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Embryo recovery rate and recipients’ pregnancy rate after nonsurgical embryo transfer in donkeys
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Theriogenology 73 (2010) 959–965 www.theriojournal.com
Embryo recovery rate and recipients’ pregnancy rate after nonsurgical embryo transfer in donkeys F. Camillo a,*, D. Panzani a, C. Scollo b, A. Rota a, A. Crisci a, I. Vannozzi a, S. Balbo b a
Dipartimento di Clinica Veterinaria, Universita` di Pisa, San Piero a Grado (PI), Italy b Azienda Regionale Foreste Demaniali Regione Siciliana, Palermo, Italy
Received 7 August 2009; received in revised form 18 November 2009; accepted 29 November 2009
Abstract Sixty-three embryos were recovered out of 83 estrous cycles (75.9%) and 98 ovulations (64.3%) of five Pantesca jennies, 2 to 5 yr old, naturally mated or artificially inseminated with fresh semen. Embryo recovery rate was influenced by number of ovulations per cycle (133% and 63% for double and single ovulations, respectively), by the day of embryo recovery attempt (12%, 83%, and 75% at Days 7, 8, and 9 after ovulation, respectively), and by the repetition of the embryo recovery attempt on successive cycles (60%, 79%, and 100% for cycles 1 to 7, 8 to 14, and 15 to 24, respectively). All recovered embryos but three were classified as good or excellent. Of 58 nonsurgical embryo transfers to Ragusana jenny recipients, 13 (22.4%), 10 (17.2%), and 9 (15.5%) resulted in a pregnancy at Days 14, 25, and 50, respectively. Recipients’ pregnancy rate was not influenced by the evaluated parameters: embryo quality and age, media employed to wash embryos, days after ovulation of the recipient, experience of the operator. Between 14 and 50 d of pregnancy, 4 of 13 (30.7%) embryos were lost with an influence of the days from ovulation of the recipient: recipients at Days 5 or 6 kept all pregnancies (N = 7), whereas recipients at Days 7 or 8 lost 3 of 4 pregnancies, as one of the two recipients at Day 3. More studies are needed before embryo transfer could be considered a reliable tool to preserve endangered donkey breeds. # 2010 Elsevier Inc. All rights reserved. Keywords: Donkey; embryo transfer
1. Introduction Donkeys have been domesticated and used in farm activities mainly for transport since 5000 B.C. [1]. After the Second World War, in the industrialized countries, there was a decline in the number of donkeys, and a number of breeds became threatened with extinction [2]; donkey conservation is imperative not only for cultural reasons and for their contribution to biodiversity [2], but also because they have an important role in production of milk [3] or for pet therapy [4].
* Corresponding author. Tel.: +39 050 2210163; fax: ++39 050 2210182.. E-mail address: [email protected] (F. Camillo). 0093-691X/$ – see front matter # 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2009.11.024
The Pantesca donkey, an Italian breed that has its roots in the island of Pantelleria, Sicily, has been considered extinct since 1980. Morphologic selection and genetic analyses performed on more than 200 potential Pantesca crossbreeds found nine subjects (three males and six females) with an estimated proportion of purity between 80% and 95%, and breeding from these nine donkeys resulted in a population of 35 animals [5]. Embryo transfer (ET) has been previously employed as a tool in the conservation of endangered equid species such as Przewalski’s horses (Equus przewalskii) [6], but data concerning ET in the donkey are still scarce and not encouraging [7–9]. Allen et al. showed that it was possible to recover embryos from donkeys by nonsurgical flushing of the uterus and reported a
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pregnancy rate at 40 d of 67% after their transfer to recipient mares by midline laparotomy [7]. However, the great majority of these conceptuses were aborted within 100 d of pregnancy, and half of the remaining fetuses died in the peripartum period [8]. Allen’s works strongly suggest that mares are not suitable recipients for donkey embryos. Vendramini et al. [9] transferred donkey embryos to donkeys, both surgically and nonsurgically, but their rate of success was extremely low. In horses, surgical intrauterine ET is no longer justified, because the nonsurgical technique, resulting in similar recipients’ pregnancy rate, is more ethical, less expensive, and less time consuming. To evaluate the possibility of a future use of ET as an aid to preserve Pantesca and other donkey breeds in threat of extinction, the aim of this study was to investigate the outcome of the application of mare ET techniques to donkeys, including nonsurgical transfer of embryos. 2. Materials and methods 2.1. Animals From March to December of two consecutive years, a total of 5 Pantesca (2 to 5 yr old, 219.4 27.6 kg/bw) and 16 Ragusana (4 to 16 yr old, 304.8 45.6 kg/bw) jennies, of unknown fertility, were used as embryo donors and recipients, respectively. Four donors and five recipients, not pregnant after the first year, were used in both years. Three Pantesca jacks (20, 15, and 13 yr old, 252.3 8.1 kg/bw) known to be fertile were used to mate the donors. The jennies were maintained in paddocks, the jacks in boxes; all animals were fed with a balanced ration of hay and commercial horse feed. 2.2. Estrus control and mating Estrus was induced using 3 mg of the prostaglandin F2a (PGF2a) analogue alfaprostol (Gabbrostim; Vetem, Agrigento, Italy) intramuscularly. Ovarian activity of donors and recipients was evaluated daily during estrus by transrectal ultrasound. From the detection of both a growing follicle 30 mm in diameter and estrous behavior, donors were naturally mated or artificially inseminated with fresh semen every second day until ovulation. For artificial insemination, semen was collected using a Colorado-type artificial vagina (equipped with disposable liners and a glass collection bottle) while the jack jumped on an estrous jenny. After each donor’s ovulation, the recipient in estrus with the largest follicle was treated with 2000 IU human
