Commercial Embryo Transfer Program Research Articles

The effect of mare's age on multiple ovulation rate, embryo recovery, post-transfer pregnancy rate, and interovulatory interval in a commercial embryo transfer program in Argentina

Advanced maternal age is an important predisposing factor on the reduction of reproductive efficiency. The aim of this study was to evaluate the effect of donor's age on several reproductive parameters in a commercial equine embryo transfer program. Donors were classified into 3 age groups: Group 1=fillies (3 and 4 years old), Group 2=middle age mares (aged 5–10) and Group 3=old mares (aged 13–25). Embryo recovery, multiple ovulation and pregnancy rates and interovulatory intervals were compared amongst age groups. Group 1 (171/244, 70.1%) and Group 2 (774/1081, 71.6%) had a higher (P<0.005) embryo recovery rate than Group 3 (385/701, 54.9%). Groups 2 and 3 were 2.5 and 3.4 times more likely to have multiple ovulations than Group 1 (P<0.05), respectively. The effect of age group on pregnancy rate was not significant (P>0.05). The interovulatory intervals length was influenced by individual mare (P<0.001), age (P<0.04), Day of flushing (P=0.009) and by month (P<0.012). The overall mean interovulatory interval of Group 1 (16.4±0.17 days) and Group 2 (16.6±0.12 days) was not different (P>0.05), but was shorter than the one of Group 3 (17.4±0.15 days; P<0.04). The embryo recovery rate of flushings from Groups 1 and 2 was influenced by the length of the previous interovulatory interval (P=0.03).

Setting up equine embryo gender determination by preimplantation genetic diagnosis in a commercial embryo transfer program

Preimplantation genetic diagnosis (PGD) allows identifying genetic traits in early embryos. Because in some equine breeds, like Polo Argentino, females are preferred to males for competition, PGD can be used to determine the gender of the embryo before transfer and thus allow the production of only female pregnancies. This procedure could have a great impact on commercial embryo production programs. The present study was conducted to adapt gender selection by PGD to a large-scale equine embryo transfer program. To achieve this, we studied (i) the effect on pregnancy rates of holding biopsied embryos for 7 to 10 hours in holding medium at 32 °C before transfer, (ii) the effect on pregnancy rates of using embryos of different sizes for biopsy, and (iii) the efficiency of amplification by heating biopsies before polymerase chain reaction. Equine embryos were classified by size (≤300, 300–1000, and >1000 μm), biopsied, and transferred 1 to 2 or 7 to 10 hours after flushing. Some of the biopsy samples obtained were incubated for 10 minutes at 95 °C and the rest remained untreated. Pregnancy rates were recorded at 25 days of gestation; fetal gender was determined using ultrasonography and compared with PGD results. Holding biopsied embryos for 7 to 10 hours before transfer produced pregnancy rates similar to those for biopsied embryos transferred within 2 hours (63% and 57%, respectively). These results did not differ from pregnancy rates of nonbiopsied embryos undergoing the same holding times (50% for 7–10 hours and 63% for 1–2 hours). Pregnancy rates for biopsied and nonbiopsied embryos did not differ between size groups or between biopsied and nonbiopsied embryos within the same size group (P > 0.05). Incubating biopsy samples for 10 minutes at 95 °C before polymerase chain reaction significantly increased the diagnosis rate (78.5% vs. 45.5% for treated and nontreated biopsy samples respectively). Gender determination using incubated biopsy samples matched the results obtained using ultrasonography in all pregnancies assessed (11/11, 100%); untreated biopsy samples were correctly diagnosed in 36 of 41 assessed pregnancies (87.8%), although the difference between treated and untreated biopsy samples was not significant. Our results demonstrated that biopsied embryos can remain in holding medium before being transferred, until gender diagnosis by PGD is complete (7–10 hours), without affecting pregnancy rates. This simplifies the management of an embryo transfer program willing to incorporate PGD for gender selection, by transferring only embryos of the desired sex. Embryo biopsy can be performed in a clinical setting on embryos of different sizes, without affecting their viability. Additionally, we showed that pretreating biopsy samples with a short incubation at 95 °C improved the overall efficiency of embryo sex determination.

First field results on the use of stallion sex-sorted semen in a large-scale embryo transfer program

Flow cytometry sex-sorting technology was developed in 1989. However, it is only the bovine species in which offspring of the desired sex are obtained at a commercial level. The aim of the present work was to evaluate efficiency parameters when using fresh sexed semen in a large-scale equine commercial embryo transfer program. During the 2009, 2010 and 2011 breeding seasons, 938 synchronized cycles were artificially inseminated. One hundred (10.6%) mares failed to ovulate, and for the remaining 838 useable cycles, 887 doses of sexed semen were used, representing 1.06 doses per cycle. In general, 435 (51.9%) out of 838 flushing performed resulted in the recovery of at least one embryo and 496 (59.1%) embryos were recovered, including twins and triplets. Pregnancy rate at 25 days achieved 81.5% (one embryo transferred per recipient). Embryo recovery rate was not statistically different either between preovulatory and postovulatory artificially inseminated mares or when increased quantities of sexed sperm per dose were used (15–45 million) (P > 0.05). A broad variation in embryo recovery rate was observed between the different stallions used in this study. Sex accuracy of the sex sorting assessed by ultrasound fetal sex determination was 90.3%. Finally, overall efficiency (female embryo pregnancies per useable cycles) was 39% (325/838), meaning that to obtain a female pregnancy of at least 75 days it was necessary to perform 2.5 flushing.

