- Email: [email protected]
Embryo quality grade and recipient ovulation rate affect pregnancy rates in embryo transfer recipient mares
Abstracts / Journal of Equine Veterinary Science 41 (2016) 51e84
culture, modified so that all media used were 285±3 mOsm. The blastocyst rates were 10/43 (23%) and 18/50 (36%) respectively (P > 0.1). In summary, systems using commercial media for oocyte manipulation, a more complete cell culture medium for late embryo development, and a consistent osmolarity supported blastocyst rates not significantly different from, but numerically higher than, those of our control system. We are now evaluating these systems to determine if better control of the in vitro embryo environment may improve embryo viability after transfer. Key Words: horse, ICSI, oocytes, embryo
61
cycle. The odds of a mare providing an embryo did not decrease as the number of flushes in a given year increased (OR¼ 0.89; P<0.22). Lastly, breed of donor affected embryo recovery, with embryo recovery from Quarter Horses (41%) being less than that for Thoroughbreds (78%) and Warmbloods (57%). Over 70% of the Quarter Horses, Arabians and Warmbloods were bred with frozen semen and only 9% of the Thoroughbreds. In summary, the highest odds of recovering an embryo are from maiden or foaling mares bred with fresh or cooled semen and inseminated more than once per cycle. Key Words: embryo recovery, semen, number of inseminations, breeds
Acknowledgments This work was supported by the Clinical Equine ICSI program, Texas A&M University and the Link Equine Research Endowment Fund, Texas A&M University.
23 Factors Affecting Embryo Recovery: Retrospective Study based on Commercial Embryo Transfer E.L. Squires 1, *, P.L. Loomis 2, D. Scofield 2, J. Baumber-Skaife 2, S. Barbacini 2, K.J. McQuerry 3, B.A. Ball 1 1 Gluck Equine Research Center, University of Kentucky, Lexington, KY; 2 Select Breeders Services, Inc., Chesapeake City, Maryland; 3 Department of Statistics, University of Kentucky, Lexington, KY *Corresponding author: [email protected] Embryo recovery and transfer has increased dramatically since first introduced in the late 1970's. Knowledge of the factors that affect the success of embryo recovery is important to discuss with clients prior to enrolling a mare in an embryo transfer program. The objectives of this study were to determine if embryo recovery was altered by: 1) age, 2) breed, 3) reproductive status, 4) type of semen, 5) number of inseminations per cycle and, 6) effect of repetitive flushes in a given year. This study utilized data collected from 10 embryo transfer facilities during 2010-14. Seven of these were in the USA and one each in Germany, Italy and Ireland. Donor mares (n¼203) were inseminated with fresh, cooled or frozen semen and embryos were recovered 7-8 days after ovulation (n¼640 flushes). The breed of mares varied with the clinics; Germany and Ireland having primarily Warmbloods and two of the clinics in the USA with mostly Quarter Horses (QH). Other breeds included Arabians, Standardbreds, Warmbloods and Paints. Age, breed, reproductive status (maiden, barren, foaling), number of times inseminated per cycle, and number of embryo recovery attempts each year were recorded. Data were analyzed using a logistic regression with repeated measures. (Proc GENMOD, SAS9.4; SAS Institute Cary, NC, USA). Donor's age had no effect on embryo recovery. The odds of embryo recovery (OR¼0.58) tended to be greater for foaling mares versus barren mares (P<0 .07) and maiden mares versus barren mares (P< 0.0013; OR¼2.73). Embryo recovery using fresh, cooled and frozen semen for barren mares was 36.3% (122/336), for foaling mares 48% (48/100) and maiden mares 56.9% (87/153) . Mares bred with either fresh (P<0.0001) or cooled semen (P<0.0003) had greater odds of embryo recovery than those inseminated with frozen semen. Embryo recovery for fresh, cooled and frozen semen was 73.2% (41/50), 58.6% (84/142) and 36.5% (162/444), respectively. In addition, the odds of getting an embryo were also improved as the number of inseminations per cycle increased (OR¼1.43; P<0.03). Only a few mares were inseminated more than twice per cycle (44% embryo recovery, 4/9) but embryo recovery increased from 41.9 % (191/454) for those bred once in the cycle to 51.4% (90/175) for those inseminated twice in the