chorionic gonadotropin (hCG; Vetecor; Bio 98, Bologna, Italy) intravenously to induce ovulation. 2.3. Embryo recovery and transfer Seven to 9 d after ovulation, donors were flushed three times for embryo recovery using a total of 2 L flushing media kept at 37 8C. The flushings were performed using either DPBS (ZE067; I.M.V., L’Aigle, France) and a one-way tubing system without filter or lactated Ringer (Galenica Senese, Siena, Italy) and a self-built ‘‘Y’’ tubing system using an EZ Way filter (PETS, Canton, TX, USA). For both techniques, an Equine Lavage Catether 32 F (AB Technology, Pullman, WA, USA) was used. Each recovered embryo was evaluated for morphology [10], washed 10 times, placed in a 0.25-mL French straw with a small amount of the washing medium, and transferred transcervically to a recipient treated intravenously with 3.3 mg/100 kg acepromazine (Prequillan; Fatro, Bologna, Italy) using a guarded French gun [11]. Some of the embryos were measured. Washing medium was either PBS (ZE067, N = 14 embryos), lactated Ringer (N = 33 embryos), or Emcare Holding Solution (Bio98 Srl, Milano, Italy; N = 11 embryos). Embryos were transferred by four operators, one experienced (with much experience both in mare palpation and equine ET) and three inexperienced: A (with much experience in mare palpation and no experience in equine ET) and B and C (with moderate experience in mare palpation and no experience in equine ET). 2.4. Ultrasound for pregnancy Pregnancy diagnosis was performed by transrectal ultrasound at 14 and 16 d after the donor’s ovulation. Pregnant jennies were scanned again at 25 and 50 d to diagnose a normal pregnancy or an eventual embryo loss. 2.5. Statistical analyses The effects of year of the study, donor jenny, number of ovulations, day of flushing, successive cycle within donor, and season of the year on positive uterine flushing rates (flushings where at least one embryo was recovered) and on embryo recovery rates (embryos recovered per cycle) were evaluated by the chi-square test and by the Kruskal-Wallis one-way ANOVA, respectively. When the effect was statistically
F. Camillo et al. / Theriogenology 73 (2010) 959–965
significant, multiple comparisons were performed by the chi-square test for positive uterine flushings and by the Mann-Whitney test for embryo recovery rates. The effects of year of the study, day of flushing (8 or 9), washing medium, successive cycle within recipient (1 to 2 vs. 3 to 7), season of the year, and operator (experienced vs. inexperienced) on recipients’ pregnancy rates were evaluated by the chi-square test. The differences between donor jennies, recipient’s day after ovulation at transfer, successive cycle within recipient, and operators (experienced and inexperienced) on recipients’ pregnancy rates were evaluated by Fisher’s exact test. Also, the differences in pregnancy losses between recipients transferred at Days 3, 5 to 6, or 7 to 8 after their ovulation were evaluated by Fisher’s exact test. Analyses were performed using the statistical package Minitab 14.1 (Minitab Inc., State College, PA, USA). Difference were considered statistically significant when P < 0.05. 3. Results A total of 63 embryos were recovered out of 83 estrous cycles (75.9%) and 98 ovulations (64.3%). Factors affecting the rate of positive flushings (when at least one embryo was recovered) and the embryo
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recovery rate (the number of embryos recovered per cycle) are reported in Table 1. All recovered embryos but three were classified as good or excellent. Mean diameter of 8-d-old donkey embryos was 720 217 mm (N = 8). Fifty-nine embryos were transferred to recipients at Days 3 to 9 after ovulation; on one occasion two embryos were transferred simultaneously to one recipient (as no other recipient was available). Of 58 transfers, 13 (22.4%), 10 (17.2%), and 9 (15.5%) recipients were pregnant at Days 14, 25, and 50, respectively. The transfer of two embryos to the same recipient resulted in a single pregnancy. The factors affecting 14-d recipients’ pregnancy rate are reported in Table 2. When the outcome of the first two successive transfers to the same recipients and in the following ones (3 to 7) were compared, no statistical difference was observed. No statistical difference was observed also in pregnancy rates obtained by the experienced compared with the three inexperienced operators. Four of 13 (30.7%) embryos were lost between 14 and 50 d of pregnancy with an influence of the day following recipient’s ovulation in which ET was done: recipients at Day 5 or 6 (N = 7) had no pregnancy losses while recipients at Day 7 or 8 lost 3 of 4 pregnancies (P = 0.024); one of the two recipients at Day 3 lost her pregnancy.
Table 1 Factors affecting the rate of positive flushings and embryo recovery rate in Pantesca jennies. Factor Year First Second Donor A B C D E Number of ovulations 1 2 Day of flushing (N = 82) 7 8 9 Successive cycle in the same donor 1–7 8–14 15–24 Time of the year March–September October–December NS, not significant. A 6¼ B = P < 0.05.
Positive flushings
P value
Embryo recovery rate
P value
27/43 (62.8%) 30/40 (75.0%)
NS
29/43 (0.67) 34/40 (0.85)
NS
18/24 (75%) 17/23 (73.9%) 9/14 (63.3%) 6/13 (46.15%) 7/9 (77.8%)
NS
21/24 (0.87) 19/23 (0.83) 10/14 (0.71) 6/13 (0.46) 7/9 (0.78)
NS
43/68 (63.2%) 14/15 (93.3%)
0.023
43/68 (0.63) 20/15 (1.33)
0.000
1/8 (12.5%)A 43/58 (79.1%)B 12/16 (75%)B
0.002
1/8 (0.12)A 48/58 (0.83)B 12/16 (0.75)B
0.003
19/35 (54.3%)A 22/29 (75.9%) 16/19 (84.2%)B
0.045
21/35 (0.60)A 23/29 (0.79) 19/19 (1.00)B
0.044
41/64 (64.1%) 16/19 (84.2%)
NS
47/64 (0.73) 16/19 (0.84)
NS
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Table 2 Factors affecting 14-d recipient pregnancy in jennies. Factor Year
Pregnancy rates
P value
First Second
5/27 (18.5%) 8/31 (25.8%)
NS
A B C D E
6/19 (31.6%) 4/19 (21%) 2/8 (25%) 0/6 (0) 1/6 (16.7%)
NS
7 8 9
0/1 (0) 9/44 (20.4%) 4/13 (30.8%)
PBS Ringer lactate Emcare
1/14 (7.1%) 9/33 (27.2%) 3/11 (27.2%)
NS
3 4 5 6 7 8 9
2/6 (33.3%) 0/6 (0) 5/13 (38.5%) 2/10 (20%) 3/12 (25%) 1/6 (16.6%) 0/3 (0)
NS
1 2 3 4 5 6 7*
4/16 (25%) 1/11 (9%) 2/9 (22.2%) 2/7 (28.5%) 2/5 (40%) 0/3 (0) 2/3 (66.7%)
NS
March–September October–December
8/43 (18.6%) 5/15 (33.3%)
NS
Experienced Inexperienced A Inexperienced B Inexperienced C
6/23 (26%) 4/15 (26.6%) 2/6 (33.3%) 0/4 (0)
NS
Donor
Day of flushing NS
Washing medium
Day following ovulation (N = 56)
Successive cycle in the same recipient
Time of the year
Operator (N = 48)
NS, not significant. *One recipient was transferred four more times (cycles 8 to 11) with no pregnancy.
