- Email: [email protected]
In vitro growth of small equine embryos after vitrification
74
Abstracts / Journal of Equine Veterinary Science 41 (2016) 51e84
51 Effect of firocoxib on ovulation and fertility rates of embryo donor mares A.M. Friso 1, *, M.A. Cyrino 1, M . T. Teoro 2, M.A. Alvarenga 1 1 Departament of Animal Reproduction and Veterinary Radiology, ~o Paulo, Brazil; 2 Haras Universidade Estadual Paulista (UNESP), Sa rio Lange, Sa ~o Paulo, Brazil LUB Breeding, Cesa *Corresponding author: [email protected], [email protected] It well known that non-steroidal anti-inflammatory drugs (NSAID) can disturb the ovulation process. The goal of this study was to evaluate the effect of a firocoxib a NSAID on ovulation and embryo recovery rates of mares. Twelve donors mares ranging from 5 to 15 years of age were used during two estrus cycles. The mares were used randomly in the treated and untreated cycle and inseminated whith the same stallion in both cycles. Their estrus cycles were monitored daily using transrectal ultrasonography until the large follicle reach 35 mm and uterine edema graded as 2 or 3. At this point, 1 mg of deslorelin acetate (Sincrorrelin®, Ourofino, SP, Brazil) was injected IM to induce ovulation. Mares were artificially inseminated 36 hours after ovulation induction with 500 million viable cooled sperm from fertile stallions. Semen was extended with BotuSemen® (Botupharma, Botucatu, SP, Brazil). Treated mares received 0.2 mg / kg / VO of firocoxib (Previcox®, Merial Animal Health LTD, SP, Brazil) at the time of ovulation induction and 24 and 48h after. In order to confirm the ovulation, the mares were examined 24 and 48 hours after ovulation induction. Embryo flushings were performed 8 days postovulation using ringer lactate in a closed system. Data were analyzed using CHI-SQUARE test. No differences were observed on ovulation time and ovulation rates between treated and untreated cycles, also no diferences were observed on embryo recovery rates from treated cycle 58% (7/12) and untreated cycle 50% (6/12). Based these results and following the protocol of the present study, we can concluded that the NSAID firocoxib can be safely used during the estrus cycles of mares in embryo transfer programmes without interfer on ovulation and embryo recovery rates. Key Words: Mare, embryo, ovulation, firocoxib
Acknowledgements Haras LUB Breeding Unesp-FMVZ
52 Successful in vitro production of mammalian embryos: a strict quality management approach C. Herrera 1, 2, E. Jeannerat 2, S. Wyck 1, L. Bittner 1, M. Van den Bergh 3, F. Janett 1, D. Burger 2, H. Bollwein 1 1 Clinic for Animal Reproduction Medicine, Vetsuisse - Faculty, University of Zürich, Switzerland; 2 Swiss Institute of Equine Medicine, Agroscope and University of Berne, Avenches, Switzerland; 3 Quartec GmbH Switzerland Continuous successful in vitro production of mammalian embryos depends largely on a stable cell-culture system, characterized by minimal variations in evident physical and chemical conditions (CO2, O2, pH, Temperature, Osmolality) and the less evident prevention of infiltration of embryo toxic pollutants (Volatile Organic Compounds VOCs, Residual Disinfectants, cleaning agents etc.). We report here about an efficient quality management (QM) of
those parameters in newly established equine and bovine in vitro embryo production laboratories. The QM consisted in a preliminary control and tuning of the basic parameters, temperature, % CO2 % O2% of all incubators and heating stages and an assessment of passive absorption and gas-chromatographic analysis of VOCs in the laboratory environment. Where needed, data loggers were installed to estimate the fluctuations in temperature and gas atmosphere in the incubators during intensive working periods. Infra-Red photography was applied to control homogenous heating of microscope heating stages. Based on the collected data, all parameters were adjusted to obtain the ideal culture conditions for every step during the embryo manipulations. The second series consisted in a weekly monitoring of the performances of the “In House Made” culture media by pH measurement after overnight equilibration and the % blastocyst formation in a bovine In Vitro Production System using oocytes collected from ovaries obtained from the slaughterhouse. When the pH was out of the range 7.42 -7.44 and/or the blastocyst formation was low, corrective measures were taken. The third surveillance consisted in a quarterly monitoring by an external company of the VOCs together, with a control of the culture media by means of a blood gas analyser. Once a stable system for in vitro production of bovine embryos was produced, we were also able to produce equine blastocyst after ICSI on oocytes obtained from the slaughterhouse or after direct oocyte collection from mares by OPU. The collected data of 72 pH measurements during the first year led to 17 adjustments of the % CO2 settings of the incubators to achieve the correct pH for our media with 2.1 mg/m NaHCO3. The temperature surveillance revealed a difference of -2 C between the indicated temperature on the display of the heating stages and the real temperature in the culture dishes, obliging to adjust the settings to 40.1 C. A constant difference of +0.8 C exists between the upper and lower shelves of the incubators, but without affecting embryo development. VOCs surveillance resulted in the replacement of ethanol as a surface disinfectant by a quaternary ammonium compound free from VOCs. The Total VOCs levels in the laboratory could be reduced below the alarm level of 0.5 ppm. A strict control of all culture conditions and the exclusion of any negative environmental factors is essential for the establishment of successful laboratories producing in vitro bovine and equine embryos.
