Theriogenology 81 (2014) 832–839
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The effects of superovulation of donor sows on ovarian response and embryo development after nonsurgical deep-uterine embryo transfer M.A. Angel a, M.A. Gil a, C. Cuello a, J. Sanchez-Osorio a, J. Gomis a, I. Parrilla a, J. Vila b, I. Colina b, M. Diaz b, J. Reixach b, J.L. Vazquez a, J.M. Vazquez a, J. Roca a, E.A. Martinez a, * a b
Department of Animal Medicine and Surgery, University of Murcia, Murcia, Spain Department of Research and Development Selección Batallé S.A., Girona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 November 2013 Received in revised form 21 December 2013 Accepted 22 December 2013
This study aimed to evaluate the effectiveness of superovulation protocols in improving the efficiency of embryo donors for porcine nonsurgical deep-uterine (NsDU) embryo transfer (ET) programs. After weaning (24 hours), purebred Duroc sows (2–6 parity) were treated with 1000 IU (n ¼ 27) or 1500 IU (n ¼ 27) of eCG. Only sows with clear signs of estrus 4 to 72 hours after eCG administration were treated with 750 IU hCG at the onset of estrus. Nonhormonally treated postweaning estrus sows (n ¼ 36) were used as a control. Sows were inseminated and subjected to laparotomy on Days 5 to 6 (Day 0 ¼ onset of estrus). Three sows (11.1%) treated with the highest dosage of eCG presented with polycystic ovaries without signs of ovulation. The remaining sows from nonsuperovulated and superovulated groups were all pregnant, with no differences in fertilization rates among groups. The number of CLs and viable embryos was higher (P < 0.05) in the superovulated groups compared with the controls and increased (P < 0.05) with increasing doses of eCG. There were no differences among groups in the number of oocytes and/or degenerated embryos. The number of transferable embryos (morulae and unhatched blastocysts) obtained in pregnant sows was higher (P < 0.05) in the superovulated groups than in the control group. In all groups, there was a significant correlation between the number of CLs and the number of viable and transferable embryos, but the number of CLs and the number of oocytes and/or degenerated embryos were not correlated. A total of 46 NsDU ETs were performed in nonhormonally treated recipient sows, with embryos (30 embryos per transfer) recovered from the 1000-IU eCG, 1500-IU eCG, and control groups. In total, pregnancy and farrowing rates were 75.1% and 73.2%, respectively, with a litter size of 9.4 0.6 piglets born, of which 8.8 0.5 were born alive. There were no differences for any of the reproductive parameters evaluated among groups. In conclusion, our results demonstrated the efficiency of eCG superovulation treatments in decreasing the donor-to-recipient ratio. Compared with nonsuperovulated sows, the number of transferable embryos was increased in superovulated sows without affecting their quality and in vivo capacity to develop to term after transfer. The results from this study also demonstrate the effectiveness of the NsDU ET procedure used, making possible the commercial use of ET technology by the pig industry. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Pig Embryo Nonsurgical embryo transfer Gonadotropin Ovarian response
* Corresponding author. Tel.: þ34 868884734; fax: þ34 868887069. E-mail address: [email protected] (E.A. Martinez). 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2013.12.017
M.A. Angel et al. / Theriogenology 81 (2014) 832–839
1. Introduction The use of embryo transfer (ET) has numerous important applications in pig production, including the movement of genetic resources with minimal risk of disease transmission, reduced transportation costs, and the absence of an effect on animal welfare during transport, compared with the transport of live animals. Despite the enormous interest shown by the pig industry in the development of this technology, its commercial use was very limited, mainly because of the requirement of surgical transfer procedures. Nonsurgical ET was considered an impossible technique for many years because of the complex anatomy of the swine genital tract (reviewed in [1–3]). The cervical folds and the length and coiled nature of the uterine horns were the main barriers to the insertion of a catheter in gilts or sows during metaestrus. However, in the last decade, new perspectives have arisen with the development of a successful procedure for nonsurgical deeputerine (NsDU) ET (reviewed in [4,5]). The procedure is simple, safe, and well tolerated by the recipients. During the first attempt at NsDU transfer using fresh embryos, an acceptable reproductive performance (71.4% farrowing rate and 6.9 piglets born) was achieved [6], opening new possibilities for the commercial use of ET technology in the pig industry. A high number of fresh, good-quality embryos per transfer are necessary to achieve optimal reproductive performance in the porcine recipients. Although no studies have compared surgical and nonsurgical ET procedures, it seems that the number of embryos that can be effectively transferred ranges between 15 and 23 for surgical transfers [7–9] and between 24 and 30 for NsDU transfers [6]. Although ovulation rates in pigs vary greatly among breeds and among animals of different ages (gilts and sows) within a breed [10,11], 15 to 25 oocytes can be proposed as typical in this species. These data indicate that the embryos collected from one donor would be enough to perform one ET with a donor-to-recipient ratio very close to 1:1. However, in practice, a percentage of donors do not become pregnant after insemination, some oocytes from pregnant donors are not fertilized, some embryos collected are nontransferable, and the embryo recovery rates are approximately 90%. All these factors together cause the actual donor-to-recipient ratio to be closer to 2:1, resulting in a high cost per transferable embryo. To reduce this ratio, a lower number of embryos per transfer could be used, although this possibility has not yet been evaluated. Another possibility is the superovulation of the donors through the use of eCG. When prepubertal gilts are used as donors, superovulation treatments not only produce a higher ovulation rate and number of embryos compared with nonstimulated mature gilts [12] but also a high percentage (w25%–50%) of unfertilized oocytes and/or degenerated embryos [12–15] and a high individual variability in the ovulatory response [12,16]. For these reasons, the use of prepubertal gilts as embryo donors should be considered carefully. Mature gilts and sow donors can also be stimulated to increase their ovulation rate through the use of gonadotropins after synchronization treatment or