24 Embryo quality grade and recipient ovulation rate affect pregnancy rates in embryo transfer recipient mares H.G. Pedersen 1, *, M. Niklasson 1, A. Vullers 2, M. Christoffersen 1 1 Section for Veterinary Reproduction and Obstetrics, Department of Large Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Dyrlægevej 68, DK-1870 Frederiksberg, Denmark; 2 Animal Embryo Center, Boekhorstweg 2, 6105 AD Maria-Hoop, Holland *Corresponding author: [email protected] In order to maximize the success of equine embryo transfer, factors affecting recipient pregnancy rates and embryo loss are important. The components of an embryo transfer program include the semen/stallion factor, the donor, the recipient, the embryo and the transfer procedure, where each component is influenced by different factors such as age and reproductive history of embryo donors, synchrony between donor and recipient and embryo quality. The aims of the study were to investigate whether age of the donor mare, embryo quality and number of recipient mare ovulations influenced recipient pregnancy rate on day 18 in an embryo transfer program. Embryos (n ¼ 181) from 76 donor mares aged 3-25 years were recovered 8-9 days after ovulation at the Animal Embryo Center, Holland. The embryos were classified according to the International Embryo Transfer Society (IETS) (4 morula, 5 - early blastocyst, 6 e blastocyst and 7 - expanded blastocyst; quality grades: 1 - excellent/good, 2 e fair). Warmblood recipient mares aged 4-15 years were ultrasound scanned for day of ovulation. Embryos were transferred on day 2-7 post ovulation. Recipient mares were scanned for pregnancy on day 18th after donor ovulation. The overall pregnancy rate was 74% (134/181). Pregnancy rates with embryos from donor mares aged 3-15 years (77%; 87/113) were not statistically different to pregnancy rates from donor mares aged >15 years (69%; 47/68). Pregnancy rates in recipient mares that had embryos transferred on day 2 post ovulation was 100% (1/1), on day 3: 63% (5/8), on day 4: 73% (32/44), on day 5: 74% (42/57), on day 6: 77% (47/61) and on day 7: 70% (7/ 10) (P>0.05). Embryos graded (Stage/Quality) as 5.1, 6.1 or 7.1 resulted in higher pregnancy rates (79%; 110/139) than embryos graded as 4.2, 5.2, 6.2, 7.2 (57%; 24/42) (P ¼ 0.008). Recipient mares with double or multiple ovulations became pregnant at a higher rate (86%; 37/43) than recipients with a single ovulation (70%; 97/ 138) (P ¼ 0.047). Embryos from mares aged more than 15 years survived to day 18 at a similar rate than embryos from younger mares. Data on pregnancy rates later than day 18 was not available, and hence we do not know whether pregnancy loss rates were similar later in the pregnancy. Recipient mares ovulating more than one follicle were pregnant at a higher rate on day 18 compared to mares ovulating one follicle only. The reason may be increased concentrations of progesterone, but no progesterone measurements were evaluated in the present study. In conclusion, donor mare age did not influence recipient mare pregnancy rate
62
Abstracts / Journal of Equine Veterinary Science 41 (2016) 51e84
on day 18, embryos graded as excellent to good resulted in higher pregnancy rates than embryos graded as fair, and recipient mares with more than one ovulation became pregnant at a higher rate than mares with one ovulation. Key Words: Embryo transfer, mare, double ovulation