53 In vitro growth of small equine embryos after vitrification Celina M. Checura*, Alexis Powers School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA, 53706 *Corresponding author: [email protected] Some laboratory procedures, like the derivation of embryonic stem cells (ESC), benefit from the availability of embryos in groups to optimize the use of time and supplies. Due to the low efficiency of superovulation in mares, cryopreservation of embryos becomes an indispensable tool for grouping embryos for these type of procedures. However, cryopreservation of equine embryos is challenging due to embryo size and permeability properties, resulting in low survival of the embryonic inner cell mass. This study was designed to compare two vitrification procedures for small equine embryos (<300 mm) for post-warming viability under in vitro conditions, measured as diameter growth. Fourteen embryos were recovered by uterine lavage from light-horse mares at days 6 and 6.5 after ovulation. Embryos were randomly assigned to one of two vitrification/warming protocols: A) a commercial vitrification kit (SYNGRO® Equine Vitrification Kit, Bioniche Animal Health) was
Abstracts / Journal of Equine Veterinary Science 41 (2016) 51e84
used for straw vitrification as recommended by the manufacturer: equilibration in vitrification solution 1 (VS1) for 5 min; VS2 for 5 min; and VS3 45-60 sec; cooling in liquid-nitrogen vapor for 1 min and then plunged into liquid nitrogen. For warming, straws were held in the air for 10 sec, placed into a 37 C water bath for 20 sec; contents of the straw were mixed by flicking the straw and then emptied into 400ml of 0.5 M Galactose in DPBS+20% FCS for 6 min, moved to DPBS+20% FCS (Holding) for 6 min; and placed in culture. B) Embryos were incubated in VS1 (1.5 M Ethylene Glycol -EG-) for 3 min; and in VS2 (7 M EG and 0.6 M galactose) for 45-60 seconds; cooled in liquid-nitrogen vapor for 1 minute, and then plunged into liquid nitrogen. For warming, straws were held in the air for 10 sec, placed into a 37 C water bath for 20 sec; flicked, and then emptied into 400ml of 0.5 M Galactose in Holding for 3 min; moved to 0.25; and 0.125 M Galactose in Holding for 3 min each, to Holding for 6 min; and placed in culture. Culture was in DMEM/ Ham's F12 (1:1) with 10% FCS at 38.5ºC, in 5% O2, 5% CO2 and 90% N2. Embryo diameter was calculated by averaging two perpendicular diameters on saved images taken before vitrification and at 24 and 48 hours post-warming. Data was log transformed for normality, analyzed as repeated measures, and compared by LSM. The main effect of treatment was significant (p<0.05), with larger diameters for treatment A, but there was no effect of time or treatment by time interaction. Means and SEM for the non-transformed data were 193.8±15.0; 239.3±45.1; and 266.6±68.3 mm, and 173.5±5.8; 158.2±8.1; 159.4±8.7 mm for 0; 24; and 48 hours for groups A and B respectively. In conclusion, treatment A was better than treatment B in maintaining embryo growth in vitro after vitrification/warming. Further studies are needed to determine the integrity of the inner cell mass of group-A embryos and to improve culture conditions.
75
was done in duplicate, using a male-specific and male-female, autosomal primer in separate tubes. DNA amplification was performed in 500 ml micro centrifuge tubes with 25 ml reaction mixture containing: 1.6 mM inner primers, 0.2 mM outer primers, 0.8 mM loop primers, 2.5 ml of 10X Bst Isothermal Amplification Buffer, 1.6 mM dNTPs and 4 ml of sample. Reaction tubes were placed in dry bath incubator at 95 C for 5 min then 2U Bst 2.0 of WarmStart DNA polymerase was added to each tube. DNA amplification proceeded in a dry bath incubator at 66 C for 40 min. Subsequently, 0.5 ml of 1000X SYBR green was added to the amplification tube and color change was recorded within 5 min (Picture 1). For each test, two tubes containing no DNA template had been used as a negative control. Serial dilution (1 ng/ml to 1 pg/ml) of genomic DNA extracted from stallions and mares confirmed that as little as 40 pg total volume of genomic DNA could be detected using LAMP. Blastocoel fluid (containing some loose cells) from Day 8, in vivo produced equine embryos were aspirated and discharged in a 4 mL microdroplet of DNAse-free water then diluted up to 10 ml. Harvested specimens were divided equally into two tubes, one for male-specific reaction and one for male-female common reaction and analyzed using LAMP. Results were compared to conventional PCR using extracted DNA from the lysed whole embryo. In 5 out of 6 embryos the same gender was detected with the LAMP and PCR techniques and in one embryo the blastocoel fluid sample no amplification was obtained with the LAMP method.