4. Discussion Although a direct comparison is not possible, the embryo recovery rate obtained in this study, 75.9%, was higher than that previously reported in donkeys, 53.3% [7] and 63.6% [8], and in commercial ET programs in horses, around 50% [12]. It was, however, lower than that obtained when young mares were inseminated with fresh semen, 87% [13]. These results confirm that, in spite of the small dimensions of donkey donors and of their tight cervixes, nonsurgical embryo recovery is not a problem in this species.
Factors affecting embryo recovery in donkeys have never been evaluated, whereas in horses the day of recovery, the number of ovulations, the age of the donor mare, and the quality of the semen have all been reported to influence embryo recovery rate [14]. In this study, more embryos were recovered when flushing of the uterus was performed on Days 8 and 9 compared with that after flushing on Day 7. This could be due either to the larger diameter of older embryos, resulting in an easier recovery, or to a delay in the descent in the uterus of donkey embryos compared with that for horse and cow embryos, although Allen’s results, about 53%
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of embryo recovery rate at Day 7 [7], make this theory less probable. Moreover, it is necessary to take into account that uterine flushings for embryo recovery were performed at Day 7 only at the beginning of the study, when some failures could have been due to the inexperience with these procedures in donkeys. In mares, embryo recovery rate was similar flushing the uterus 7 to 9 d after ovulation [15]. As in horses, the number of ovulations positively affected the possibility to recover an embryo. In particular, the embryo recovery rate after single or double ovulations in the studied jennies, 63% and 133%, was very close to that reported in mares, 65% and 116% [16]. Differences in embryo recovery rate due to year of the study, donors, and season were not observed. In spite of the absence of statistical significance, the embryo recovery rate obtained in the first year, 67%, was lower than that obtained in the second year, 85%: This could be explained both by the need to be familiar with the reproductive apparatus of the jenny and by the choice of flushing the uterus at Day 7 at the beginning of the study. Even though only fresh semen from stallions of good fertility was employed and all donors were young, some individual, non-statistical differences were observed: One jenny provided embryos in less than 50% of the cycles, whereas two jennies were excellent donors, with an embryo recovery rate higher than 80%. No differences were observed in donors’ fertility between time periods, March–September or October– December. This was not unattended based on the continuous ovarian activity of the donkey breeds studied in Italy, Martinafranca and Amiata [17,18], and previously observed in Pantesca and Ragusana jennies (F. Camillo, unpublished data). This could extend the possibility to apply ET in donkeys compared with horses, where both the seasonality and the rules of the stud books limit the ET activity to spring and summer. Carnevale et al [19] reported that repeated inseminations and embryo collections in mares, approximately seven in one breeding season, were associated with increased chronic inflammation of the uterus. This negative effect was not observed in this study as the proportion of recovered embryos on successive attempts from 8 to 14 and from 15 to 24 was higher than in attempts from 1 to 7. The differences between horses and donkeys of the two studies may be explained by several factors such as age of the animals, differences in methods used to evaluate uterine damage, and selection of donors. Age of mares was not reported [19] while jennies were very young, and it is well known that
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young age is generally associated with a condition of resistance to endometritis [20]. Uterine score biopsy, used in mares by Carnevale and co-workers, is probably a much more sensitive method than embryo recovery rate for the evaluation of the possible damage of repeated uterine lavages. Moreover, in this study a selection of the jennies was made, as only the two excellent donors were employed during the entire period, for 23 and 24 cycles, respectively. The recipients’ pregnancy rate obtained in this study, about 22% at 14 d and 15% at 50 d, confirmed the extremely poor results reported in the only study available on transfer of donkey embryos to donkeys, where only 5.5% of recipients were pregnant after surgical or nonsurgical transfer [9]. These percentages are lower than that observed after both surgical or nonsurgical transfer of horse embryos to horses (50% to 75%) [14] and than that described after surgical transfer of donkey embryos to mares or of horse embryos to jennies (67% and 55%, respectively) [7]. In an equine ET program, recipients’ pregnancy rate is influenced mostly by embryo quality and management of recipients [12]. Embryos graded 3 or 4 or with a delayed development resulted in a lower recipient pregnancy rate than embryos graded 1 or 2 and with a normal development [21]; moreover, embryos larger than 2 mm were reported to be difficult to manipulate and susceptible to damage resulting in a low recipients’ pregnancy rate [21]. In the current study, all donkey embryos were smaller than 2 mm, only three appeared smaller based on estimated age, and all but three were classified as good or excellent (score 1 and 2): for this reason, it seems difficult to relate the poor pregnancy rates with embryo quality. However, it is not possible to exclude some embryo defects or damage caused by flushing or by washing procedures or media and not appreciable with the simple morphologic evaluation. Although there was not an overall significant effect of embryo washing media, PBS resulted in a pregnancy rate about 4 times lower than that with lactated Ringer or Emcare. Quality of recipients, based on uterine and cervical tone at Day 5 postovulation, significantly affected pregnancy rate in mares [12,21]; in the current study, recipient jennies were not examined before transfer as, due to their small number, most of the time only one recipient was available. Moreover, in an equine ET program, recipients are excluded if they are not pregnant on two consecutive occasions: This rule could not be applied to the current study. In mares, embryos are generally transferred to recipients ovulating from 1 d before to 3 d after the