25 Association between equine sperm parameters and ICSI outcome Raul Gonzalez-Castro, Elaine Carnevale* Equine Reproduction Laboratory, Colorado State University, Fort Collins Colorado, 80521, USA *Corresponding author: [email protected] Intracytoplasmic sperm injection (ICSI) is used to produce offspring from stallions that have limited or poor quality semen. Sperm fertilizing potential, embryo development, and pregnancy outcome depend on sperm integrity and functionality. We hypothesized that sperm parameters would be associated with ICSI success. Aims of the study were to evaluate sperm parameters in samples used for ICSI in an equine clinical program and to elucidate which sperm parameters were related to ICSI outcome. Oocytes (n¼159) from light-horse mares aged 7 to 26 yr (n¼40, mean±SD, 17.8±5.5 yr) were collected and injected with frozen-thawed sperm (n¼119 samples) from 40 stallions. Semen samples were frozen at various facilities using different cryodiluents. Sperm samples were obtained and evaluated immediately after sperm selection for ICSI. Semen analyses included percentages of: 1) normal morphology (Hancock stain, Animal Reproduction System, Chino, CA), 2) live sperm (Hancock stain, ARS), 3) functional membranes (hypoosmotic swelling test, HOS), and 4) DNA fragmentation (sperm chromatin dispersion). Pearson's correlation was used to make comparisons among sperm parameters, and generalized linear model was used to compare sperm parameters with ICSI outcomes (rates of cleavage, blastocyst or pregnancy at 25 days). Sperm parameters for all samples were: normal morphology, 38.1±13.7%; live sperm, 41.3±15.2%; functional membranes, 29.4±11.1%, and DNA fragmentation, 9.2±7.1%. When sperm parameters were related with each other, the only significant associations were moderate positive correlations between percentages of live sperm and sperm with normal morphology (r¼0.40, P<0.0001) or functional membranes (r¼0.37, P<0.0001). When sperm parameters and ICSI outcome were analyzed, normal morphology and DNA fragmentation were not significantly associated with any ICSI outcome. Cleavage and blastocyst formation increased with percentage of live sperm (P¼0.02 and P¼0.03, respectively). A higher percentage of sperm with functional membranes was positively associated with cleavage, blastocyst and pregnancy rates (P¼0.01, P¼0.08 and P¼0.009, respectively). From the samples analyzed, the percentage of live sperm and sperm with functional membranes were most predictive of ICSI outcome.
26 Assessment of fetal sex determination in early equine pregnancy by isolation of fetal cells from the maternal circulation € hne 1, *, Michaela Prchal-Murphy 2, Martin Hofer 3, Ralf Martin Ko Steinborn 3, Christine Aurich 4 1 Obstetrics, Gynecology, Andrology, Vetmeduni, Vienna, Austria; 2 Institute of Pharmacology and Toxicology, Vetmeduni, Vienna, Austria; 3 Genomics Core Facility, VetCore, Vetmeduni, Vienna,
Austria; 4 Centre for Artificial Insemination and Embryo Transfer, Vetmeduni, Vienna, Austria *Corresponding author: [email protected] In humans, fetal cells cross the placenta [Tsuji T et. Al . Cytometry 1999; 37 (4): 291e301] and can be used for sex determination already in the first trimester] Fiddler M J Clin Med 2014; 3 (3): 972e985]. Despite the epitheliochorial nature of the equine placenta, fetal DNA is able to pass this barrier during the last three months of pregnancy [de Leon P et.al Theriogenology 2012; 77 (3): 694e698]. Because equine chorion girdle cells perform invasive growth into the maternal endometrium, we hypothesized that fetal cells are detectable in the maternal regulation after formation of endometrial cups. It was the aim of the study to detect and isolate fetal nucleated red blood cells (NRBC) from the maternal circulation in the first 100 days of pregnancy. We assumed that approximately 50% of mares would bear a male fetus which would allow proof the presence of fetal cells by determination of male-associated genes. Blood samples were taken from four pregnant ponies (gestation days 83, 90, 96, 102) and