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after weaning (reviewed in [17]). In addition to the high ovulatory response variability and the increased ovulation rate, it has been shown that the administration of eCG increases embryonic losses in gilts [18,19] and sows [20] at Days 24 to 40 of pregnancy. A limited number of studies have investigated the quality of 5- to 6-day-old embryos collected from superovulated gilts [21] and sows [22] and the subsequent reproductive performance of recipients transferred with this type of embryos. Because these studies, and others using superovulation of the donors for ET, did not include a control (nonsuperovulated) group [8,21–30] making comparisons is impossible, and the efficacy of superovulation has not yet been clearly established. The objective of the present study was to determine (1) the effect of two doses of eCG to induce superovulation in sows on the number and quality of 5- to 6-day-old embryos and (2) the reproductive performance of recipients after NsDU transfer of embryos collected from superovulated and nonsuperovulated donors. 2. Materials and methods All chemicals were purchased from Sigma-Aldrich Co. (Alcobendas, Madrid, Spain) unless otherwise stated. All experimental procedures used in the present study were carried out in accordance with the 2010/63/EU EEC Directive for animal experiments and were reviewed and approved by the Ethical Committee for Experimentation with Animals of the University of Murcia, Spain. 2.1. Animals Experiments were conducted under field conditions at a commercial pig-breeding farm (Selección Batallé S.A., Girona, Spain). Purebred Duroc sows (2–6 parity) were used as donors and recipients. The sows were allocated individually to crates in a mechanically ventilated confinement facility and were fed a commercial ration twice a day. Water was provided ad libitum. 2.2. Detection of estrus Estrus detection was performed by experienced personnel twice a day (7 AM and 5 PM) beginning 2 days after weaning by allowing nose-to-nose contact of females with a vasectomized mature boar and by applying back pressure. Sows exhibiting a standing heat reflex in the presence of the boar were considered to be in estrus. The first day of onset of estrus was designated as Day 0. 2.3. Estrous synchronization and superovulation treatments Weaning was used to synchronize estrus between donors and recipients. To standardize the schedule of ETs, only sows with a weaning to estrus interval of 3 to 4 days were selected as donors or recipients. Superovulation of the donors was induced by an intramuscular administration of different doses of eCG (Folligon; Intervet International B.V., Boxmeer, The Netherlands) 24 hours after weaning. Only sows with clear signs of estrus
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48 to 72 hours post-eCG administration were treated with 750 IU, intramuscular, of hCG (Veterin Corion; Divasa Farmavic S.A., Barcelona, Spain) at the onset of estrus. 2.4. Insemination of donors Donors were inseminated using semen from healthy, mature (2–3 years of age) Duroc boars of proven fertility housed at the insemination station of the company. The sperm-rich fraction of each ejaculate was diluted in Beltsville thawing solution and stored at 18 C for a maximum of 72 hours. Postcervical inseminations were performed at 0, 24, and 36 hours after the onset of estrus with doses of 1.5 109 spermatozoa in 45 mL. 2.5. Embryo recovery and evaluation Embryos were collected using laparotomy on Days 5 and 6 of the cycle. Donors were sedated with administration of azaperone (2 mg/kg of body weight, intramuscular). General anesthesia was induced with sodium thiopental (7 mg/kg of body weight, intravenous) and maintained with isoflurane (3.5%–5%). The reproductive tract was exposed via midline incision, and CLs were counted on the ovaries to evaluate the ovulatory response. Embryos were recovered by flushing the tip of each uterine horn with 30 mL of recovery medium. The recovery medium used was protein-free Tyrode’s lactate (TL)-HEPES-polyvinyl alcohol (PVA) (TL-HEPES-PVA) [31] with some modifications. This medium was composed of 124.3 mM NaCl, 3.2 mM KCl, 2 mM NaHCO3, 0.34 mM KH2PO4, 10 mM Nalactate, 0.5 mM MgCl2$6H2O, 2 mM CaCl2$2H2O, 10 mM HEPES, 0.2 mM Na-pyruvate, 12 mM sorbitol, 0.1% (wt/vol) polyvinyl alcohol, 75 mg/mL potassium penicillin G, and 50 mg/mL streptomycin sulfate. Recovered embryos were evaluated under a stereomicroscope at a magnification of 60 to grade their developmental stage and quality according to the morphological criteria determined by the International Embryo Transfer Society [32]. Morulae and blastocysts classified as grade 1 and 2 (excellent or good quality, respectively) were considered viable. The rest of structures collected, which included oocytes or one-cell eggs, poor-quality or poorly developed embryos, and degenerating and degenerated embryos, were classified as oocytes and/or degenerated embryos. Morulae were classified as uncompacted (blastomeres had begun to compact tightly, forming a cluster cell mass; each individual blastomere was identifiable but not distinct) and compacted (blastomeres compacted completely; the cell boundary was not visible). Embryos with an incipient visible blastocoel or with a well-defined blastocoel, inner cell mass, and trophoblast completely discernible were classified as blastocysts. Blastocysts with partial or complete loss of the zona pellucida were defined as hatched embryos. Only viable, compacted morulae and unhatched blastocysts were considered transferable and used for ET. Transferable embryos were washed six times in embryo recovery medium and placed in sterilized Eppendorf tubes containing 1.5 mL of the same medium in a thermostatically controlled incubator (39 C) and maintained for up to 6 hours before transfer.
2.6. Nonsurgical ET The transfers were performed in a small room (12 crates) exclusively used for that purpose. The NsDU ETs were conducted in nonhormonally treated recipient sows on Days 4 to 6 of the cycle. Sows were housed in gestation crates and were not sedated. The transfer procedure was performed as described by Martinez, et al. [6]. The perineal area of the recipients was thoroughly cleaned with soap and water using a different sponge for each sow. The tail of each recipient was covered with a latex glove to protect the vulva from possible contamination. The vulva was then washed and decontaminated (inside and outside) using sterile gauze soaked with chlorhexidine. Commercial nonsurgical ET catheters (Deep Blue ET catheter; Minitüb, Tiefenbach, Germany), individually packaged and sterilized, were used for the transfers. The ET catheter is composed of an artificial insemination spirette containing a flexible catheter (FC; 1.8 m length) inside and a protective sanitary sheath outside. Before insertion, the inner tubing of the flexible catheter was rinsed with 0.3 mL of TL-HEPESPVA medium at 39 C and the protective sheath was lubricated with silicone (Rüsch silkospray; Willy Rüsch GMBH, Kernen-Rommelshausen, Germany). Then, the spirette was inserted through the vulva into the first 20 to 25 cm of the vagina. In this region, the spirette tip was pushed through the sheath and inserted into the cervix. The FC was then moved through the cervical canal and propelled forward along one uterine horn until the length of the FC outside the recipient was approximately 30 to 40 cm. The FC was flushed with 0.3 mL of medium at 39 C using a 1 mL disposable syringe when the tip of the FC reached the uterine body. When the FC was completely inserted into one uterine horn, a 1-mL syringe containing 30 embryos in 0.1 mL TL-HEPES-PVA medium was connected to the FC, and the contents were introduced into the FC. Finally, an additional volume of 0.3 mL TL-HEPES-PVA medium was used to force the embryos out of the FC into the uterus. Correct positioning of the FC was assumed if no bends or kinks in the catheter were present after its removal. 2.7. Experimental design A total of 90 donor sows of proven fertility and satisfactory reproductive characteristics were superovulated with 1000-IU and 1500-IU eCG. Nonhormonally treated sows were used as a control. The reproductive history of donors in each group was similar (fertility [range: 95.3 1.6%–97.2 1.8%], litter size [range: 11.0 0.2–11.2 0.2 piglets], parity number [range: 4.4 0.4–5.0 0.4], and lactation length [range: 21.3 0.2–21.7 0.2 days]). Transferable embryos (compacted morulae and unhatched blastocysts) from each group were nonsurgically transferred (30 embryos per transfer) to a total of 46 recipient sows. Recipients were selected based on their reproductive history and body condition. There were no differences in the reproductive history of recipients assigned to each group (fertility [range: 95.1 2.6%–99.9 2.5%], litter size [range: 9.3 0.5–10.5 0.5 piglets], parity number [range: 2.3 0.4–2.9 0.4], and lactation length [range: 21.9 0.6–22.3 0.6 days]). All NsDU ETs were performed 4 to