154 EMBRYO RESPONSE OF SUPEROVULATED HOLSTEIN HEIFERS INSEMINATED WITH X-SORTED FROZEN SPERM

A study was conducted within a commercial embryo transfer program to assess the appropriateness of X-sorted semen in superovulated heifers. Commercial X-sorted semen frozen at a dose of 2 million sperm/straw and unsorted semen frozen at a dose of 20 to 30 million sperm/straw was used in donors according to the owner’s genetic criteria. One-hundred 15- to 18-month-old Holstein heifers were superovulated as follows: Animals received a CIDR device (Pfizer Animal Health, New York, NY, USA) for 13 days. At the time of CIDR insertion and 5 days later, heifers received 2 mL (0.15 mg) of d-cloprostenol (Veteglan, Laboratorios Calier, Spain). Gonadotropin-releasing hormone (GnRH), 200 mcg IM (Gonasyl, SIVA, Spain) was administered twice, 36 hours after the second prostaglandin (PGF) and 24 hours after CIDR removal. Superovulatory treatments with commercial FSH (Pluset, Laboratorios Calier) were initiated 36 hours after the first GnRH, with a total dose of 11.5 mL (575 IU), given twice daily in 10 daily decreasing-dose injections. Luteolysis was induced with 0.15 mg PGF given twice, 12 h before and at the time of CIDR removal. Heifers were randomly assigned to two groups for insemination in the uterine body: Group A (n = 31) heifers were inseminated with 5 straws of X-sorted semen in three inseminations (2 straws at 12 h, 2 straws at 18 h, and 1 straw at 24 h after the last GnRH); Group B (n = 69) heifers were inseminated twice with unsorted semen (one straw at each of 12 and 24 h after the last GnRH). A total of 614 embryos (153 in Group A and 461 in Group B) were collected and frozen. There were no significant differences between groups (Mann–Whitney rank sum test, SigmaStat, Systat Software Inc., San Jose, CA, USA) in the embryo production obtained (Table 1). Eighty-four embryos frozen with ethylene glycol were thawed and transferred; 34 from Group A, where all births were females and 50 from Group B with a 0.54 sex ratio. Pregnancy rates were 55 and 54% in Groups A and B, respectively. In conclusion, with the superovulation and AI schedule utilized in this experiment, the use of X-sorted semen in heifers can achieve reproductive outcomes similar to those obtained with unsexed semen, with the benefit of an increase in females offspring. Table 1.Mean number of recovered ova/embryos, transferable embryos, and unfertilized ova obtained in superovulated Holstein heifers inseminated with X-sorted (Group A) and unsorted (Group B) semen

Interruption of gestation and reuse of recipient mares in a commercial embryo transfer program

Interruption of gestation and reuse of recipient mares in a commercial embryo transfer program

Effect of hCG concentration on embryo recovery rate in a commercial equine embryo transfer program in Brazil

Injections were administered intramuscularly. Animals were split into five groups (n 1⁄4 25). G1: Acyclic recipients showing endometrial edema two days after treatment with 5.0 mg of estradiol benzoate (Estrogin , Farmavet, Sao Paulo, Brazil) were injected with 5.0 mL of P4LA300. G2: Cyclic mares showing one 35mm follicle and endometrial edema were treated with 2,000IU of hCG (Vetecor , Hertape Calier, Brazil) and 5.0 mL of P4LA300. Progesterone injections were discontinued at ET if an ovulation was detected. G3: Estrous recipients showing 25 mm follicles and endometrial edema were treated with 5.0 mL of P4LA300. G4: Diestrous recipients five to fourteen days post-ovulation, showing no endometrial edema and a visible corpus luteum were simultaneously treated with 5.0 mg of estradiol benzoate and dinoprost tromethamine (Lutalyse , Pfizer, Sao Paulo, Brazil). If considerable endometrial edema was detected two days after treatment, 5.0 mL of P4LA300 were administered. G5: Cyclic recipients whose ovulations were induced with 2.000 IU of hCG, serving as control. Progesterone-treated recipients received 5.0 mL of P4LA300 at ET, and, upon pregnancy diagnosis, were injected at fourteen-day intervals with 10.0 mL, until 120 days. Pregnancy at 15 days and loss rates at 120 days were statistically evaluated through Fisher's exact tests (P 0.05), respectively, 76.0% (19/25) and 10.5% (2/19) (G1); 68.0% (17/ 25) and 5.9% (1/17) (G2); 80.0% (20/25) and 10.0% (2/20) (G3); 60.9% (14/23) and 0% (0/14) (G4) and 60.0% (15/25) and 13.3% (2/15) (G5). In G2, 72.0% (18/25) showed a corpus luteum at ET, with a 72.2% (13/18) of pregnancy rate. Regardless of their estrous cycle phase or ovarian activity, recipients with either natural or pharmacologically induced endometrial edema, submitted to ET four days after treatment with long-acting progesterone, showed satisfactory pregnancy and embryo loss rates, allowing simple and effortless synchronization between donor and recipients.

