible to penetrate the cervix. No embryos or ova were recovered in the ¯ushings of two does in theÿ8 h PG group, four does in theÿ24 h PG group and two does in theÿ24 h PGOXT group. As shown in Table 2, the number of embryos or ova recovered and the recovery rate in does yielding embryos or ova were similar for all groups. Of the embryos or ova recov-ered, in the ÿ8 h PG-group 10% were blastocysts, 52% morulae, 17% degenerate embryos and 21% uncleaved ova. The corresponding values for the
ÿ24 h PG-group are 56, 31, 4 and 8%, respectively, and for the ÿ24 h PGOXT-group, 50, 44, 4 and 1%, respectively. The number of transferable em-bryos was higher in the ÿ24 h PG and ÿ24 h PGOXT groups, compared to theÿ8 h PG group, although the difference was not signi®cant. The dura-tion of the ¯ushing process was signi®cantly shorter (P<0.05) in both groups treated 24 h before ¯ushing, compared to the group treated 8 h beforehand. The injection of 1 IU of oxytocin prior to ¯ushing had very little effect on the recovery rate or duration of the ¯ushing process.
Table 3 differentiates between the 10 ¯ushes of the uterine horn ¯ushed ®rst, the 10 ¯ushes of the uterine horn ¯ushed second and the ®nal 10 ¯ushes (®ve on each side). The proportion of embryos or ova recov-ered from the ®rst 10 ¯ushes in the ÿ8 h GP group was signi®cantly (P<0.05) lower than that in the
ÿ24 h PG groups (50 versus 82 and 81%, respectively) (Fig. 2).
Table 1
Effect of PGF2ainjection 8 h before uterine ¯ushing(ÿ8 h PG) on number of embryos or ova recovered and duration of the ¯ushing procedure
(30 ¯ushes with 40 ml medium each)a
Treatment No. of does
Number of CL Embryos or ova recovered
Recovery rate (%)
Duration of flushing (min)
Mean SEM Mean SEM Mean SEM Mean SEM
ÿ8 h PG 9 10.8 1.4 8.4 a 1.4 79 a 8 77 6
Control 8 10.4 1.1 4.4 b 1.0 43 b 11 80 5
4. Discussion
When attempting to establish a technique for trans-cervical embryo collection in Boer goats, as a ®rst step a suitable catheter and a way of introducing it had to be found. Once this was accomplished, it was possible to collect embryos from does in a standing position at recovery rates of 50%. When preceding the ¯ushing process by treatment with a luteolytic dose of pros-taglandin F2a, the recovery rate was increased to 90%
(Pereira et al., 1998). In all likelihood, the decrease in plasma progesterone concentration accompanying luteolysis promotes uterine contractility in response to infusion of the ¯ushing medium. The main dis-advantage of the technique published is the substantial amount of time and labor required.
In the present investigation the use of a restraining device reduced the required personnel from the ori-ginal four to two persons and minimized strain on the animals. The larger bore catheter enhanced the unin-terrupted drainage of infused media. The increased volume (40 ml at a time) of medium infused during Experiment 1, resulted in leakage past the catheter and was discontinued in Experiment 2.
Experiment 1, which was conducted with the inten-tion to re-examine the necessity of the luteolytic prostaglandin treatment prior to ¯ushing, unequivo-cally con®rmed the ®ndings of Pereira et al. (1998). The prostaglandin treatment increased the proportion of embryos recovered from 43 to 79%. The study of Pereira et al. (1998) showed that by 16 h after pros-taglandin treatment, plasma progesterone concentra-tions had not quite reached basal levels. Therefore, in Experiment 2 the prostaglandin treatment was advanced to 24 h before embryo collection. As a consequence, the time required for collection was reduced and the proportion of embryos recovered from the ®rst 10 ¯ushes was increased, compared to the group treated 8 h before collection. More than 80% of the embryos recovered from the ¯ushings of the ®rst uterine horn indicate that embryos from both uterine horns are involved. The subsequent ¯ushing of the other uterine horn added another 12% of the total embryo yield. The corresponding values from the group treated 8 h before ¯ushing were 50 and 31% (P<0.05), respectively. Presumably this has to do with increased myometrial contractility, associated with a lower concentration of plasma progesterone.
Table 2
Effect of time of PGF2ainjection before uterine ¯ushing (8 or 24 h) and oxytocin (OXT) administration on embryos or ova recovered and
duration of the ¯ushing procedure (30 ¯ushes with 20 ml medium each)a
Treatment No. of does
Number of CL Embryos or ova recovered
Recovery rate (%)
Duration of flushing (min)
Mean SEM Total Transferable Mean SEM Mean SEM
Mean SEM Mean SEM %
ÿ8 h PG 10 11.0 1.3 7.2 1.5 2.9 1.1 40 67 11 83 a 8
ÿ24 h PG 16 11.8 1.3 6.9 1.2 5.4 0.9 78 58 13 67 b 2
ÿ24 h PGOXT 18 13.3 1.4 8.6 1.6 6.4 1.3 74 63 9 63 b 2
aValues in the same column with different alphabets differ (P<0.05).
Table 3
Distribution of embryos or ova recovered from the ®rst, second and last 10 ¯ushes (Experiment 2)a
Treatment First 10 flushes Second 10 flushes Third 10 flushes
Mean SEM % Mean SEM % Mean SEM %
ÿ8 h PG 3.6 1.5 50 a 2.2 0.5 31 a 1.4 0.3 19 a
ÿ24 h PG 5.5 1.1 82 b 0.9 0.2 13 b 0.2 0.1 4 b
ÿ24 h PGOXT 7.2 1.5 81 b 0.9 0.4 11 b 0.4 0.2 6 b
If operational time is a concern, e.g. when ¯ushing large numbers of donors, it might be justi®ed to waive the last 10 ¯ushes. This would mean sacri®cing of about 5% of the potential embryo yield, while saving time. As a consequence, the duration of embryo collection would amount to less than 45 min per doe. A renewed attempt to improve the ef®ciency of embryo collection by administration of a small dose of oxytocin at the onset of ¯ushing was made, although this treatment proved to be only marginally effective in the investigation by Pereira et al. (1998). The reason being that a better response was expected due to the lower plasma progesterone concentration. This did not materialize, however. In accordance with the previous study, the advantage of the oxytocin-treatment was marginal, not statistically signi®cant and therefore not justi®ed.
In this investigation it was succeeded in simplifying and accelerating the procedure of non-surgical embryo collection in superovulated Boer goats as described by Pereira and Holtz (1996) and Pereira et al. (1998). The main improvements involved the invention of a restraining device, the use of a wider-bore catheter, the earlier induction of luteolysis (24 h before ¯ushing compared to 8 or 16 h) and a modi®ed ¯ushing mode. These adjustments were instrumental in saving labor and time, without impairing recovery rate or embryo yield. The potential of an oxytocin treatment to further
improve upon the ef®ciency of embryo collection, e.g. by applying it at different dosages, should not be completely ruled out. The suitability of the embryo collection technique for breeds other than the Boer goat should be addressed in subsequent studies. It should be mentioned in this context, that, in a separate trial, nulliparous Boer goat does, although fully grown, gave most unsatisfactory embryo recovery rates.
Acknowledgements
The authors owe thanks to J.F. Beckers (Faculty of Veterinary Medicine, LieÂge, Belgium) for supplying FSH, Intervet (Boxmeer, Netherlands) and Pharma-ciaUpjohn (Erlangen, Germany) for donating pro-gestin implants and prostaglandin F2a, respectively.
The senior author was supported by a DAAD-fellow-ship.
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