M.A. Angel et al. / Theriogenology 81 (2014) 832–839
6 hours after embryo recovery by the same operator. The experiment was conducted in a total of 10 trials. Each trial was conducted in separate sessions over a 1-year period and consisted of eight to 12 donors and four to six ETs. Donors from the three treatment groups were inseminated with sperm doses from the same boar and included in each trial. Recipients with transferred embryos collected from the three groups were also included in each trial. The ovulatory response of the donors was determined by counting the number of CLs in both ovaries. To evaluate the effectiveness of the superovulation treatments, the number of viable and transferable embryos and oocytes and/or degenerated embryos was counted in each donor. The recovery rate was defined as the ratio of the number of embryos and oocytes and/or degenerated embryos recovered to the number of CLs present. The fertilization rate was defined as the ratio of the number of viable embryos to the total number of embryos and oocytes and/or degenerated embryos collected. In addition, the presence of follicular cysts (ovarian structures filled with a transparent liquid, without ovulation signs, and with a diameter greater than 2 cm at the moment of laparotomy) and polycystic ovaries (ovaries with more than eight follicular cysts) was recorded in each donor. Starting 12 days after NsDU ET, the recipients were checked daily for signs of estrus. Pregnancy was diagnosed using ultrasonography on Day-20 posttransfer. All pregnant sows were allowed to carry litters to term, and farrowing rates, and litter sizes were recorded. Embryo survival rate was calculated as the ratio of the number of piglets born alive to the number of embryos transferred to all recipients. 2.8. Statistical analysis The data were analyzed using the IBM SPSS 19 Statistics package (SPSS, Chicago, IL, USA). Percentage data were compared using Fisher’s exact test. The continuous variables were evaluated using the Kolmogorov–Smirnov test to check the assumption of normality and compared among groups with ANOVA. Post hoc analysis was performed with Bonferroni’s test. Within each treatment, correlations between donor parity and CLs, viable embryos, transferable embryos, and oocytes and/or degenerated embryos, and between CLs and oocyte or degenerated embryos were analyzed with the nonparametric Spearman rank-order coefficient (rs). Pearson’s correlation coefficient (r) was used to evaluate the associations between the CLs and the other continuous variables (viable embryos, transferable embryos). The coefficient of variation (CV ¼ standard deviation/mean) was used as a measure of variability of the ovulatory response and was calculated within each group. Differences were considered significant at P < 0.05. Differences among values with 0.05 < P < 0.10 were accepted as representing tendencies toward differences. The results are expressed as percentages and means standard error of the mean. 3. Results The effects of superovulation treatment on pregnancy rates and the incidence of cysts are shown in Table 1. The
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Table 1 Effect of the superovulation treatment on pregnancy rates and incidence of ovarian cysts in donor sows at Days 5 and 6 after insemination. Characteristic
Treatment (IU of eCG) 0 (Control) 1000
1500
Sows, n Pregnancy rate, n (%) Sows with cysts, n (%) Number of cysts in sows with cysts, (mean SEM) Sows with polycystic ovaries, n (%)
36 36 (100) 14 (38.9) 2.4 0.4
27 27 (100) 9 (33.3) 2.9 0.6
27 24 (88.9)a 12 (44.4) 3.6 1.0
0 (0.0)
0 (0.0)
3 (11.1)a
Abbreviation: SEM, standard error of the mean. a Tendency with respect to control group (P ¼ 0.07).
potential pregnancy rate (percentage of sows with more than four viable embryos) tended (P ¼ 0.07) to be lower in sows superovulated with the highest dosage of eCG compared with the controls. This result was because of the presence of polycystic ovaries in three sows (11.1%). None of these sows had CLs on their ovaries, and the size of the majority of cysts was greater than 3 cm. In the control and 1000-IU eCG groups, there was no incidence of polycystic ovaries. A high proportion of donors (range: 33.3%–44.4%) had cysts in the ovaries, with a mean number of cysts ranging between 2.4 0.4 and 3.6 1.0 per sow, with no differences among groups. There were no differences in the recovery rates (range: 89.8%–94.6%) or fertilization rates (range: 91.5%–94.3%) among groups. A higher variability among donors in the ovulatory response to gonadotropins (range: 11 to 36 and 17 to 51 CLs, CV ¼ 21.4% and 26.5%, for the 1000- and 1500IU eCG groups, respectively) compared with the controls (15–28 CLs, CV ¼ 15.9%) was observed. The reproductive parameters obtained in pregnant sows from the three groups are presented in Table 2. The mean number of CLs and viable embryos was higher (P < 0.05) in the superovulated groups compared with the controls and increased (P < 0.05) with increasing doses of eCG. There were no differences among groups in the mean number of oocytes and/or degenerated embryos, which ranged between 1.1 0.3 and 2.4 0.8. The mean number of transferable embryos obtained in pregnant sows was higher (P < 0.05) in the superovulated groups than in the control group and tended (P ¼ 0.07) to be greater in the 1500 IU eCG group than in the 1000-IU eCG group. However, this tendency disappeared when the total number of sows superovulated (pregnant and nonpregnant) was considered (20.6 1.0 and 22.4 2.1 transferable embryos for the 1000-IU and 1500-IU eCG groups, respectively). In general, the proportion of transferable embryos in relation to the number of viable embryos was high in all three groups, although it was higher (P < 0.03) in the superovulated groups (94.1% and 96.8% for the 1000- and 1500-IU eCG groups, respectively) compared with the control group (89.7%). At Day 5 of pregnancy, the percentages of nontransferable viable embryos were different (P < 0.01) among groups (3.5%, 0.0%, and 23.1% for the 1000-IU eCG, 1500-IU eCG, and control groups, respectively) due exclusively to the presence of uncompact morulae. At Day 6 of pregnancy, a higher (P < 0.02) percentage of nontransferable viable embryos (8.1%) was recorded in the 1500 IU eCG group
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Table 2 Influence of superovulation treatment on the reproductive parameters in pregnant donor sows. Characteristic
Treatment (IU of eCG)
Pregnant sows, n CLs, (mean SEM) Viable embryos collected, (mean SEM) Oocytes and/or degenerated embryos, (mean SEM) Recovery rate (%) Transferable embryos, (mean SEM)
36 27 24 19.7 0.5a 24.5 1.0b 31.0 1.7c a b 16.5 0.6 21.3 1.0 26.8 1.6c
0 (Control) 1000
1.1 0.3
1.3 0.5
1500
2.4 0.8
89.8 90.5 94.6 14.8 1.1a 20.6 1.0b 25.2 1.9b,d
Abbreviation: SEM, standard error of the mean. a,b,c Different superscripts within the same row differ (P < 0.05). d Tendency with respect to the 1000-IU eCG group (P ¼ 0.07).