Use of long-acting progesterone to acyclic embryo recipient mares

The objective of this study was to evaluate the efficiency of cyclic and acyclic progesterone-treated recipient mares in a commercial embryo transfer program. Acyclic recipients were treated with decreasing amounts of estradiol cypionate (ECP® - Pfizer Saude Animal, Sao Paulo, Brazil), followed by 1500 mg of a sustained-release progesterone injection (P4LA150® - Laboratorios B.E.T., Rio de Janeiro, Brazil). Out of all 1506 embryo recovery attempts performed, 59.36% were positive (894/1506). Pregnancy rates of cyclic and acyclic mares were, respectively, 44.12% (319/723) and 57.68% (139/241) - higher in acyclic recipients under progesterone treatment. Embryo loss was similar between groups (9.72% 31/319 vs. 11.51% - 16/139). When fertility data on acyclic mares treated with long-action progesterone were grouped in relation to the interval of the beginning of the treatment with progesterone to the embryo transfer, there was no difference in pregnancy rates at 12 days (51.28% - 20/39; 58.92% - 33/56; 60.52% - 46/76 and 58.57% - 41/70 for embryos transferred 3 (P3), 4 (P4), 5 (P5) and 6 (P6) days after the beginning of the treatment with progesterone). Groups P3, P4, P5 and P6 showed embryo loss rates of 20.00% (4/20), 12.12% (4/33), 2.17% (1/46) and 17.07% (7/41), respectively. These rates were similar in groups P3, P4 and P6. Pregnant recipients submitted to an embryo transferring procedure on P5 showed lower embryo mortality rates than on P3 and P6. P4 recipients tended to suffer higher embryo mortality rates than P5. Acyclic recipient mares under progesterone treatment can be satisfactoryly used in embryo transfer programs.

A Short and Simple Improved-Primer Extension Preamplification (I-PEP) Procedure for Whole Genome Amplification (WGA) of Bovine Cells

Embryo transfer is a reproductive technique that has a major impact on the dissemination of economically important genes and the rate of genetic gain in breeding schemes. In recent years, there has been increasing interest in the use of sexed and genotyped embryos in commercial embryo transfer programs. Marker/gene assisted selection (MAS / GAS) projects can be performed in the pre-implantation stage through mass production of characterized embryos. Biopsy of a few cells in the morulla stage is essential for pre-implantation genetic diagnosis (PGD), in which sex determination, evaluation of disease genes, and genotyping for candidate genes are performed. Limited quantity of cells and low amount of DNA restrict the use of multiple molecular analyses in PGD programs. Recently, whole genome amplification (WGA) techniques promise to overcome this problem by providing sufficient input DNA for analysis. Among several techniques proposed for WGA, the primer extension pre-amplification (PEP) and the improved-primer extension pre-amplification (I-PEP) methods are the most commonly used. However, these methods are time-consuming and need more than 12 h amplification cycles. Since the time is a critical parameter in the successful characterized embryo transfer, the shortening of diagnosis time is highly desirable. In this study, we developed a short and simple I-PEP procedure (∼3 h) and evaluated its performance for the amplification of bovine genomic DNA. We assessed short WGA procedure by polymerase chain reaction (PCR) amplification of 7 specific loci. The results indicated that the short procedure possesses enough sensitivity for the molecular genetic analysis of 1 input cell. Although the efficiency of the method was 100%, there was an inconsistency between genomic DNA (gDNA) and whole genome amplification product (wgaDNA) genotypes for kappa-casein locus; that is, however, most likely due to allele drop-out (ADO) or false homozigocity. The results of this study indicate that with the application of reliable methods, WGA-amplified bovine DNA will be a useful source for sexing and genotyping bovine embryos in several quantitative trait locus (QTL) markers.

110 PREGNANCY RATES OF BOVINE EMBRYOS AND HEMI-EMBRYOS TRANSFERENCE

Embryo transfer (ET) in cattle speeds up genetic gains, but its use is limited by cost-benefit analysis. In addition other techniques can be developed, such as sexing and bipartition of embryos. An alternative to improve the economic viability of ET in cattle would be to bipartition embryos in order to increase the yield of the technique. The aim of this study was to investigate the operational viability of embryo bipartition technique in an ET program in cattle and to study aspects related to the viability and development of bovine hemi-embryos (HE) compared with intact ones. The embryos were collected by nonsurgical technique 7 days after the onset of oestrus. Viable structures (130) from 49 embryo collections from 25 Aberdeen and Simmental donor cows and heifers were used. Only embryos in compact morula, early blastocyst and blastocyst stage, with a morphological range from excellent to good grade (IETS Grade 1), were split. The embryos, split (78) or not (52), were transferred into the uterine horn ipsilateral to the corpus luteum of the recipients 7 days after oestrus. The treatment groups evaluated were T1 (intact embryo, n = 52), T2 (1 HE, n = 27) and T3 (2 HE, n = 51). Crossbred heifers were used as recipients and pregnancy diagnosis was done at 60 to 80 days of gestation. The embryos of T1, T2 and T3 were classified morphologically as excellent or good and by developmental stage as morula, early blastocyst, or blastocyst, distributed as follows: T1: 30, 22 and 16, 17 and 19; T2: 15, 12 and 7, 9 and 11; and T3: 29, 22 and 15, 15 and 21, respectively. The birth rate per original embryos was greater for T2 than for T1 and T3 (Table 1). The pregnancy rates for excellent and good embryos and morulae, early blastocysts and blastocysts were not different (P &gt; 0.05). The T1, T2 and T3 twin births were 0, 1 and 5, respectively. It is concluded that embryo bipartition technique applied in a commercial ET program is a viable operational technique. Table 1.Pregnancy at 60 to 80 days and birth rate per original embryos used Supported by CAPES, CNPq, FAPEMIG and BIOTRAN.