five pregnant Warmblood mares (gestation days 45, 56, 77, 88, 91). For fluorescence-activated cell sorting (FACS) blood samples were incubated with anti-mouse CD45 antibody distinguishing leukocytes from erythrocytes. Subsequently, cell nuclei were labelled using a hypertonic propidium iodide solution. The NRBC fraction was obtained by CD45-negative and propidium iodide-positive sorting. Only samples of pregnant mares contained 3,275 to 12,106 cells per 100 million sorted events, but these were absent in the blood of a non-pregnant mare and a stallion that served as negative controls. For sex assignation DNA was extracted and amplified by a qPCR assay targeting autosomal and Y chromosome-specific single copy genes, MC1R and SRY, respectively. Two DNA samples of male and female horses served as positive and negative controls. Under this setup, we were unable to detect the two target sequences. Considering the extended DNA fragmentation as evidenced by electrophoretic separation on an agarose gel and on the Agilent 4200 TapeStation, we tried to increase the number of intact target copies by performing whole genome amplification. This improved qPCR detection by at least one order of magnitude in case of MC1R (fold change 10 to 250), but all samples remained negative for SRY. Because it is not feasible that all fetuses in our study are female, results confirm that fetal cells are not able to pass the placental barrier in horses at least during the first 100 days of pregnancy. Key Words: horse, fetal sex determination, FACS, qPCR
27 Rescuing equine cloned embryos by aggregation s Gambini 1, 2, *, Jennifer M. Kelly 3, Angus O. Andre McKinnon 4, Richard Fry 5, 6, Daniel F. Salamone 1, 2, Paul J. Verma 3, 7 1 n Animal, Catedra de Fisiología Animal, Departamento Produccio Facultad de Agronomía, Universidad de Buenos Aires, Ciudad noma de Buenos Aires, Argentina; 2 Consejo Nacional de Auto noma Investigaciones Científicas y T ecnicas (CONICET), Ciudad Auto de Buenos Aires, Argentina; 3 South Australian Research and Development Institute (SARDI), Turretfield Research Centre, Rosedale, South Australia, Australia; 4 Goulburn Valley Equine Hospital, Victoria, Australia; 5 Clone International Pty Ltd, Victoria, Australia; 6 University of Melbourne, Victoria, Australia; 7 Stem Cells & Reprogramming Group, Monash University, Victoria, Australia *Corresponding author: [email protected] In vitro development of equine pre-implantation embryos and especially cloned embryos remains inefficient, often resulting in
culture, modified so that all media used were 285±3 mOsm. The blastocyst rates were 10/43 (23%) and 18/50 (36%) respectively (P > 0.1). In summary, systems using commercial media for oocyte manipulation, a more complete cell culture medium for late embryo development, and a consistent osmolarity supported blastocyst rates not significantly different from, but numerically higher than, those of our control system. We are now evaluating these systems to determine if better control of the in vitro embryo environment may improve embryo viability after transfer. Key Words: horse, ICSI, oocytes, embryo
61
cycle. The odds of a mare providing an embryo did not decrease as the number of flushes in a given year increased (OR¼ 0.89; P<0.22). Lastly, breed of donor affected embryo recovery, with embryo recovery from Quarter Horses (41%) being less than that for Thoroughbreds (78%) and Warmbloods (57%). Over 70% of the Quarter Horses, Arabians and Warmbloods were bred with frozen semen and only 9% of the Thoroughbreds. In summary, the highest odds of recovering an embryo are from maiden or foaling mares bred with fresh or cooled semen and inseminated more than once per cycle. Key Words: embryo recovery, semen, number of inseminations, breeds
Acknowledgments This work was supported by the Clinical Equine ICSI program, Texas A&M University and the Link Equine Research Endowment Fund, Texas A&M University.