compared with the control (2.4%) and 1000-IU eCG (2.9%) groups as a result of the presence of hatched blastocysts. In all groups, there was a significant linear correlation between the number of CLs and the total number of viable
embryos and transferable embryos, but the number of CLs and the number of oocytes and/or degenerated embryos were not correlated (Fig. 1). The donor parity was not correlated in any group with the number of CLs, viable embryos, transferable embryos, or oocytes and/or degenerated embryos. A total of 1693 transferable embryos were collected, of which 1283 (75.8%) and 410 (24.2%) were classified as morulae or early blastocysts and full blastocysts, respectively. Of these embryos, 1369 were used for ET and the remaining were used for other experiments. The total number of transferable embryos collected from the donors inseminated in the 1000-IU eCG (n ¼ 27), 1500-IU eCG (n ¼ 27), and the control (n ¼ 36) groups was 556, 604, and 533, respectively, resulting in a donor-to-recipient ratio of 1.4:1, 1.3:1, and 2:1, respectively. Five out of 46 ETs (10.9%) were eliminated from the study because of incorrect insertions of the NsDU ET catheter. The transfers were mainly performed in each group with embryos at the morula or early blastocyst (range: 60.0%–63.3%) and full blastocyst stages (range: 27.7%–36.4%) on recipients during Day 5 of the cycle (>80%
Fig. 1. Relationship between the number of CLs and the number of viable and transferable embryos and unfertilized oocytes and/or degenerated embryos on Days 5 and 6 after the onset of estrus in Duroc donor sows treated 24 hours after weaning with 0 (control), 1000-, and 1500-IU eCG. N.S., nonsignificant; r, Pearson coefficient; rs, Spearman coefficient.
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of the transfers). The number of transferred embryos per recipient was 29.5 0.3, 29.9 0.1, and 29.9 0.1 (range: 27–30) for the 1000-IU eCG, 1500-IU eCG, and control groups, respectively, with no differences among groups. The reproductive performance of recipients after transfers is shown in Table 3. In total, pregnancy and farrowing rates were 75.1% and 73.2%, respectively, and the litter size was 9.4 0.6 piglets born, of which 8.8 0.5 were born alive, with an average weight of 1.5 0.6 kg. The percentage of female offspring was 51.4. There were no differences for any of the reproductive parameters evaluated among groups. Additionally, there were no differences among groups in embryo survival rates (range: 20.9%–22.4%). 4. Discussion The results from this experiment clearly demonstrate that the embryos collected from superovulated donor sows with eCG present similar morphological quality at Days 5 and 6 of the cycle and similar in vivo ability to develop to term after transfers as those collected from nonsuperovulated donors. In addition, this study confirms previous reports on the effectiveness of the NsDU ET technology. Regardless of the superovulation treatment (1000- or 1500-IU eCG), we found a high proportion of donors (33%– 44%) with cysts in the ovaries, numbering 3 to 3.5 cysts per sow. These data were similar to those found in nonsuperovulated sows, indicating that the superovulation treatment was not implicated in the cyst incidence. In all of these cases, the cysts were unilateral or bilateral and originated from follicles, which do not ovulate, and coexisted with normal CLs. A much lower incidence of ovarian cysts in sows with previous reproductive failures (between 3.3% and 13.0%) [33–35] or in farms without any particular reproductive problems (2.4%) [36] has been reported. Although the presence of ovarian cysts has been associated with a greater return to estrus rate, a decreased farrowing rate, and an increased percentage of anestral sows [36], in our study, these cysts did not interfere with the reproductive cycles, as can be demonstrated by the excellent reproductive history and by the excellent quality of the embryos collected. Most likely, these cysts were nonfunctional, causing no interference with the reproductive cycle, as has been reported for single cysts [37]. Although the reason for the high percentage of sows with cysts found in our study is unclear, we cannot dismiss the possibility that the high cyst incidence might be an innate characteristic of Table 3 Reproductive performance of the recipients after nonsurgical deeputerine transfer of embryos collected from superovulated purebred Duroc sows. Characteristic
Treatment (IU of eCG) 0 (Control)
1000
1500
Recipients, n Pregnancy rate, n (%) Farrowing rate, n (%) Total born, (mean SEM) Born alive, (mean SEM) Stillborn, (mean SEM)
11 8 (72.7) 8 (72.7) 9.0 0.9 8.6 0.8 0.4 0.3
15 12 (80.0) 11 (73.3) 9.3 1.2 8.6 1.1 0.6 0.2
15 11 (73.3) 11 (73.3) 9.7 1.0 9.2 0.9 0.5 0.3
Abbreviation: SEM, standard error of the mean.