37 EFFECT OF EMBRYO STAGE ON PREGNANCY RATE FOLLOWING DIRECT TRANSFER OF BOVINE EMBRYOS FROZEN IN ETHYLENE GLYCOL

Annually, more than 400 000 in vivo-recovered bovine embryos are officially reported by members of the Canadian and American Embryo Transfer Associations. Between 65 and 70% of these embryos are cryopreserved and more than 95% are frozen in ethylene glycol (EG). Statistics on factors affecting embryo freezing are difficult to obtain because many cattle breeders/farmers no longer report pregnancy rates back to embryo transfer (ET) practitioners. Concerns are often expressed as to the optimal stage at which to freeze bovine in vivo-derived embryos. This is a retrospective analysis of results from 5 commercial ET programs (1 in the United States, 3 in Canada and 1 in the Netherlands) for which pregnancy data relative to embryo stage at freezing were made available. Embryos representing 4 stages of development, as defined by the IETS (4 = late morula, 5 = early blastocyst, 6 = mid blastocyst and 7 = expanded blastocyst) are included in the data. The number of embryos thawed and transferred ranged from 3954 to 24 827 for the 5 programs, with a total of 72 828. Embryos were frozen in either 1.5 M EG or 1.5 M EG + 0.1 M sucrose and exposure time to cryoprotectant before cooling ranged from 4 to 40 min. Pregnancy rates are shown in Table 1. Although the pregnancy rate for stage 6 embryos was only 2.6 and 3.2 percentage points lower than stages 4 and 5, respectively, these differences were highly significant and pregnancy rates for stage 6 embryos were lower than those for stages 4 and 5 in 4 of the 5 ET programs. The small decreased survival of stage 6 embryos is probably only moderately important in a commercial context. However, the pregnancy rate of stage 7 embryos was lower than all other stages for the combined dataset as well as in all 5 ET programs, with the difference between stages 5 and 7 ranging from 6.5 to 16.4 percentage points. Clearly, stage 7 embryos survive freezing at a significantly lower rate than stages 4, 5 and 6 and neither time of exposure to EG nor inclusion of sucrose in the freezing medium provided an obvious improvement. Although bovine ET practitioners routinely attempt to collect embryos on day 7 post-oestrus, recovery of stage 7 embryos cannot always be avoided. Further investigation into factors contributing to the decreased survival of stage 7 embryos is warranted. Table 1.Effect of embryo stage on pregnancy rate of bovine embryos frozen in EG

230 SUPEROVULATORY RESPONSE OF BEEF COWS WITH OR WITHOUT A CORPUS LUTEUM AT THE TIME OF INSERTION OF A PROGESTERONE RELEASING DEVICE

Commercial embryo transfer programs require frequent superovulation of embryo donors. Although early reports suggested that donor cows require 60 to 90 days to recover from superovulation, recent information suggests that this interval could be reduced to 25 to 30 days. Because donor cows reovulate at variable intervals after embryo collection, some donors do not have an ultrasonically detectable corpus luteum (CL) when frequent superovulation programs are initiated. A retrospective study was performed to evaluate the influence of the presence of a CL at the time of initiating treatments on superovulatory response of beef donors that had been previously collected once and then were collected every 28 to 35 days 2 or 3 times. The data were from 190 collections performed in 48 Angus, 36 in 10 Brangus and 74 in 20 Bonsmara donors. On Day 0, all cows were ultrasonically examined for the presence of a CL (Falco 100 Vet, 8-MHz transducer, Pie Medical, the Netherlands) and were treated with 5 mg of oestradiol-17β and 50 mg of progesterone IM and a progesterone-releasing intravaginal device (Cue-Mate, Bioniche Animal Health, Belleville, Ontario, Canada). On Day 4, donors were superstimulated with Folltropin-V (Bioniche Animal Health), in twice-daily injections over 4 days or diluted in 20 mg mL–1 hyaluronan and given by a single IM injection. Folltropin-V dosages used were 300 or 400 mg (Angus), 260 or 300 mg (Brangus), or 200 or 300 mg (Bonsmara). On Day 6, all cows received 2 doses of PGF2α 12 h apart and Cue-Mates were removed in the p.m. In the a.m. of Day 8, cows received 12.5 mg pLH (Lutropin-V; Bioniche Animal Health) and were inseminated 12 and 24 h later. Ova/embryos were collected nonsurgically on Day 15 and evaluated following IETS guidelines. For each breed, superovulatory response (i.e. mean number of CL) and embryo data were evaluated by ANOVA for mixed models, using CL, FSH dose and treatment as fixed variables and cow identification as a random variable. Forty-six (24.2%) Angus donors, 4 (11.1%) Brangus donors and 5 (6.8%) Bonsmara donors did not have a CL at the time of initiating FSH treatment. There was no significant effect of FSH dose or treatment protocol on superovulatory response and the presence or absence of a CL did not influenced embryo production (P &gt; 0.31). Mean (± SEM) number of CL, ova/embryos and transferable embryos were 13.2 ± 0.5, 12.4 ± 0.7 and 6.1 ± 0.4 in Angus donors with a CL vs 13.0 ± 0.7, 11.5 ± 0.9 and 5.6 ± 0.7 for those without a CL (P &gt; 0.37); 14.8 ± 1.2, 14.2 ± 1.6 and 8.4 ± 1.1 for Brangus donors with a CL vs 15.2 ± 3.1; 11.2 ± 3.3 and 5.7 ± 2.8 for those without a CL (P &gt; 0.34); and 13.2 ± 0.8, 11.2 ± 1.0 and 7.4 ± 0.5 for Bonsmara donors with a CL vs 17.4 ± 3.7, 13.6 ± 6.5 and 9.2 ± 4.3 for those without a CL (P &gt; 0.16). In summary, the presence or absence of a CL did not affect embryo production in donor cows superstimulated every 28 to 35 days using a progesterone-releasing device and FSH. Study was supported by Bioniche Animal Health, Belleville, Ontario, Canada.