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donor, corresponding with Days 5 to 9 after their ovulation [21]. In the current study, again due to the small number of recipients, embryos were transferred not only to jennies at Days 5 to 9 but also at Day 3 or 4 after ovulation, without differences in 14-d pregnancy rates; however, it is possible that the overall very small number of pregnancies obtained may account for the lack of statistical differences between recipients at different days after ovulation. The donkey embryo loss observed in the current study, 30.7% between 14 and 50 d of pregnancy, was higher than that normally observed in mares after natural mating or artificial insemination [22] or employed as embryo recipients [21]. The pregnancy loss rate was higher for embryos transferred to jennies at Day 7 or 8 than for embryos transferred to jennies at Day 5 or 6 after ovulation. Similar findings were reported in a large number of mares where embryo loss rate was 7.3% to 8.6%, 15.1% to 22.4%, and 30.8% in recipients at Days 5 to 6, 7 to 8, and 9 after ovulation, respectively [21]. To explain this observation, a more efficient maternal recognition of pregnancy in mares at an earlier stage after ovulation or the need of an embryonic signal earlier than at Days 7 to 9 after ovulation was advocated [21]. Nonsurgical transfer of embryos to jennies was more difficult than it is in mares due to the donkey cervix, which appeared smaller, longer, and tighter than the equine cervix. In most cases, the cervixes of the jennies were so contracted that a cranial deviation of the distal portion of this structure was appreciable, making the cervical ostium looking cranially and not caudally. In spite of the expected difficulty to catheterize the cervixes of jennies, the recipients’ pregnancy rate achieved by the only experienced operator was the same as that achieved by the three inexperienced operators, 6 of 23 and 6 of 25, respectively. In summary, the current study resulted in a good embryo recovery rate, comparable with or even higher than that normally observed in mares, but in an unacceptable low recipients’ pregnancy rate. The reasons for these poor results remain to be understood as, in the current study, the ET techniques routinely employed in horses were simply transferred to the donkey, with the possibility that several factors had a negative effect on the outcome. For instance, it is possible that donkey embryos have different requirements with respect to flushing and washing media: from this point of view, this study seems to indicate that Ringer lactate and Emcare Holding Solution could be a better choice for embryos than PBS. In particular, it is possible that donkey embryos are more susceptible to
the potential inhibition of embryo development by phosphate [23]. Other critical points could be the donkey cervix and uterus. Transcervical ET in jennies required more manipulations than that in mares, thus, release of PGF2a, as reported during cervical manipulation in some mares [24], followed by a decrease in progesterone plasma concentration and embryo death, can be hypothesized. Moreover, it is not possible to exclude an irritating action on the recipients’ uterus induced by the holding media, similar to the one hypothesized to explain the poor results of artificial insemination with frozen semen in jennies [25,26]. In the current study, a critical evaluation of PGF2a or progesterone levels was not performed, and clinical signs of endometritis or PGF2a release after transfer with an early return to estrus were not recorded. Finally, recipients may have had an important role in the outcome of ET but it was neither possible to know their reproductive histories nor to clinically evaluate their quality before transfer. In conclusion, the results of this study suggest that other investigations are needed before ET could be considered a reliable tool to preserve endangered donkey breeds. Acknowledgments This study was funded and managed by the Azienda Regionale Foreste Demaniali Regione Siciliana with the cooperation of Istituto Incremento Ippico per la Sicilia. References [1] Rossel S, Marshall F, Peters J, Pilgram T, Adams MD, O’Connor D. Domestication of the donkey: timing, processes, and indicators. Proc Natl Acad Sci USA 2008;105:3715–20. [2] Scherf BD. World Watch List for Domestic Animal Diversity. FAO 2000. [3] Vita D, Passalacqua G, Di Pasquale G, Caminiti L, Crisafulli G, Rulli I, Pajno GB. Ass’s milk in children with atopic dermatitis and cow’s milk allergy: crossover comparison with goat’s milk. Pediatr Allergy Immunol 2007;18:594–8. [4] Feather PM. Riding for the disabled at the Slade Centre. In: The Donkey Sanctuary, The Professional Handbook of the Donkey, 2nd Edition, Svendsen ED (Ed.), Sovereign Printing Group, 1989, pp. 247-258. [5] Regione Siciliana Azienda Foreste Demaniali. Allevamento degli asini di Pantelleria. Sicilia Foreste Suppl 2004. [6] Summers PM, Shephard AM, Hodges JK, Kydd J, Boyle MS, Allen WR. Successful transfer of the embryos of Przewalski’s Horses (Equus przewalskii) and Grant’s Zebra (E. burchelli) to domestic mares (Equus caballus). J Reprod Fertil 1987;80:13–20. [7] Allen WR, Kydd J, Boyle MS, Antczac DF. Between-species transfer of horse and donkey embryos: a valuable research tool. Equine Vet J 1985;3:53–62.