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purebred Duroc sows. The potential pregnancy rate tended to be lower in sows superovulated with the highest dose of eCG. This fact was exclusively because of the presence in three sows (11.1%) of multiple large follicular cysts with no sign of ovulation and without additional CLs on the ovaries, which is associated with infertility [35]. The remaining sows from nonsuperovulated and superovulated groups were all pregnant at Days 5 and 6 after insemination, with no differences in fertilization rates among groups. Therefore, it seems clear that the superovulation treatment had no adverse effect on oocyte maturation, the transport of the gametes, fertilization, or early embryo development. Superovulation treatments are characterized by high variability in the ovulatory response [38]. Our study provides additional reference data concerning intersow variability in the ovulatory response in nonsuperovulated and superovulated sows. We found that the variability was high in nonsuperovulated sows (CV ¼ 15.9%). Similar variation in the ovulation rates (CV range, 16%–20%) has been reported in untreated gilts [12,39,40] and sows [20]. As expected, the ovulatory response variability found in the present study increased in the superovulated sows (CV ¼ 21.4% and 26.5% for the 1000-IU and 1500-IU eCG groups, respectively). However, this variation was slightly more than half of the previously reported values, with similar superovulation treatments (800–1500 IU eCG) for cyclic gilts and sows, in which the CVs were approximately 40% [20,22,24,38]. Because ovulation rates differ among purebred, hyperprolific, and crossbred sows [41–44], this apparent discrepancy may be explained by the different lines and breeds used in these studies, for which the superovulatory response can differ widely [5]. Superovulation treatments have also been associated with high percentages of oocytes and/or degenerated embryos. In our study, we not only obtained an increased number of viable and transferable embryos in the superovulated groups when compared with those in nonsuperovulated sows, but the number of oocytes and/or degenerated embryos was not different among groups and represented 7% of the total structures collected. In addition, only a very low proportion (
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The effects of superovulation of donor sows on ovarian response and embryo development after nonsurgical deep-uterine embryo transfer M.A. Angel a, M.A. Gil a, C. Cuello a, J. Sanchez-Osorio a, J. Gomis a, I. Parrilla a, J. Vila b, I. Colina b, M. Diaz b, J. Reixach b, J.L. Vazquez a, J.M. Vazquez a, J. Roca a, E.A. Martinez a, * a b
Department of Animal Medicine and Surgery, University of Murcia, Murcia, Spain Department of Research and Development Selección Batallé S.A., Girona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 November 2013 Received in revised form 21 December 2013 Accepted 22 December 2013
This study aimed to evaluate the effectiveness of superovulation protocols in improving the efficiency of embryo donors for porcine nonsurgical deep-uterine (NsDU) embryo transfer (ET) programs. After weaning (24 hours), purebred Duroc sows (2–6 parity) were treated with 1000 IU (n ¼ 27) or 1500 IU (n ¼ 27) of eCG. Only sows with clear signs of estrus 4 to 72 hours after eCG administration were treated with 750 IU hCG at the onset of estrus. Nonhormonally treated postweaning estrus sows (n ¼ 36) were used as a control. Sows were inseminated and subjected to laparotomy on Days 5 to 6 (Day 0 ¼ onset of estrus). Three sows (11.1%) treated with the highest dosage of eCG presented with polycystic ovaries without signs of ovulation. The remaining sows from nonsuperovulated and superovulated groups were all pregnant, with no differences in fertilization rates among groups. The number of CLs and viable embryos was higher (P < 0.05) in the superovulated groups compared with the controls and increased (P < 0.05) with increasing doses of eCG. There were no differences among groups in the number of oocytes and/or degenerated embryos. The number of transferable embryos (morulae and unhatched blastocysts) obtained in pregnant sows was higher (P < 0.05) in the superovulated groups than in the control group. In all groups, there was a significant correlation between the number of CLs and the number of viable and transferable embryos, but the number of CLs and the number of oocytes and/or degenerated embryos were not correlated. A total of 46 NsDU ETs were performed in nonhormonally treated recipient sows, with embryos (30 embryos per transfer) recovered from the 1000-IU eCG, 1500-IU eCG, and control groups. In total, pregnancy and farrowing rates were 75.1% and 73.2%, respectively, with a litter size of 9.4 0.6 piglets born, of which 8.8 0.5 were born alive. There were no differences for any of the reproductive parameters evaluated among groups. In conclusion, our results demonstrated the efficiency of eCG superovulation treatments in decreasing the donor-to-recipient ratio. Compared with nonsuperovulated sows, the number of transferable embryos was increased in superovulated sows without affecting their quality and in vivo capacity to develop to term after transfer. The results from this study also demonstrate the effectiveness of the NsDU ET procedure used, making possible the commercial use of ET technology by the pig industry. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Pig Embryo Nonsurgical embryo transfer Gonadotropin Ovarian response
* Corresponding author. Tel.: þ34 868884734; fax: þ34 868887069. E-mail address: [email protected] (E.A. Martinez). 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2013.12.017
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1. Introduction The use of embryo transfer (ET) has numerous important applications in pig production, including the movement of genetic resources with minimal risk of disease transmission, reduced transportation costs, and the absence of an effect on animal welfare during transport, compared with the transport of live animals. Despite the enormous interest shown by the pig industry in the development of this technology, its commercial use was very limited, mainly because of the requirement of surgical transfer procedures. Nonsurgical ET was considered an impossible technique for many years because of the complex anatomy of the swine genital tract (reviewed in [1–3]). The cervical folds and the length and coiled nature of the uterine horns were the main barriers to the insertion of a catheter in gilts or sows during metaestrus. However, in the last decade, new perspectives have arisen with the development of a successful procedure for nonsurgical deeputerine (NsDU) ET (reviewed in [4,5]). The procedure is simple, safe, and well tolerated by the recipients. During the first attempt at NsDU transfer using fresh embryos, an acceptable reproductive performance (71.4% farrowing rate and 6.9 piglets born) was achieved [6], opening new possibilities for the commercial use of ET technology in the pig industry. A high number of fresh, good-quality embryos per transfer are necessary to achieve optimal reproductive performance in the porcine recipients. Although no studies have compared surgical and nonsurgical ET procedures, it seems that the number of embryos that can be effectively transferred ranges between 15 and 23 for surgical transfers [7–9] and between 24 and 30 for NsDU transfers [6]. Although ovulation rates in pigs vary greatly among breeds and among animals of different ages (gilts and sows) within a breed [10,11], 15 to 25 oocytes can be proposed as typical in this species. These data indicate that the embryos collected from one donor would be enough to perform one ET with a donor-to-recipient ratio very close to 1:1. However, in practice, a percentage of donors do not become pregnant after insemination, some oocytes from pregnant donors are not fertilized, some embryos collected are nontransferable, and the embryo recovery rates are approximately 90%. All these factors together cause the actual donor-to-recipient ratio to be closer to 2:1, resulting in a high cost per transferable embryo. To reduce this ratio, a lower number of embryos per transfer could be used, although this possibility has not yet been evaluated. Another possibility is the superovulation of the donors through the use of eCG. When prepubertal gilts are used as donors, superovulation treatments not only produce a higher ovulation rate and number of embryos compared with nonstimulated mature gilts [12] but also a high percentage (w25%–50%) of unfertilized oocytes and/or degenerated embryos [12–15] and a high individual variability in the ovulatory response [12,16]. For these reasons, the use of prepubertal gilts as embryo donors should be considered carefully. Mature gilts and sow donors can also be stimulated to increase their ovulation rate through the use of gonadotropins after synchronization treatment or