151 PREGNANCY RATES AFTER A NONSTEROIDAL ANTI-INFLAMMATORY (FLUNIXIN MEGLUMINE) AND PROGESTERONE (P4) ADMINISTRATION TO MANGALARGA MARCHADOR RECIPIENT MARES

The aim was to evaluate whether pregnancy rates could be improved by randomly injecting recipient mares with flunixin meglumine (FM; Banamine®, Schering-Plough, São Paulo, Brazil) on the day of embryo transfer combined or not with long-acting progesterone (P4) in commercial embryo transfer programs. One hundred sixty-five Mangalarga Marchador recipient mares from the 2006–2007 breeding season of 4 different commercial programs were used. Ages ranged from 4 to 15 years old. In 86 mares, 1.1 mg kg–1 of BW of FM was given intravenously immediately after embryo transfer, and in 79 mares, only saline was injected. Additionally, 59 mares receiving saline were injected with 1 800.0 mg of progesterone at embryo transfer and every week thereafter until 110 days of gestation. Of the 86 receiving FM, 17 received the same progesterone protocol. Embryos were transferred nonsurgically in Ham’s F-10 or Encare medium. Pregnancy was checked by ultrasonography at 15, 30, and 60 days after transfer, and only a positive diagnosis on the latter was considered pregnancy in the statistical analysis. In a 2 × 2 factorial design (2 levels of FM and P4), pregnancy rates were analysed using the GENMOD procedure considering a binomial distribution (SAS®, Cary NC), and means were compared by orthogonal contrasts. Pregnancy rates were lower (P &lt; 0.0001) in FM-treated (50.2 ± 5.3%) compared with control (88.2 ± 5.1%) mares and in P4-treated (85.9 ± 5.0%) compared with control (52.4 ± 5.5%) mares. An interaction effect between FM and P4 was not observed (P &lt; 0.49). For mares receiving or not receiving FM, the addition of the P4 protocol lowered pregnancy rates. In mares not treated with FM, pregnancy rates were 81.4 ± 5.1% and 95.0 ± 8.8% for P4-treated and nontreated mares, respectively (P &lt; 0.05). In FM-treated mares that did not receive P4, pregnancy rates were 76.8 ± 4.8% compared with 23.5 ± 9.6% of those treated with P4 (P &lt; 0.0001). In conclusion, the indiscriminate use of progesterone and nonsteroidal anti-inflammatory drugs may actually decrease reproductive efficiency and even act synergistically to potentiate their effect. These results must be interpreted with caution, considering that the data are derived from field observations, although sources are from very reliable and well-controlled operations. Nevertheless, the authors consider it imperative to have a proper diagnosis before generalizing the use of medical tools to improve pregnancy rates in horses. Financial support from CNPQ–Conselho Nacional de Desenvolvimento Científico e Tecnológico, CAPES–Coordenação de Aperfeiçoamento de Pessoal de nível Superior. Schering/Plough, JOFADEL, and ABCCMM–Associação Brasileira dos Criadores do Cavalo Mangalarga Marchador.

Influence of environmental temperature, exercise, semen type and ovulation characteristics on reproductive performance in a commercial embryo transfer program

Influence of environmental temperature, exercise, semen type and ovulation characteristics on reproductive performance in a commercial embryo transfer program