F. Camillo et al. / Theriogenology 73 (2010) 959–965 [8] Allen WR, Kydd DF, Antczak DF. Intraspecies and extraspecies equine pregnancies. In: Equine Reproduction, McKinnon AO, Voss JL (Eds.), Lea & Febiger, 1993, pp. 536-553. [9] Vendramini OM, Bruyas JF, Fieni F, Battut I, Tainturier D. Embryo transfer in Poitou donkeys, preliminary results. Theriogenology 1997;47:409. [10] McKinnon AO, Squires EL. Equine embryo transfer. Vet Clin North Am Equine Pract 1988;4:305–33. [11] Lagneaux D, Duchamp G, Palmer E. La transplantation embryonnaire chez la jument. In : 148 CEREOPA, 1988, pp. 163-181. [12] Squires EL, Carnevale EM, McCue PM, Bruemmer JE. Embryo technologies in the horse. Theriogenology 2003;59:151–70. [13] Camillo F, Vannozzi I, Rota A, Romagnoli S, Aria G. Comparison of embryo recovery rates from two years old and mature mares. In: Proceedings 5th InternationalSymposium on Equine Embryo Transfer, Havemeyer Foundation Monograph Series 3; 2001. p. 86–7. [14] Squires EL, McCue PM, Vanderwall D. The current status of equine embryo transfer. Theriogenology 1999;51:91–104. [15] Fleury JJ, Alvarenga MA. Effect of collection day on embryo recovery and pregnancy rates in a nonsurgical equine embryo transfer program. Theriogenology 1999;51:261. [16] Losinno L, Aguillar JJ, Lisa H. Impact of multiple ovulations in a commercial embryo transfer programme. In: Proceedings 5th International Symposium on Equine Embryo Transfer, Havemeyer Foundation Monograph Series 3; 2001. p. 81–3. [17] Carluccio A, Pau S, Picci G, Tosi U. De Amicis I Zedda MT, et al. Il ciclo estrale nell’asina di Martina Franca. Veterinaria Pratica Equina 2003;3:41. [18] Panzani D, Crisci A, Viggiani R, Rota A, Camillo F. Clinical, behavioural and endocrinological characteristics of the Amiata
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Theriogenology 73 (2010) 959–965 www.theriojournal.com
Embryo recovery rate and recipients’ pregnancy rate after nonsurgical embryo transfer in donkeys F. Camillo a,*, D. Panzani a, C. Scollo b, A. Rota a, A. Crisci a, I. Vannozzi a, S. Balbo b a
Dipartimento di Clinica Veterinaria, Universita` di Pisa, San Piero a Grado (PI), Italy b Azienda Regionale Foreste Demaniali Regione Siciliana, Palermo, Italy
Received 7 August 2009; received in revised form 18 November 2009; accepted 29 November 2009
Abstract Sixty-three embryos were recovered out of 83 estrous cycles (75.9%) and 98 ovulations (64.3%) of five Pantesca jennies, 2 to 5 yr old, naturally mated or artificially inseminated with fresh semen. Embryo recovery rate was influenced by number of ovulations per cycle (133% and 63% for double and single ovulations, respectively), by the day of embryo recovery attempt (12%, 83%, and 75% at Days 7, 8, and 9 after ovulation, respectively), and by the repetition of the embryo recovery attempt on successive cycles (60%, 79%, and 100% for cycles 1 to 7, 8 to 14, and 15 to 24, respectively). All recovered embryos but three were classified as good or excellent. Of 58 nonsurgical embryo transfers to Ragusana jenny recipients, 13 (22.4%), 10 (17.2%), and 9 (15.5%) resulted in a pregnancy at Days 14, 25, and 50, respectively. Recipients’ pregnancy rate was not influenced by the evaluated parameters: embryo quality and age, media employed to wash embryos, days after ovulation of the recipient, experience of the operator. Between 14 and 50 d of pregnancy, 4 of 13 (30.7%) embryos were lost with an influence of the days from ovulation of the recipient: recipients at Days 5 or 6 kept all pregnancies (N = 7), whereas recipients at Days 7 or 8 lost 3 of 4 pregnancies, as one of the two recipients at Day 3. More studies are needed before embryo transfer could be considered a reliable tool to preserve endangered donkey breeds. # 2010 Elsevier Inc. All rights reserved. Keywords: Donkey; embryo transfer
1. Introduction Donkeys have been domesticated and used in farm activities mainly for transport since 5000 B.C. [1]. After the Second World War, in the industrialized countries, there was a decline in the number of donkeys, and a number of breeds became threatened with extinction [2]; donkey conservation is imperative not only for cultural reasons and for their contribution to biodiversity [2], but also because they have an important role in production of milk [3] or for pet therapy [4].
* Corresponding author. Tel.: +39 050 2210163; fax: ++39 050 2210182.. E-mail address: [email protected] (F. Camillo). 0093-691X/$ – see front matter # 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2009.11.024
The Pantesca donkey, an Italian breed that has its roots in the island of Pantelleria, Sicily, has been considered extinct since 1980. Morphologic selection and genetic analyses performed on more than 200 potential Pantesca crossbreeds found nine subjects (three males and six females) with an estimated proportion of purity between 80% and 95%, and breeding from these nine donkeys resulted in a population of 35 animals [5]. Embryo transfer (ET) has been previously employed as a tool in the conservation of endangered equid species such as Przewalski’s horses (Equus przewalskii) [6], but data concerning ET in the donkey are still scarce and not encouraging [7–9]. Allen et al. showed that it was possible to recover embryos from donkeys by nonsurgical flushing of the uterus and reported a
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pregnancy rate at 40 d of 67% after their transfer to recipient mares by midline laparotomy [7]. However, the great majority of these conceptuses were aborted within 100 d of pregnancy, and half of the remaining fetuses died in the peripartum period [8]. Allen’s works strongly suggest that mares are not suitable recipients for donkey embryos. Vendramini et al. [9] transferred donkey embryos to donkeys, both surgically and nonsurgically, but their rate of success was extremely low. In horses, surgical intrauterine ET is no longer justified, because the nonsurgical technique, resulting in similar recipients’ pregnancy rate, is more ethical, less expensive, and less time consuming. To evaluate the possibility of a future use of ET as an aid to preserve Pantesca and other donkey breeds in threat of extinction, the aim of this study was to investigate the outcome of the application of mare ET techniques to donkeys, including nonsurgical transfer of embryos. 