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after weaning (reviewed in [17]). In addition to the high ovulatory response variability and the increased ovulation rate, it has been shown that the administration of eCG increases embryonic losses in gilts [18,19] and sows [20] at Days 24 to 40 of pregnancy. A limited number of studies have investigated the quality of 5- to 6-day-old embryos collected from superovulated gilts [21] and sows [22] and the subsequent reproductive performance of recipients transferred with this type of embryos. Because these studies, and others using superovulation of the donors for ET, did not include a control (nonsuperovulated) group [8,21–30] making comparisons is impossible, and the efficacy of superovulation has not yet been clearly established. The objective of the present study was to determine (1) the effect of two doses of eCG to induce superovulation in sows on the number and quality of 5- to 6-day-old embryos and (2) the reproductive performance of recipients after NsDU transfer of embryos collected from superovulated and nonsuperovulated donors. 2. Materials and methods All chemicals were purchased from Sigma-Aldrich Co. (Alcobendas, Madrid, Spain) unless otherwise stated. All experimental procedures used in the present study were carried out in accordance with the 2010/63/EU EEC Directive for animal experiments and were reviewed and approved by the Ethical Committee for Experimentation with Animals of the University of Murcia, Spain. 2.1. Animals Experiments were conducted under field conditions at a commercial pig-breeding farm (Selección Batallé S.A., Girona, Spain). Purebred Duroc sows (2–6 parity) were used as donors and recipients. The sows were allocated individually to crates in a mechanically ventilated confinement facility and were fed a commercial ration twice a day. Water was provided ad libitum. 2.2. Detection of estrus Estrus detection was performed by experienced personnel twice a day (7 AM and 5 PM) beginning 2 days after weaning by allowing nose-to-nose contact of females with a vasectomized mature boar and by applying back pressure. Sows exhibiting a standing heat reflex in the presence of the boar were considered to be in estrus. The first day of onset of estrus was designated as Day 0. 2.3. Estrous synchronization and superovulation treatments Weaning was used to synchronize estrus between donors and recipients. To standardize the schedule of ETs, only sows with a weaning to estrus interval of 3 to 4 days were selected as donors or recipients. Superovulation of the donors was induced by an intramuscular administration of different doses of eCG (Folligon; Intervet International B.V., Boxmeer, The Netherlands) 24 hours after weaning. Only sows with clear signs of estrus
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48 to 72 hours post-eCG administration were treated with 750 IU, intramuscular, of hCG (Veterin Corion; Divasa Farmavic S.A., Barcelona, Spain) at the onset of estrus. 2.4. Insemination of donors Donors were inseminated using semen from healthy, mature (2–3 years of age) Duroc boars of proven fertility housed at the insemination station of the company. The sperm-rich fraction of each ejaculate was diluted in Beltsville thawing solution and stored at 18 C for a maximum of 72 hours. Postcervical inseminations were performed at 0, 24, and 36 hours after the onset of estrus with doses of 1.5 109 spermatozoa in 45 mL. 2.5. Embryo recovery and evaluation Embryos were collected using laparotomy on Days 5 and 6 of the cycle. Donors were sedated with administration of azaperone (2 mg/kg of body weight, intramuscular). General anesthesia was induced with sodium thiopental (7 mg/kg of body weight, intravenous) and maintained with isoflurane (3.5%–5%). The reproductive tract was exposed via midline incision, and CLs were counted on the ovaries to evaluate the ovulatory response. Embryos were recovered by flushing the tip of each uterine horn with 30 mL of recovery medium. The recovery medium used was protein-free Tyrode’s lactate (TL)-HEPES-polyvinyl alcohol (PVA) (TL-HEPES-PVA) [31] with some modifications. This medium was composed of 124.3 mM NaCl, 3.2 mM KCl, 2 mM NaHCO3, 0.34 mM KH2PO4, 10 mM Nalactate, 0.5 mM MgCl2$6H2O, 2 mM CaCl2$2H2O, 10 mM HEPES, 0.2 mM Na-pyruvate, 12 mM sorbitol, 0.1% (wt/vol) polyvinyl alcohol, 75 mg/mL potassium penicillin G, and 50 mg/mL streptomycin sulfate. Recovered embryos were evaluated under a stereomicroscope at a magnification of 60 to grade their developmental stage and quality according to the morphological criteria determined by the International Embryo Transfer Society [32]. Morulae and blastocysts classified as grade 1 and 2 (excellent or good quality, respectively) were considered viable. The rest of structures collected, which included oocytes or one-cell eggs, poor-quality or poorly developed embryos, and degenerating and degenerated embryos, were classified as oocytes and/or degenerated embryos. Morulae were classified as uncompacted (blastomeres had begun to compact tightly, forming a cluster cell mass; each individual blastomere was identifiable but not distinct) and compacted (blastomeres compacted completely; the cell boundary was not visible). Embryos with an incipient visible blastocoel or with a well-defined blastocoel, inner cell mass, and trophoblast completely discernible were classified as blastocysts. Blastocysts with partial or complete loss of the zona pellucida were defined as hatched embryos. Only viable, compacted morulae and unhatched blastocysts were considered transferable and used for ET. Transferable embryos were washed six times in embryo recovery medium and placed in sterilized Eppendorf tubes containing 1.5 mL of the same medium in a thermostatically controlled incubator (39 C) and maintained for up to 6 hours before transfer.
2.6. Nonsurgical ET The transfers were performed in a small room (12 crates) exclusively used for that purpose. The NsDU ETs were conducted in nonhormonally treated recipient sows on Days 4 to 6 of the cycle. Sows were housed in gestation crates and were not sedated. The transfer procedure was performed as described by Martinez, et al. [6]. The perineal area of the recipients was thoroughly cleaned with soap and water using a different sponge for each sow. The tail of each recipient was covered with a latex glove to protect the vulva from possible contamination. The vulva was then washed and decontaminated (inside and outside) using sterile gauze soaked with chlorhexidine. Commercial nonsurgical ET catheters (Deep Blue ET catheter; Minitüb, Tiefenbach, Germany), individually packaged and sterilized, were used for the transfers. The ET catheter is composed of an artificial insemination spirette containing a flexible catheter (FC; 1.8 m length) inside and a protective sanitary sheath outside. Before insertion, the inner tubing of the flexible catheter was rinsed with 0.3 mL of TL-HEPESPVA medium at 39 C and the protective sheath was lubricated with silicone (Rüsch silkospray; Willy Rüsch GMBH, Kernen-Rommelshausen, Germany). Then, the spirette was inserted through the vulva into the first 20 to 25 cm of the vagina. In this region, the spirette tip was pushed through the sheath and inserted into the cervix. The FC was then moved through the cervical canal and propelled forward along one uterine horn until the length of the FC outside the recipient was approximately 30 to 40 cm. The FC was flushed with 0.3 mL of medium at 39 C using a 1 mL disposable syringe when the tip of the FC reached the uterine body. When the FC was completely inserted into one uterine horn, a 1-mL syringe containing 30 embryos in 0.1 mL TL-HEPES-PVA medium was connected to the FC, and the contents were introduced into the FC. Finally, an additional volume of 0.3 mL TL-HEPES-PVA medium was used to force the embryos out of the FC into the uterus. Correct positioning of the FC was assumed if no bends or kinks in the catheter were present after its removal. 