172 PREGNANCY RATES AND EARLY PREGNANCY LOSSES IN POSTPARTUM EQUINE EMBRYO RECIPIENTS

In commercial equine embryo transfer programs, the cost of recipient mares represents one of the major economical items that increases the final cost of the pregnancies (Douglas 1985; Losinno and Alvarenga 2006). Due to the risks to newborn foal, it is uncommon to use foaling recipients in the large equine embryo transfer programs in Argentina. The objective of this study was to evaluate pregnancy and early embryonic loss rates between foaling and non-foaling recipients in a commercial embryo transfer program. A total of 173 embryos were collected from Arabian donor mares during 3 consecutive breeding seasons, 2006-2009. Donor mare’s ages ranged from 2 to 22 years, and donors were inseminated with either fresh or frozen semen from 8 stallions 2-17 years old and of proved fertility. Uterine flushing was performed on Day 8 to 9 post-ovulation by standard techniques (Douglas 1985) and recovered embryos were transferred nonsurgically. Recipient mares 3-12 years old were selected clinically by rectal palpation and ultrasound. All embryos were transferred to synchronized recipient mares (-1 to +1 according to donor’s ovulation day) and randomly assigned to non-foaling (n = 84) or foaling (n = 89) recipient groups. For the analysis, we first considered pregnancy and early pregnancy loss rates in both groups, and second, the foaling mares were divided in 3 groups according to the days between foaling and transfer: 1) before 30 days postpartum (n = 10), 2) between 31 and 60 days postpartum (n = 30), and 3) after 61 days postpartum (n = 49). Pregnancy diagnosis was performed by ultrasonography on Days 14, 30, and 60 post-ovulation of the donor. Results were evaluated by chi-square test. No statistical differences (P &gt; 0.05) were observed in pregnancy rates between non-foaling and foaling recipients (76.2% and 80.9%, respectively) and in post-foaling groups (80% for recipients transferred before 30 days postpartum, 73.3% for recipients transferred between 31 to 60 days postpartum, and 85.7% for recipients transferred after 61 days postpartum). No statistical differences (P &gt; 0.05) were found in pregnancy loss rates between recipients transferred before 30 and after 61 days postpartum (25% v. 4.8%, respectively) and between recipients transferred before 30 days postpartum and the non-foaling group (25% v. 10.9%, respectively). Pregnancy loss rates for recipients transferred after 31 and 61 days postpartum (9.1% and 4.8%, respectively) were not significantly different with the non-foaling group, and both are within previously reported values. Our results show that postpartum recipients can be used 30 days after normal foaling in a commercial embryo transfer program with similar pregnancy and early pregnancy loss rates and can be a valuable tool maximizing the use of the recipient mares during consecutive breeding seasons.

176 PREGNANCY AND PREGNANCY LOSS RATES OF ACYCLIC RECIPIENT MARES TREATED WITH TWO DIFFERENT LONG-ACTING PROGESTERONE CONCENTRATIONS

Long-acting progesterone compounds containing concentrations of 150 mg mL-1 (P4LA150) have been routinely used in Brazil, especially at the beginning and end of the breeding season when the percentage of recipient mares showing normal estrous cycles is low compared to donors (Greco GM et al. 2008 Proceedings 7th International Symposium on Equine Embryo Transfer, 59-60). However, recipients treated with P4LA150 must receive weekly administrations until pregnancies achieve 100 days. The present study aimed to evaluate pregnancy and pregnancy loss rates from acyclic mares treated at 7-day intervals with P4LA150 and 14-day intervals with a long-acting progesterone formulation containing 300 mg mL-1 (P4LA300). Acyclic Campolina breed mares (n = 80) ranging from 4 to 13 years old showing considerable endometrial edema 2 days after estradiol benzoate injection (5 mg, i.m.) were split in 2 groups. Group 1 mares (n = 40) received 10 mL of P4LA150 (1500 mg) i.m. every 7 days, whereas Group 2 mares (n = 40) were treated with 10 mL of P4LA300 (3000 mg) i.m. every 14 days. Steroid hormone formulations were produced in our research laboratory, at São Paulo State University, Botucatu, Brazil. Embryos 7 to 8 days of age were transferred into the uteri of recipients between 4 and 7 days after the first progesterone administration. Following a positive pregnancy diagnosis at 14 to 15 days, mares were examined at Days 50 and 100, when progesterone treatment was discontinued. These evaluations were conducted in order to determine pregnancy loss rates during both periods. Results were analyzed through the chi-square test and significance was set at P &lt; 0.05. Pregnancy rates and pregnancy loss rates at Day 50 from mares treated with P4LA150 (Group 1) and P4LA300 (Group 2) did not differ. Pregnancy rates were 60.0% (24/40) and 75.0% (30/40) at Day 50 for mares treated with P4LA150 (Group 1) and P4LA300 (Group 2), respectively (P &gt; 0.10). Pregnancy loss rates at Day 50 were 8.3% (2/24) and 10.0% (3/30) for mares treated with P4LA150 and P4LA300, respectively (P &gt; 0.10). Pregnancy losses were not identified at Day 100. Based on the results from the present study, we concluded that acyclic recipient mares treated with P4LA150 or P4LA300 are able to obtain satisfactory pregnancy rates with low pregnancy losses. Moreover, the use of 3000 mg of long-acting progesterone every 14 days reduces overall progesterone administrations, minimizes stress in horse handling, and makes the choice for acyclic recipient mares more appealing in commercial embryo transfer programs. Authors would like to thank FAPESP for financial support.

Use of a Low Dose of Equine Purified FSH to Induce Multiple Ovulations in Mares

The effects of a low dose of equine purified FSH (eFSH) on incidence of multiple ovulations and embryo recovery rate in mares were studied. During the physiological breeding season in Brazil (19 degrees 45'45'S), 14 Mangalarga Marchador donor mares were used in a crossover study and another 25 mares of the same breed, between 3 years and 12 years of age were used as recipients for the embryo transfers. Donors were monitored during two consecutive oestrus cycles, an untreated control cycle followed by a treated cycle, when eFSH was administered. In both cycles, after an embryo collection attempt on day 8 post-ovulation all mares received 7.5 mg dinoprost and had their two largest follicles tracked daily by ultrasonography until the period of ovulation. Mares were inseminated every 48 h with extended fresh semen from a single stallion after the identification of a 35-mm follicle until the period of ovulation. Ovulations were induced by intravenous administration of 2.500 IU of human chorionic gonadotropin, upon detection of a 35- to 40-mm follicle. In the treated cycle, 5 mg eFSH was given intramuscularly once a day, from day 8 post previous ovulation until at least one follicle reached 35 mm in diameter. Embryo flushes were performed on day 8 of dioestrus (day 0 = ovulation). Treatment with eFSH resulted in higher (p < 0.05) ovulation rate and incidence of multiple ovulations compared to the control (1.6 vs 1.0 and 50% vs 0%, respectively--one mare had triple ovulation). However, embryo recovery rates in the control and treated cycles were similar (0.8 and 1.0, respectively; p > 0.05). Pregnancy rates in the recipient mares following embryo transfer were similar for the control and eFSH cycles (11/11 and 10/14, respectively). Additional studies are necessary in order to develop a low-dose protocol for the use of eFSH that brings a more consistent contribution to the efficiency of commercial equine embryo transfer programs.