23 Factors Affecting Embryo Recovery: Retrospective Study based on Commercial Embryo Transfer E.L. Squires 1, *, P.L. Loomis 2, D. Scofield 2, J. Baumber-Skaife 2, S. Barbacini 2, K.J. McQuerry 3, B.A. Ball 1 1 Gluck Equine Research Center, University of Kentucky, Lexington, KY; 2 Select Breeders Services, Inc., Chesapeake City, Maryland; 3 Department of Statistics, University of Kentucky, Lexington, KY *Corresponding author: [email protected] Embryo recovery and transfer has increased dramatically since first introduced in the late 1970's. Knowledge of the factors that affect the success of embryo recovery is important to discuss with clients prior to enrolling a mare in an embryo transfer program. The objectives of this study were to determine if embryo recovery was altered by: 1) age, 2) breed, 3) reproductive status, 4) type of semen, 5) number of inseminations per cycle and, 6) effect of repetitive flushes in a given year. This study utilized data collected from 10 embryo transfer facilities during 2010-14. Seven of these were in the USA and one each in Germany, Italy and Ireland. Donor mares (n¼203) were inseminated with fresh, cooled or frozen semen and embryos were recovered 7-8 days after ovulation (n¼640 flushes). The breed of mares varied with the clinics; Germany and Ireland having primarily Warmbloods and two of the clinics in the USA with mostly Quarter Horses (QH). Other breeds included Arabians, Standardbreds, Warmbloods and Paints. Age, breed, reproductive status (maiden, barren, foaling), number of times inseminated per cycle, and number of embryo recovery attempts each year were recorded. Data were analyzed using a logistic regression with repeated measures. (Proc GENMOD, SAS9.4; SAS Institute Cary, NC, USA). Donor's age had no effect on embryo recovery. The odds of embryo recovery (OR¼0.58) tended to be greater for foaling mares versus barren mares (P<0 .07) and maiden mares versus barren mares (P< 0.0013; OR¼2.73). Embryo recovery using fresh, cooled and frozen semen for barren mares was 36.3% (122/336), for foaling mares 48% (48/100) and maiden mares 56.9% (87/153) . Mares bred with either fresh (P<0.0001) or cooled semen (P<0.0003) had greater odds of embryo recovery than those inseminated with frozen semen. Embryo recovery for fresh, cooled and frozen semen was 73.2% (41/50), 58.6% (84/142) and 36.5% (162/444), respectively. In addition, the odds of getting an embryo were also improved as the number of inseminations per cycle increased (OR¼1.43; P<0.03). Only a few mares were inseminated more than twice per cycle (44% embryo recovery, 4/9) but embryo recovery increased from 41.9 % (191/454) for those bred once in the cycle to 51.4% (90/175) for those inseminated twice in the
24 Embryo quality grade and recipient ovulation rate affect pregnancy rates in embryo transfer recipient mares H.G. Pedersen 1, *, M. Niklasson 1, A. Vullers 2, M. Christoffersen 1 1 Section for Veterinary Reproduction and Obstetrics, Department of Large Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Dyrlægevej 68, DK-1870 Frederiksberg, Denmark; 2 Animal Embryo Center, Boekhorstweg 2, 6105 AD Maria-Hoop, Holland *Corresponding author: [email protected] In order to maximize the success of equine embryo transfer, factors affecting recipient pregnancy rates and embryo loss are important. The components of an embryo transfer program include the semen/stallion factor, the donor, the recipient, the embryo and the transfer procedure, where each component is influenced by different factors such as age and reproductive history of embryo donors, synchrony between donor and recipient and embryo quality. The aims of the study were to investigate whether age of the donor mare, embryo quality and number of recipient mare ovulations influenced recipient pregnancy rate on day 18 in an embryo transfer program. Embryos (n ¼ 181) from 76 donor mares aged 3-25 years were recovered 8-9 days after ovulation at the Animal Embryo Center, Holland. The embryos were classified according to the International Embryo Transfer Society (IETS) (4 morula, 5 - early blastocyst, 6 e blastocyst and 7 - expanded blastocyst; quality grades: 1 - excellent/good, 2 e fair). Warmblood recipient mares aged 4-15 years were ultrasound scanned for day of ovulation. Embryos were transferred on day 2-7 post