2. Materials and methods 2.1. Animals From March to December of two consecutive years, a total of 5 Pantesca (2 to 5 yr old, 219.4 27.6 kg/bw) and 16 Ragusana (4 to 16 yr old, 304.8 45.6 kg/bw) jennies, of unknown fertility, were used as embryo donors and recipients, respectively. Four donors and five recipients, not pregnant after the first year, were used in both years. Three Pantesca jacks (20, 15, and 13 yr old, 252.3 8.1 kg/bw) known to be fertile were used to mate the donors. The jennies were maintained in paddocks, the jacks in boxes; all animals were fed with a balanced ration of hay and commercial horse feed. 2.2. Estrus control and mating Estrus was induced using 3 mg of the prostaglandin F2a (PGF2a) analogue alfaprostol (Gabbrostim; Vetem, Agrigento, Italy) intramuscularly. Ovarian activity of donors and recipients was evaluated daily during estrus by transrectal ultrasound. From the detection of both a growing follicle 30 mm in diameter and estrous behavior, donors were naturally mated or artificially inseminated with fresh semen every second day until ovulation. For artificial insemination, semen was collected using a Colorado-type artificial vagina (equipped with disposable liners and a glass collection bottle) while the jack jumped on an estrous jenny. After each donor’s ovulation, the recipient in estrus with the largest follicle was treated with 2000 IU human
chorionic gonadotropin (hCG; Vetecor; Bio 98, Bologna, Italy) intravenously to induce ovulation. 2.3. Embryo recovery and transfer Seven to 9 d after ovulation, donors were flushed three times for embryo recovery using a total of 2 L flushing media kept at 37 8C. The flushings were performed using either DPBS (ZE067; I.M.V., L’Aigle, France) and a one-way tubing system without filter or lactated Ringer (Galenica Senese, Siena, Italy) and a self-built ‘‘Y’’ tubing system using an EZ Way filter (PETS, Canton, TX, USA). For both techniques, an Equine Lavage Catether 32 F (AB Technology, Pullman, WA, USA) was used. Each recovered embryo was evaluated for morphology [10], washed 10 times, placed in a 0.25-mL French straw with a small amount of the washing medium, and transferred transcervically to a recipient treated intravenously with 3.3 mg/100 kg acepromazine (Prequillan; Fatro, Bologna, Italy) using a guarded French gun [11]. Some of the embryos were measured. Washing medium was either PBS (ZE067, N = 14 embryos), lactated Ringer (N = 33 embryos), or Emcare Holding Solution (Bio98 Srl, Milano, Italy; N = 11 embryos). Embryos were transferred by four operators, one experienced (with much experience both in mare palpation and equine ET) and three inexperienced: A (with much experience in mare palpation and no experience in equine ET) and B and C (with moderate experience in mare palpation and no experience in equine ET). 2.4. Ultrasound for pregnancy Pregnancy diagnosis was performed by transrectal ultrasound at 14 and 16 d after the donor’s ovulation. Pregnant jennies were scanned again at 25 and 50 d to diagnose a normal pregnancy or an eventual embryo loss. 2.5. Statistical analyses The effects of year of the study, donor jenny, number of ovulations, day of flushing, successive cycle within donor, and season of the year on positive uterine flushing rates (flushings where at least one embryo was recovered) and on embryo recovery rates (embryos recovered per cycle) were evaluated by the chi-square test and by the Kruskal-Wallis one-way ANOVA, respectively. When the effect was statistically
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significant, multiple comparisons were performed by the chi-square test for positive uterine flushings and by the Mann-Whitney test for embryo recovery rates. The effects of year of the study, day of flushing (8 or 9), washing medium, successive cycle within recipient (1 to 2 vs. 3 to 7), season of the year, and operator (experienced vs. inexperienced) on recipients’ pregnancy rates were evaluated by the chi-square test. The differences between donor jennies, recipient’s day after ovulation at transfer, successive cycle within recipient, and operators (experienced and inexperienced) on recipients’ pregnancy rates were evaluated by Fisher’s exact test. Also, the differences in pregnancy losses between recipients transferred at Days 3, 5 to 6, or 7 to 8 after their ovulation were evaluated by Fisher’s exact test. Analyses were performed using the statistical package Minitab 14.1 (Minitab Inc., State College, PA, USA). Difference were considered statistically significant when P < 0.05. 3. Results A total of 63 embryos were recovered out of 83 estrous cycles (75.9%) and 98 ovulations (64.3%). Factors affecting the rate of positive flushings (when at least one embryo was recovered) and the embryo
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recovery rate (the number of embryos recovered per cycle) are reported in Table 1. All recovered embryos but three were classified as good or excellent. Mean diameter of 8-d-old donkey embryos was 720 217 mm (N = 8). Fifty-nine embryos were transferred to recipients at Days 3 to 9 after ovulation; on one occasion two embryos were transferred simultaneously to one recipient (as no other recipient was available). Of 58 transfers, 13 (22.4%), 10 (17.2%), and 9 (15.5%) recipients were pregnant at Days 14, 25, and 50, respectively. The transfer of two embryos to the same recipient resulted in a single pregnancy. The factors affecting 14-d recipients’ pregnancy rate are reported in Table 2. When the outcome of the first two successive transfers to the same recipients and in the following ones (3 to 7) were compared, no statistical difference was observed. No statistical difference was observed also in pregnancy rates obtained by the experienced compared with the three inexperienced operators. Four of 13 (30.7%) embryos were lost between 14 and 50 d of pregnancy with an influence of the day following recipient’s ovulation in which ET was done: recipients at Day 5 or 6 (N = 7) had no pregnancy losses while recipients at Day 7 or 8 lost 3 of 4 pregnancies (P = 0.024); one of the two recipients at Day 3 lost her pregnancy.
Table 1 Factors affecting the rate of positive flushings and embryo recovery rate in Pantesca jennies. Factor Year First Second Donor A B C D E Number of ovulations 1 2 Day of flushing (N = 82) 7 8 9 Successive cycle in the same donor 1–7 8–14 15–24 Time of the year March–September October–December NS, not significant. A 6¼ B = P < 0.05.