298 COMPARISON OF THREE DIFFERENT PROTOCOLS FOR SUPERSTIMULATION OF DAIRY CATTLE

Traditionally, successful superstimulation of cattle depended on initiating injections of gonadotrophin at mid-cycle, approximately at second follicular wave emergence. This approach limited the convenience of scheduling donors for superstimulation. With the use of intravaginal progesterone-releasing devices and estradiol 17β, superstimulation can be initiated successfully at any time of the estrous cycle. However, because estradiol cannot be legally injected into cattle in an increasing number of countries, the efficacy of GnRH as an estradiol substitute was investigated. A retrospective analysis was performed on data collected in a commercial bovine embryo transfer program over a period of several years. All donors were lactating dairy cows at least two years of age; approximately 75% were comprised of Holstein and the remainder of Jersey, Guernsey, or Brown Swiss breeds. The three treatments employed were (1) Controls injected twice daily for 4 days with a total of 240 to 400 mg of porcine FSH (Folltropin-V, Bioniche Animal Health, Inc.) in decreasing doses starting between day 7 and day 14 of diestrus, with PG (Lutalyse, Pfizer Animal Health) given at the time of FSH injections no. 5 (35 mg) and 6 (25 mg); (2) Estradiol females received a CIDR (Pfizer Animal Health), 5.0 mg estradiol 17β and 100 mg progesterone in oil on random days of the estrous cycle; FSH was initiated 4 days later as described for controls with CIDR removal at the time of FSH injection no. 6; (3) GnRH females received a CIDR on random days of the estrous cycle and 100 μg GnRH on day 1.5 following CIDR insertion; FSH was initiated 60 h after GnRH injection as described for controls with CIDR removal at the time of FSH injection no. 6. All donors were inseminated with one straw of frozen semen 12 and 24 h after the onset of estrus. Embryos were nonsurgically recovered 7 to 8 days after onset of estrus. Only embryos of grades 1 to 3 (IETS classification) were included in the data. Data were analyzed by ANOVA and Tukey’s hsd test was used to distinguish significance among means as shown in Table 1. Estradiol females produced approximately 2 more ova/embryos per procedure than Control and GnRH groups and an average of 0.8 more embryos per female than did the Control group, but there was no difference compared to the GnRH group. Similar to what has been shown in other commercial embryo transfer data sets, nearly 25% of the donors in each group failed to produce at least one good embryo. Clearly, all three treatments resulted in efficacious superstimulation. In light of the legality issues surrounding the use of estradiol, this study shows that GnRH can be used quite successfully to superstimulate dairy cattle at random times of the estrous cycle. Table 1.Average numbers of ova and embryos recovered from dairy cows superstimulated with three different protocols We thank G.E. Seidel, Jr. and S.C. Purcell for assistance with statistical analysis.

Effects of Bovine Somatotropin (bST) Administration Combined with Controlled Internal Drug Release (CIDR) on Embryo Quality and Pregnancy of Hanwoo (Korean Native Beef Cattle) during Commercial Embryo Transfer Program

Effects of recombinant bovine somatotropin (bST) on plasma hormonal concentration, embryo quality, and pregnancy rate were examined during the superovulation and synchronization treatment in donor and recipient cows. Hanwoos (Korean native beef cattle) were treated with controlled internal drug release (CIDR) combined with bST (CIDR+bST) or without bST (CIDR) as donor cows. The embryos recovered from donors were transferred into Holstein recipient heifers treated with bST (CIDR+bST) or without bST (CIDR) for synchronization. The correlation between IGF-I and P4 showed a positive pattern in the CIDR+bST group (r = 0.44, p<0.01), but a negative pattern was shown in the CIDR group (r = -0.59, p<0.02) at day 7 of estrous cycles. Although the number of recovered, transferable, and degenerated embryos was not different, quantities of grade 1 (excellent) embryos in CIDR+bST group were significantly higher than those of the CIDR group (p<0.01). The pregnancy rate was higher in the CIDR+bST recipient group compared to CIDR group (p<0.05), when the embryos were recovered from the donors treated with CIDR. However, the pregnancy was maintained highly in both recipient groups, when the embryos were produced by CIDR+bST treated donors. It can be concluded that bST administration combined with CIDR is an effective method for superovulation and synchronization treatment to stabilize plasma hormonal levels, to obtain excellent quality of embryos, and to get higher pregnancy rate.