2.7. Experimental design A total of 90 donor sows of proven fertility and satisfactory reproductive characteristics were superovulated with 1000-IU and 1500-IU eCG. Nonhormonally treated sows were used as a control. The reproductive history of donors in each group was similar (fertility [range: 95.3 1.6%–97.2 1.8%], litter size [range: 11.0 0.2–11.2 0.2 piglets], parity number [range: 4.4 0.4–5.0 0.4], and lactation length [range: 21.3 0.2–21.7 0.2 days]). Transferable embryos (compacted morulae and unhatched blastocysts) from each group were nonsurgically transferred (30 embryos per transfer) to a total of 46 recipient sows. Recipients were selected based on their reproductive history and body condition. There were no differences in the reproductive history of recipients assigned to each group (fertility [range: 95.1 2.6%–99.9 2.5%], litter size [range: 9.3 0.5–10.5 0.5 piglets], parity number [range: 2.3 0.4–2.9 0.4], and lactation length [range: 21.9 0.6–22.3 0.6 days]). All NsDU ETs were performed 4 to
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6 hours after embryo recovery by the same operator. The experiment was conducted in a total of 10 trials. Each trial was conducted in separate sessions over a 1-year period and consisted of eight to 12 donors and four to six ETs. Donors from the three treatment groups were inseminated with sperm doses from the same boar and included in each trial. Recipients with transferred embryos collected from the three groups were also included in each trial. The ovulatory response of the donors was determined by counting the number of CLs in both ovaries. To evaluate the effectiveness of the superovulation treatments, the number of viable and transferable embryos and oocytes and/or degenerated embryos was counted in each donor. The recovery rate was defined as the ratio of the number of embryos and oocytes and/or degenerated embryos recovered to the number of CLs present. The fertilization rate was defined as the ratio of the number of viable embryos to the total number of embryos and oocytes and/or degenerated embryos collected. In addition, the presence of follicular cysts (ovarian structures filled with a transparent liquid, without ovulation signs, and with a diameter greater than 2 cm at the moment of laparotomy) and polycystic ovaries (ovaries with more than eight follicular cysts) was recorded in each donor. Starting 12 days after NsDU ET, the recipients were checked daily for signs of estrus. Pregnancy was diagnosed using ultrasonography on Day-20 posttransfer. All pregnant sows were allowed to carry litters to term, and farrowing rates, and litter sizes were recorded. Embryo survival rate was calculated as the ratio of the number of piglets born alive to the number of embryos transferred to all recipients. 2.8. Statistical analysis The data were analyzed using the IBM SPSS 19 Statistics package (SPSS, Chicago, IL, USA). Percentage data were compared using Fisher’s exact test. The continuous variables were evaluated using the Kolmogorov–Smirnov test to check the assumption of normality and compared among groups with ANOVA. Post hoc analysis was performed with Bonferroni’s test. Within each treatment, correlations between donor parity and CLs, viable embryos, transferable embryos, and oocytes and/or degenerated embryos, and between CLs and oocyte or degenerated embryos were analyzed with the nonparametric Spearman rank-order coefficient (rs). Pearson’s correlation coefficient (r) was used to evaluate the associations between the CLs and the other continuous variables (viable embryos, transferable embryos). The coefficient of variation (CV ¼ standard deviation/mean) was used as a measure of variability of the ovulatory response and was calculated within each group. Differences were considered significant at P < 0.05. Differences among values with 0.05 < P < 0.10 were accepted as representing tendencies toward differences. The results are expressed as percentages and means standard error of the mean. 3. Results The effects of superovulation treatment on pregnancy rates and the incidence of cysts are shown in Table 1. The
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Table 1 Effect of the superovulation treatment on pregnancy rates and incidence of ovarian cysts in donor sows at Days 5 and 6 after insemination. Characteristic
Treatment (IU of eCG) 0 (Control) 1000
1500
Sows, n Pregnancy rate, n (%) Sows with cysts, n (%) Number of cysts in sows with cysts, (mean SEM) Sows with polycystic ovaries, n (%)
36 36 (100) 14 (38.9) 2.4 0.4
27 27 (100) 9 (33.3) 2.9 0.6
27 24 (88.9)a 12 (44.4) 3.6 1.0
0 (0.0)
0 (0.0)
3 (11.1)a
Abbreviation: SEM, standard error of the mean. a Tendency with respect to control group (P ¼ 0.07).
potential pregnancy rate (percentage of sows with more than four viable embryos) tended (P ¼ 0.07) to be lower in sows superovulated with the highest dosage of eCG compared with the controls. This result was because of the presence of polycystic ovaries in three sows (11.1%). None of these sows had CLs on their ovaries, and the size of the majority of cysts was greater than 3 cm. In the control and 1000-IU eCG groups, there was no incidence of polycystic ovaries. A high proportion of donors (range: 33.3%–44.4%) had cysts in the ovaries, with a mean number of cysts ranging between 2.4 0.4 and 3.6 1.0 per sow, with no differences among groups. There were no differences in the recovery rates (range: 89.8%–94.6%) or fertilization rates (range: 91.5%–94.3%) among groups. A higher variability among donors in the ovulatory response to gonadotropins (range: 11 to 36 and 17 to 51 CLs, CV ¼ 21.4% and 26.5%, for the 1000- and 1500IU eCG groups, respectively) compared with the controls (15–28 CLs, CV ¼ 15.9%) was observed. The reproductive parameters obtained in pregnant sows from the three groups are presented in Table 2. The mean number of CLs and viable embryos was higher (P < 0.05) in the superovulated groups compared with the controls and increased (P < 0.05) with increasing doses of eCG. There were no differences among groups in the mean number of oocytes and/or degenerated embryos, which ranged between 1.1 0.3 and 2.4 0.8. The mean number of transferable embryos obtained in pregnant sows was higher (P < 0.05) in the superovulated groups than in the control group and tended (P ¼ 0.07) to be greater in the 1500 IU eCG group than in the 1000-IU eCG group. However, this tendency disappeared when the total number of sows superovulated (pregnant and nonpregnant) was considered (20.6 1.0 and 22.4 2.1 transferable embryos for the 1000-IU and 1500-IU eCG groups, respectively). In general, the proportion of transferable embryos in relation to the number of viable embryos was high in all three groups, although it was higher (P < 0.03) in the superovulated groups (94.1% and 96.8% for the 1000- and 1500-IU eCG groups, respectively) compared with the control group (89.7%). At Day 5 of pregnancy, the percentages of nontransferable viable embryos were different (P < 0.01) among groups (3.5%, 0.0%, and 23.1% for the 1000-IU eCG, 1500-IU eCG, and control groups, respectively) due exclusively to the presence of uncompact morulae. At Day 6 of pregnancy, a higher (P < 0.02) percentage of nontransferable viable embryos (8.1%) was recorded in the 1500 IU eCG group
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Table 2 Influence of superovulation treatment on the reproductive parameters in pregnant donor sows. Characteristic
Treatment (IU of eCG)
Pregnant sows, n CLs, (mean SEM) Viable embryos collected, (mean SEM) Oocytes and/or degenerated embryos, (mean SEM) Recovery rate (%) Transferable embryos, (mean SEM)
36 27 24 19.7 0.5a 24.5 1.0b 31.0 1.7c a b 16.5 0.6 21.3 1.0 26.8 1.6c
0 (Control) 1000
1.1 0.3
1.3 0.5
1500
2.4 0.8
89.8 90.5 94.6 14.8 1.1a 20.6 1.0b 25.2 1.9b,d
Abbreviation: SEM, standard error of the mean. a,b,c Different superscripts within the same row differ (P < 0.05). d Tendency with respect to the 1000-IU eCG group (P ¼ 0.07).