ovulation. Recipient mares were scanned for pregnancy on day 18th after donor ovulation. The overall pregnancy rate was 74% (134/181). Pregnancy rates with embryos from donor mares aged 3-15 years (77%; 87/113) were not statistically different to pregnancy rates from donor mares aged >15 years (69%; 47/68). Pregnancy rates in recipient mares that had embryos transferred on day 2 post ovulation was 100% (1/1), on day 3: 63% (5/8), on day 4: 73% (32/44), on day 5: 74% (42/57), on day 6: 77% (47/61) and on day 7: 70% (7/ 10) (P>0.05). Embryos graded (Stage/Quality) as 5.1, 6.1 or 7.1 resulted in higher pregnancy rates (79%; 110/139) than embryos graded as 4.2, 5.2, 6.2, 7.2 (57%; 24/42) (P ¼ 0.008). Recipient mares with double or multiple ovulations became pregnant at a higher rate (86%; 37/43) than recipients with a single ovulation (70%; 97/ 138) (P ¼ 0.047). Embryos from mares aged more than 15 years survived to day 18 at a similar rate than embryos from younger mares. Data on pregnancy rates later than day 18 was not available, and hence we do not know whether pregnancy loss rates were similar later in the pregnancy. Recipient mares ovulating more than one follicle were pregnant at a higher rate on day 18 compared to mares ovulating one follicle only. The reason may be increased concentrations of progesterone, but no progesterone measurements were evaluated in the present study. In conclusion, donor mare age did not influence recipient mare pregnancy rate
62
Abstracts / Journal of Equine Veterinary Science 41 (2016) 51e84
on day 18, embryos graded as excellent to good resulted in higher pregnancy rates than embryos graded as fair, and recipient mares with more than one ovulation became pregnant at a higher rate than mares with one ovulation. Key Words: Embryo transfer, mare, double ovulation
25 Association between equine sperm parameters and ICSI outcome Raul Gonzalez-Castro, Elaine Carnevale* Equine Reproduction Laboratory, Colorado State University, Fort Collins Colorado, 80521, USA *Corresponding author: [email protected] Intracytoplasmic sperm injection (ICSI) is used to produce offspring from stallions that have limited or poor quality semen. Sperm fertilizing potential, embryo development, and pregnancy outcome depend on sperm integrity and functionality. We hypothesized that sperm parameters would be associated with ICSI success. Aims of the study were to evaluate sperm parameters in samples used for ICSI in an equine clinical program and to elucidate which sperm parameters were related to ICSI outcome. Oocytes (n¼159) from light-horse mares aged 7 to 26 yr (n¼40, mean±SD, 17.8±5.5 yr) were collected and injected with frozen-thawed sperm (n¼119 samples) from 40 stallions. Semen samples were frozen at various facilities using different cryodiluents. Sperm samples were obtained and evaluated immediately after sperm selection for ICSI. Semen analyses included percentages of: 1) normal morphology (Hancock stain, Animal Reproduction System, Chino, CA), 2) live sperm (Hancock stain, ARS), 3) functional membranes (hypoosmotic swelling test, HOS), and 4) DNA fragmentation (sperm chromatin dispersion). Pearson's correlation was used to make comparisons among sperm parameters, and generalized linear model was used to compare sperm parameters with ICSI outcomes (rates of cleavage, blastocyst or pregnancy at 25 days). Sperm parameters for all samples were: normal morphology, 38.1±13.7%; live sperm, 41.3±15.2%; functional membranes, 29.4±11.1%, and DNA fragmentation, 9.2±7.1%. When sperm parameters were related with each other, the only significant associations were moderate positive correlations between percentages of live sperm and sperm with normal morphology (r¼0.40, P<0.0001) or functional membranes (r¼0.37, P<0.0001). When sperm parameters and ICSI outcome were analyzed, normal morphology and DNA fragmentation were not significantly associated with any ICSI outcome. Cleavage and blastocyst formation increased with percentage of live sperm (P¼0.02 and P¼0.03, respectively). A higher percentage of sperm with functional membranes was positively associated with cleavage, blastocyst and pregnancy rates (P¼0.01, P¼0.08 and P¼0.009, respectively). From the samples analyzed, the percentage of live sperm and sperm with functional membranes were most predictive of ICSI outcome.