368 FIELD STUDIES ON THE EFFECTIVENESS OF THE YCD EMBRYO SEXING TECHNIQUE IN BOVINE

The YCD sexing technique is applied in many commercial breeding programs due to its ease of operation in the field and its reliable and accurate sex determination results. Less equipment requirements and more simplified protocols provide great convenience for field applications of this method, and processing errors and chances of assay contamination are reduced. To increase the effectiveness of this technique in future field applications, field data collected from commercial embryo transfer programs in 63 farms in China during June 2002 to December 2005 were analyzed. Embryos were flushed from superovulated dairy cattle and subjected to a biopsy for sex determination using protocols and YCD sexing kits supplied by AB Technology Ltd. (Pullman, WA, USA). Embryos were implanted fresh on the same day or frozen with AG freeze medium (AB Technology Ltd.) for later transfer. The chi-square test was used for statistic analysis. This study indicates that sampling with a piece of zona pellucida (ZP) during embryo sexing increased the incidence of no response (NR) but did not alter the accuracy of determining sex of offspring. Larger biopsies significantly reduced the incidence of NR after DNA amplification and electrophoresis separation, and increased the accuracy of sex determination of calves (from 88.1%, 111/126 to 98.5%, 129/131; P &amp;lt; 0.01). Biopsies containing dead and/or detached cells did not increase the NR incidence, but decreased the accuracy of sex determination of calves from 93.4% (240/257) to 86.6% (58/67; P &amp;lt; 0.05). There were no relationships between NR incidences and embryo quality, nor between the sex determination accuracy of calves and embryo quality or stages. However, the NR incidence increased for PCR analyses of earlier development stages (5.1% at morula to early blastocyst) compared to blastocyst (3.2%) and expanded blastocyst (2.2%; P &amp;lt; 0.05). The sex determination accuracy decreased to 77.4% (65/84) when embryos with unclear or expanded and faint female electrophoresis bands were transferred compared to those with clear bands (97.1%, 233/240). Field data suggest that larger biopsies during embryo sexing favor the effectiveness and accuracy of the YCD technique; however, increased sample size may affect the embryo survival, resulting in a decrease in the pregnancy success of embryo transfer. Reduced accuracy in YCD analyses using dead or detached blastomeres may be caused by the loss of male genes in dead or degenerated cells. Increased NR incidence in sampling of ZP for analysis may be due to biopsy loss when transferring the biopsy at the sampling step of the YCD method. In addition, the accuracy of sexing will be lower when embryos with unclear electrophoresis results are used. It is concluded that a proper and stage-dependent size of biopsy should be collected and used to ensure the accuracy of sex determination without reducing embryo survival, and the inclusion of a few live cells in a biopsy is important to the success of the sexing determination of this technique.

363 FACTORS INFLUENCING PREGNANCY RATES FOLLOWING TRANSFER OF BOVINE IN VIVO EMBRYOS BIOPSIED FOR SEX DETERMINATION

Data collected from commercial embryo transfer programs in 63 farms in China during June 2002 to December 2005 was analyzed to examine the effects of various factors (biopsy, freezing, sample size, embryo development and quality, in vitro culture, and recipient quality) on pregnancy rates of in vivo-biopsied embryos. Embryos were flushed from superovulated dairy cattle and subjected to a biopsy for sexing determination using protocols and sexing kits supplied by AB Technology Ltd. Fresh embryos were implanted on the same day or frozen with AG freeze medium (AB Technology Ltd., Pullman, WA, USA) for later transfer. Recipients were synchronized with CIDA + PG protocols. Embryos were cultured in 6-well dishes containing 1.3 mL of holding medium (AB Technology Ltd.) in each well at room temperature (20–25�C) for examination of embryo survival in vitro. The chi-square test was used in statistic analysis. The implantation of fresh embryos after biopsy did not affect pregnancy rates (49.6%, 257/518) compared to that of non-biopsied fresh and frozen–thawed embryo groups (52.9%, 47/140 and 46.6%, 177/380, respectively). However, for biopsied embryos subjected to frozen and thawed procedures before implantation, particularly for those subjected to the removal of a larger biopsy, a reduced pregnancy rate was observed (41.8%, 297/710; P &amp;lt; 0.01). Pregnancy rates among biopsied embryos at 3 different development stages (morula-early blastocyst, blastocyst, and expanded blastocyst) were not different. Similar results were found between embryo groups of grade 1 and 2. A significant decrease in pregnancy rate (0/10) was observed with embryos held in vitro for a longer period of time (&amp;gt;5 h), suggesting detrimental effects of in vitro conditions on embryo survival. The highest pregnancy rate (68.0%) was observed in recipients synchronized for the first time before being implanted with biopsied embryos. Significant decreases in such rates were found in recipients synchronized for the second or third times or those with an abortion history at the first or second synchronization-implantation treatment (P &amp;lt; 0.01). Better pregnancy rates (45.6%, 41/90; 46.1%, 76/165; and 45.5%, 5/11) were obtained for recipients implanted with biopsied embryos at Days 7.5, 8.0, and 8.5 post-heat detection, respectively, compared to 16% at Day 7 (3/18, P &amp;lt; 0.05). It is concluded that mechanical treatment (cutting) does not reduce the survival of biopsied embryos; however, cryopreservation reduces their ability to survive in vivo. The analyses also suggest that holding embryos in vitro should not be longer than 5 h unless more favorable in vitro conditions can be provided. To achieve better results of implantation of biopsied embryos, embryo transfer should be performed during 7.5–8.5 days post-estrus, and the healthy recipients synchronized for the first time should be used.