compared with the control (2.4%) and 1000-IU eCG (2.9%) groups as a result of the presence of hatched blastocysts. In all groups, there was a significant linear correlation between the number of CLs and the total number of viable
embryos and transferable embryos, but the number of CLs and the number of oocytes and/or degenerated embryos were not correlated (Fig. 1). The donor parity was not correlated in any group with the number of CLs, viable embryos, transferable embryos, or oocytes and/or degenerated embryos. A total of 1693 transferable embryos were collected, of which 1283 (75.8%) and 410 (24.2%) were classified as morulae or early blastocysts and full blastocysts, respectively. Of these embryos, 1369 were used for ET and the remaining were used for other experiments. The total number of transferable embryos collected from the donors inseminated in the 1000-IU eCG (n ¼ 27), 1500-IU eCG (n ¼ 27), and the control (n ¼ 36) groups was 556, 604, and 533, respectively, resulting in a donor-to-recipient ratio of 1.4:1, 1.3:1, and 2:1, respectively. Five out of 46 ETs (10.9%) were eliminated from the study because of incorrect insertions of the NsDU ET catheter. The transfers were mainly performed in each group with embryos at the morula or early blastocyst (range: 60.0%–63.3%) and full blastocyst stages (range: 27.7%–36.4%) on recipients during Day 5 of the cycle (>80%
Fig. 1. Relationship between the number of CLs and the number of viable and transferable embryos and unfertilized oocytes and/or degenerated embryos on Days 5 and 6 after the onset of estrus in Duroc donor sows treated 24 hours after weaning with 0 (control), 1000-, and 1500-IU eCG. N.S., nonsignificant; r, Pearson coefficient; rs, Spearman coefficient.
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of the transfers). The number of transferred embryos per recipient was 29.5 0.3, 29.9 0.1, and 29.9 0.1 (range: 27–30) for the 1000-IU eCG, 1500-IU eCG, and control groups, respectively, with no differences among groups. The reproductive performance of recipients after transfers is shown in Table 3. In total, pregnancy and farrowing rates were 75.1% and 73.2%, respectively, and the litter size was 9.4 0.6 piglets born, of which 8.8 0.5 were born alive, with an average weight of 1.5 0.6 kg. The percentage of female offspring was 51.4. There were no differences for any of the reproductive parameters evaluated among groups. Additionally, there were no differences among groups in embryo survival rates (range: 20.9%–22.4%). 4. Discussion The results from this experiment clearly demonstrate that the embryos collected from superovulated donor sows with eCG present similar morphological quality at Days 5 and 6 of the cycle and similar in vivo ability to develop to term after transfers as those collected from nonsuperovulated donors. In addition, this study confirms previous reports on the effectiveness of the NsDU ET technology. Regardless of the superovulation treatment (1000- or 1500-IU eCG), we found a high proportion of donors (33%– 44%) with cysts in the ovaries, numbering 3 to 3.5 cysts per sow. These data were similar to those found in nonsuperovulated sows, indicating that the superovulation treatment was not implicated in the cyst incidence. In all of these cases, the cysts were unilateral or bilateral and originated from follicles, which do not ovulate, and coexisted with normal CLs. A much lower incidence of ovarian cysts in sows with previous reproductive failures (between 3.3% and 13.0%) [33–35] or in farms without any particular reproductive problems (2.4%) [36] has been reported. Although the presence of ovarian cysts has been associated with a greater return to estrus rate, a decreased farrowing rate, and an increased percentage of anestral sows [36], in our study, these cysts did not interfere with the reproductive cycles, as can be demonstrated by the excellent reproductive history and by the excellent quality of the embryos collected. Most likely, these cysts were nonfunctional, causing no interference with the reproductive cycle, as has been reported for single cysts [37]. Although the reason for the high percentage of sows with cysts found in our study is unclear, we cannot dismiss the possibility that the high cyst incidence might be an innate characteristic of Table 3 Reproductive performance of the recipients after nonsurgical deeputerine transfer of embryos collected from superovulated purebred Duroc sows. Characteristic
Treatment (IU of eCG) 0 (Control)
1000
1500
Recipients, n Pregnancy rate, n (%) Farrowing rate, n (%) Total born, (mean SEM) Born alive, (mean SEM) Stillborn, (mean SEM)
11 8 (72.7) 8 (72.7) 9.0 0.9 8.6 0.8 0.4 0.3
15 12 (80.0) 11 (73.3) 9.3 1.2 8.6 1.1 0.6 0.2
15 11 (73.3) 11 (73.3) 9.7 1.0 9.2 0.9 0.5 0.3
Abbreviation: SEM, standard error of the mean.
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purebred Duroc sows. The potential pregnancy rate tended to be lower in sows superovulated with the highest dose of eCG. This fact was exclusively because of the presence in three sows (11.1%) of multiple large follicular cysts with no sign of ovulation and without additional CLs on the ovaries, which is associated with infertility [35]. The remaining sows from nonsuperovulated and superovulated groups were all pregnant at Days 5 and 6 after insemination, with no differences in fertilization rates among groups. Therefore, it seems clear that the superovulation treatment had no adverse effect on oocyte maturation, the transport of the gametes, fertilization, or early embryo development. Superovulation treatments are characterized by high variability in the ovulatory response [38]. Our study provides additional reference data concerning intersow variability in the ovulatory response in nonsuperovulated and superovulated sows. We found that the variability was high in nonsuperovulated sows (CV ¼ 15.9%). Similar variation in the ovulation rates (CV range, 16%–20%) has been reported in untreated gilts [12,39,40] and sows [20]. As expected, the ovulatory response variability found in the present study increased in the superovulated sows (CV ¼ 21.4% and 26.5% for the 1000-IU and 1500-IU eCG groups, respectively). However, this variation was slightly more than half of the previously reported values, with similar superovulation treatments (800–1500 IU eCG) for cyclic gilts and sows, in which the CVs were approximately 40% [20,22,24,38]. Because ovulation rates differ among purebred, hyperprolific, and crossbred sows [41–44], this apparent discrepancy may be explained by the different lines and breeds used in these studies, for which the superovulatory response can differ widely [5]. Superovulation treatments have also been associated with high percentages of oocytes and/or degenerated embryos. In our study, we not only obtained an increased number of viable and transferable embryos in the superovulated groups when compared with those in nonsuperovulated sows, but the number of oocytes and/or degenerated embryos was not different among groups and represented 7% of the total structures collected. In addition, only a very low proportion (