Positive flushings
P value
Embryo recovery rate
P value
27/43 (62.8%) 30/40 (75.0%)
NS
29/43 (0.67) 34/40 (0.85)
NS
18/24 (75%) 17/23 (73.9%) 9/14 (63.3%) 6/13 (46.15%) 7/9 (77.8%)
NS
21/24 (0.87) 19/23 (0.83) 10/14 (0.71) 6/13 (0.46) 7/9 (0.78)
NS
43/68 (63.2%) 14/15 (93.3%)
0.023
43/68 (0.63) 20/15 (1.33)
0.000
1/8 (12.5%)A 43/58 (79.1%)B 12/16 (75%)B
0.002
1/8 (0.12)A 48/58 (0.83)B 12/16 (0.75)B
0.003
19/35 (54.3%)A 22/29 (75.9%) 16/19 (84.2%)B
0.045
21/35 (0.60)A 23/29 (0.79) 19/19 (1.00)B
0.044
41/64 (64.1%) 16/19 (84.2%)
NS
47/64 (0.73) 16/19 (0.84)
NS
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Table 2 Factors affecting 14-d recipient pregnancy in jennies. Factor Year
Pregnancy rates
P value
First Second
5/27 (18.5%) 8/31 (25.8%)
NS
A B C D E
6/19 (31.6%) 4/19 (21%) 2/8 (25%) 0/6 (0) 1/6 (16.7%)
NS
7 8 9
0/1 (0) 9/44 (20.4%) 4/13 (30.8%)
PBS Ringer lactate Emcare
1/14 (7.1%) 9/33 (27.2%) 3/11 (27.2%)
NS
3 4 5 6 7 8 9
2/6 (33.3%) 0/6 (0) 5/13 (38.5%) 2/10 (20%) 3/12 (25%) 1/6 (16.6%) 0/3 (0)
NS
1 2 3 4 5 6 7*
4/16 (25%) 1/11 (9%) 2/9 (22.2%) 2/7 (28.5%) 2/5 (40%) 0/3 (0) 2/3 (66.7%)
NS
March–September October–December
8/43 (18.6%) 5/15 (33.3%)
NS
Experienced Inexperienced A Inexperienced B Inexperienced C
6/23 (26%) 4/15 (26.6%) 2/6 (33.3%) 0/4 (0)
NS
Donor
Day of flushing NS
Washing medium
Day following ovulation (N = 56)
Successive cycle in the same recipient
Time of the year
Operator (N = 48)
NS, not significant. *One recipient was transferred four more times (cycles 8 to 11) with no pregnancy.
4. Discussion Although a direct comparison is not possible, the embryo recovery rate obtained in this study, 75.9%, was higher than that previously reported in donkeys, 53.3% [7] and 63.6% [8], and in commercial ET programs in horses, around 50% [12]. It was, however, lower than that obtained when young mares were inseminated with fresh semen, 87% [13]. These results confirm that, in spite of the small dimensions of donkey donors and of their tight cervixes, nonsurgical embryo recovery is not a problem in this species.
Factors affecting embryo recovery in donkeys have never been evaluated, whereas in horses the day of recovery, the number of ovulations, the age of the donor mare, and the quality of the semen have all been reported to influence embryo recovery rate [14]. In this study, more embryos were recovered when flushing of the uterus was performed on Days 8 and 9 compared with that after flushing on Day 7. This could be due either to the larger diameter of older embryos, resulting in an easier recovery, or to a delay in the descent in the uterus of donkey embryos compared with that for horse and cow embryos, although Allen’s results, about 53%
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of embryo recovery rate at Day 7 [7], make this theory less probable. Moreover, it is necessary to take into account that uterine flushings for embryo recovery were performed at Day 7 only at the beginning of the study, when some failures could have been due to the inexperience with these procedures in donkeys. In mares, embryo recovery rate was similar flushing the uterus 7 to 9 d after ovulation [15]. As in horses, the number of ovulations positively affected the possibility to recover an embryo. In particular, the embryo recovery rate after single or double ovulations in the studied jennies, 63% and 133%, was very close to that reported in mares, 65% and 116% [16]. Differences in embryo recovery rate due to year of the study, donors, and season were not observed. In spite of the absence of statistical significance, the embryo recovery rate obtained in the first year, 67%, was lower than that obtained in the second year, 85%: This could be explained both by the need to be familiar with the reproductive apparatus of the jenny and by the choice of flushing the uterus at Day 7 at the beginning of the study. Even though only fresh semen from stallions of good fertility was employed and all donors were young, some individual, non-statistical differences were observed: One jenny provided embryos in less than 50% of the cycles, whereas two jennies were excellent donors, with an embryo recovery rate higher than 80%. No differences were observed in donors’ fertility between time periods, March–September or October– December. This was not unattended based on the continuous ovarian activity of the donkey breeds studied in Italy, Martinafranca and Amiata [17,18], and previously observed in Pantesca and Ragusana jennies (F. Camillo, unpublished data). This could extend the possibility to apply ET in donkeys compared with horses, where both the seasonality and the rules of the stud books limit the ET activity to spring and summer. Carnevale et al [19] reported that repeated inseminations and embryo collections in mares, approximately seven in one breeding season, were associated with increased chronic inflammation of the uterus. This negative effect was not observed in this study as the proportion of recovered embryos on successive attempts from 8 to 14 and from 15 to 24 was higher than in attempts from 1 to 7. The differences between horses and donkeys of the two studies may be explained by several factors such as age of the animals, differences in methods used to evaluate uterine damage, and selection of donors. Age of mares was not reported [19] while jennies were very young, and it is well known that
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young age is generally associated with a condition of resistance to endometritis [20]. Uterine score biopsy, used in mares by Carnevale and co-workers, is probably a much more sensitive method than embryo recovery rate for the evaluation of the possible damage of repeated uterine lavages. Moreover, in this study a selection of the jennies was made, as only the two excellent donors were employed during the entire period, for 23 and 24 cycles, respectively. The recipients’ pregnancy rate obtained in this study, about 22% at 14 d and 15% at 50 d, confirmed the extremely poor results reported in the only study available on transfer of donkey embryos to donkeys, where only 5.5% of recipients were pregnant after surgical or nonsurgical transfer [9]. These percentages are lower than that observed after both surgical or nonsurgical transfer of horse embryos to horses (50% to 75%) [14] and than that described after surgical transfer of donkey embryos to mares or of horse embryos to jennies (67% and 55%, respectively) [7]. In an equine ET program, recipients’ pregnancy rate is influenced mostly by embryo quality and management of recipients [12]. Embryos graded 3 or 4 or with a delayed development resulted in a lower recipient pregnancy rate than embryos graded 1 or 2 and with a normal development [21]; moreover, embryos larger than 2 mm were reported to be difficult to manipulate and susceptible to damage resulting in a low recipients’ pregnancy rate [21]. In the current study, all donkey embryos were smaller than 2 mm, only three appeared smaller based on estimated age, and all but three were classified as good or excellent (score 1 and 2): for this reason, it seems difficult to relate the poor pregnancy rates with embryo quality. However, it is not possible to exclude some embryo defects or damage caused by flushing or by washing procedures or media and not appreciable with the simple morphologic evaluation. Although there was not an overall significant effect of embryo washing media, PBS resulted in a pregnancy rate about 4 times lower than that with lactated Ringer or Emcare. Quality of recipients, based on uterine and cervical tone at Day 5 postovulation, significantly affected pregnancy rate in mares [12,21]; in the current study, recipient jennies were not examined before transfer as, due to their small number, most of the time only one recipient was available. Moreover, in an equine ET program, recipients are excluded if they are not pregnant on two consecutive occasions: This rule could not be applied to the current study. In mares, embryos are generally transferred to recipients ovulating from 1 d before to 3 d after the
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donor, corresponding with Days 5 to 9 after their ovulation [21]. In the current study, again due to the small number of recipients, embryos were transferred not only to jennies at Days 5 to 9 but also at Day 3 or 4 after ovulation, without differences in 14-d pregnancy rates; however, it is possible that the overall very small number of pregnancies obtained may account for the lack of statistical differences between recipients at different days after ovulation. The donkey embryo loss observed in the current study, 30.7% between 14 and 50 d of pregnancy, was higher than that normally observed in mares after natural mating or artificial insemination [22] or employed as embryo recipients [21]. The pregnancy loss rate was higher for embryos transferred to jennies at Day 7 or 8 than for embryos transferred to jennies at Day 5 or 6 after ovulation. Similar findings were reported in a large number of mares where embryo loss rate was 7.3% to 8.6%, 15.1% to 22.4%, and 30.8% in recipients at Days 5 to 6, 7 to 8, and 9 after ovulation, respectively [21]. To explain this observation, a more efficient maternal recognition of pregnancy in mares at an earlier stage after ovulation or the need of an embryonic signal earlier than at Days 7 to 9 after ovulation was advocated [21]. Nonsurgical transfer of embryos to jennies was more difficult than it is in mares due to the donkey cervix, which appeared smaller, longer, and tighter than the equine cervix. In most cases, the cervixes of the jennies were so contracted that a cranial deviation of the distal portion of this structure was appreciable, making the cervical ostium looking cranially and not caudally. In spite of the expected difficulty to catheterize the cervixes of jennies, the recipients’ pregnancy rate achieved by the only experienced operator was the same as that achieved by the three inexperienced operators, 6 of 23 and 6 of 25, respectively. In summary, the current study resulted in a good embryo recovery rate, comparable with or even higher than that normally observed in mares, but in an unacceptable low recipients’ pregnancy rate. The reasons for these poor results remain to be understood as, in the current study, the ET techniques routinely employed in horses were simply transferred to the donkey, with the possibility that several factors had a negative effect on the outcome. For instance, it is possible that donkey embryos have different requirements with respect to flushing and washing media: from this point of view, this study seems to indicate that Ringer lactate and Emcare Holding Solution could be a better choice for embryos than PBS. In particular, it is possible that donkey embryos are more susceptible to
the potential inhibition of embryo development by phosphate [23]. Other critical points could be the donkey cervix and uterus. Transcervical ET in jennies required more manipulations than that in mares, thus, release of PGF2a, as reported during cervical manipulation in some mares [24], followed by a decrease in progesterone plasma concentration and embryo death, can be hypothesized. Moreover, it is not possible to exclude an irritating action on the recipients’ uterus induced by the holding media, similar to the one hypothesized to explain the poor results of artificial insemination with frozen semen in jennies [25,26]. In the current study, a critical evaluation of PGF2a or progesterone levels was not performed, and clinical signs of endometritis or PGF2a release after transfer with an early return to estrus were not recorded. Finally, recipients may have had an important role in the outcome of ET but it was neither possible to know their reproductive histories nor to clinically evaluate their quality before transfer. In conclusion, the results of this study suggest that other investigations are needed before ET could be considered a reliable tool to preserve endangered donkey breeds. Acknowledgments This study was funded and managed by the Azienda Regionale Foreste Demaniali Regione Siciliana with the cooperation of Istituto Incremento Ippico per la Sicilia. References [1] Rossel S, Marshall F, Peters J, Pilgram T, Adams MD, O’Connor D. Domestication of the donkey: timing, processes, and indicators. Proc Natl Acad Sci USA 2008;105:3715–20. [2] Scherf BD. World Watch List for Domestic Animal Diversity. FAO 2000. [3] Vita D, Passalacqua G, Di Pasquale G, Caminiti L, Crisafulli G, Rulli I, Pajno GB. Ass’s milk in children with atopic dermatitis and cow’s milk allergy: crossover comparison with goat’s milk. Pediatr Allergy Immunol 2007;18:594–8. [4] Feather PM. Riding for the disabled at the Slade Centre. In: The Donkey Sanctuary, The Professional Handbook of the Donkey, 2nd Edition, Svendsen ED (Ed.), Sovereign Printing Group, 1989, pp. 247-258. [5] Regione Siciliana Azienda Foreste Demaniali. Allevamento degli asini di Pantelleria. Sicilia Foreste Suppl 2004. [6] Summers PM, Shephard AM, Hodges JK, Kydd J, Boyle MS, Allen WR. Successful transfer of the embryos of Przewalski’s Horses (Equus przewalskii) and Grant’s Zebra (E. burchelli) to domestic mares (Equus caballus). J Reprod Fertil 1987;80:13–20. [7] Allen WR, Kydd J, Boyle MS, Antczac DF. Between-species transfer of horse and donkey embryos: a valuable research tool. Equine Vet J 1985;3:53–62.
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