26 Assessment of fetal sex determination in early equine pregnancy by isolation of fetal cells from the maternal circulation € hne 1, *, Michaela Prchal-Murphy 2, Martin Hofer 3, Ralf Martin Ko Steinborn 3, Christine Aurich 4 1 Obstetrics, Gynecology, Andrology, Vetmeduni, Vienna, Austria; 2 Institute of Pharmacology and Toxicology, Vetmeduni, Vienna, Austria; 3 Genomics Core Facility, VetCore, Vetmeduni, Vienna,
Austria; 4 Centre for Artificial Insemination and Embryo Transfer, Vetmeduni, Vienna, Austria *Corresponding author: [email protected] In humans, fetal cells cross the placenta [Tsuji T et. Al . Cytometry 1999; 37 (4): 291e301] and can be used for sex determination already in the first trimester] Fiddler M J Clin Med 2014; 3 (3): 972e985]. Despite the epitheliochorial nature of the equine placenta, fetal DNA is able to pass this barrier during the last three months of pregnancy [de Leon P et.al Theriogenology 2012; 77 (3): 694e698]. Because equine chorion girdle cells perform invasive growth into the maternal endometrium, we hypothesized that fetal cells are detectable in the maternal regulation after formation of endometrial cups. It was the aim of the study to detect and isolate fetal nucleated red blood cells (NRBC) from the maternal circulation in the first 100 days of pregnancy. We assumed that approximately 50% of mares would bear a male fetus which would allow proof the presence of fetal cells by determination of male-associated genes. Blood samples were taken from four pregnant ponies (gestation days 83, 90, 96, 102) and five pregnant Warmblood mares (gestation days 45, 56, 77, 88, 91). For fluorescence-activated cell sorting (FACS) blood samples were incubated with anti-mouse CD45 antibody distinguishing leukocytes from erythrocytes. Subsequently, cell nuclei were labelled using a hypertonic propidium iodide solution. The NRBC fraction was obtained by CD45-negative and propidium iodide-positive sorting. Only samples of pregnant mares contained 3,275 to 12,106 cells per 100 million sorted events, but these were absent in the blood of a non-pregnant mare and a stallion that served as negative controls. For sex assignation DNA was extracted and amplified by a qPCR assay targeting autosomal and Y chromosome-specific single copy genes, MC1R and SRY, respectively. Two DNA samples of male and female horses served as positive and negative controls. Under this setup, we were unable to detect the two target sequences. Considering the extended DNA fragmentation as evidenced by electrophoretic separation on an agarose gel and on the Agilent 4200 TapeStation, we tried to increase the number of intact target copies by performing whole genome amplification. This improved qPCR detection by at least one order of magnitude in case of MC1R (fold change 10 to 250), but all samples remained negative for SRY. Because it is not feasible that all fetuses in our study are female, results confirm that fetal cells are not able to pass the placental barrier in horses at least during the first 100 days of pregnancy. Key Words: horse, fetal sex determination, FACS, qPCR
27 Rescuing equine cloned embryos by aggregation s Gambini 1, 2, *, Jennifer M. Kelly 3, Angus O. Andre McKinnon 4, Richard Fry 5, 6, Daniel F. Salamone 1, 2, Paul J. Verma 3, 7 1 n Animal, Catedra de Fisiología Animal, Departamento Produccio Facultad de Agronomía, Universidad de Buenos Aires, Ciudad noma de Buenos Aires, Argentina; 2 Consejo Nacional de Auto noma Investigaciones Científicas y T ecnicas (CONICET), Ciudad Auto de Buenos Aires, Argentina; 3 South Australian Research and Development Institute (SARDI), Turretfield Research Centre, Rosedale, South Australia, Australia; 4 Goulburn Valley Equine Hospital, Victoria, Australia; 5 Clone International Pty Ltd, Victoria, Australia; 6 University of Melbourne, Victoria, Australia; 7 Stem Cells & Reprogramming Group, Monash University, Victoria, Australia *Corresponding author: [email protected] In vitro development of equine pre-implantation embryos and especially cloned embryos remains inefficient, often resulting in