Key Words: equine embryo, vitrification
54 Preimplantation Gender Determination on Equine Embryos using LAMP P. Dini 1, *, M. Van Poucke 2, C. Herrera 3, 4, L. Peelman 2, Peter Daels 1 1 Department of Reproduction, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Belgium; 2 Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Belgium; 3 Clinic of Reproductive Medicine, Vetsuisse Faculty, Zürich, Switzerland; 4 Swiss Institute of Equine Medicine, Agroscope and University of Berne, Avenches, Switzerland *Corresponding author: [email protected] Prediction and selection of gender before transferring embryos to recipient mares is gaining interest in the equine embryo transfer industry. The availability of a simple, same-day test for gender determination may allow transferring fresh embryos with the desired gender and is expected to have a high economic impact on commercial programs. Gender determination based on PCR has been reported in horses and uses either embryonic cells or, more recently, cell-free genomic DNA present in blastocoel fluid (Choi et al., 2010; Herrera et al., 2015). However, these techniques remain time consuming and require sophisticated equipment and skilled personnel. LAMP is a DNA amplification method that can amplify a specific DNA sequence at constant temperature (60-65 C), in a short period of time (<1 hr), without the need for agarose gel electrophoresis (Notomi et al., 2000). In the present study, we have successfully designed a set of primers (Inner, Outer and Loop primers) for the ETY-1 gene on the male specific region of the equine Y-chromosome and autosomal specific primers as a housekeeping gene. Gender determination
Picture 1. LAMP assay on male and female genomic DNA. Top row: male, female and 2 negative controls (MQ-Water) with male specific primers. Bottom row male, female and 2 negative controls (MQ-Water) with autosomal specific primers. Green-yellow color indicates positive reaction.
55 Comparison of pregnancy rates between acyclic mules and cyclic mares as recipients for embryo transfer C.E. Camargo 1, *, R.C. Macan 2, S.F. Rechsteiner 1, 3, E.L. Gastal 4 1 Postgraduate Program in Animal Medicine: Equine, UFRGS, Porto Alegre, RS, Brazil; 2 Veterinary Medicine Faculty, PUCPR, Curitiba, PR, Brazil; 3 HISTOREP, Biology Institute, UFPel, Pelotas, RS, Brazil; 4 Department of Animal Science, Food and Nutrition, Southern Illinois University, Carbondale, IL, USA *Corresponding author: [email protected] Shortages in recipient mares are a real problem in countries where the equine embryo transfer technique has become very common. The objectives of this study were to compare pregnancy rates between acyclic mules and cyclic mares used as recipients for embryo transfer, and evaluate the number of embryo transfer attempts to achieve a pregnancy. This study was conducted at the Experimental Farm Gralha Azul of the Pontifical Catholic University of Paran a. Four embryo donor mares aged 4 to 10 years were used. To test the efficacy of the embryo transfer technique in mules and to compare it with mares, two groups of recipients
Abstracts / Journal of Equine Veterinary Science 41 (2016) 51e84
51 Effect of firocoxib on ovulation and fertility rates of embryo donor mares A.M. Friso 1, *, M.A. Cyrino 1, M . T. Teoro 2, M.A. Alvarenga 1 1 Departament of Animal Reproduction and Veterinary Radiology, ~o Paulo, Brazil; 2 Haras Universidade Estadual Paulista (UNESP), Sa rio Lange, Sa ~o Paulo, Brazil LUB Breeding, Cesa *Corresponding author: [email protected], [email protected] It well known that non-steroidal anti-inflammatory drugs (NSAID) can disturb the ovulation process. The goal of this study was to evaluate the effect of a firocoxib a NSAID on ovulation and embryo recovery rates of mares. Twelve donors mares ranging from 5 to 15 years of age were used during two estrus cycles. The mares were used randomly in the treated and untreated cycle and inseminated whith the same stallion in both cycles. Their estrus cycles were monitored daily using transrectal ultrasonography until the large follicle reach 35 mm and uterine edema graded as 2 or 3. At this point, 1 mg of deslorelin acetate (Sincrorrelin®, Ourofino, SP, Brazil) was injected IM to induce ovulation. Mares were artificially inseminated 36 hours after ovulation induction with 500 million viable cooled sperm from fertile stallions. Semen was extended with BotuSemen® (Botupharma, Botucatu, SP, Brazil). Treated mares received 0.2 mg / kg / VO of firocoxib (Previcox®, Merial Animal Health LTD, SP, Brazil) at the time of ovulation induction and 24 and 48h after. In order to confirm the ovulation, the mares were examined 24 and 48 hours after ovulation induction. Embryo flushings were performed 8 days postovulation using ringer lactate in a closed system. Data were analyzed using CHI-SQUARE test. No differences were observed on ovulation time and ovulation rates between treated and untreated cycles, also no diferences were observed on embryo recovery rates from treated cycle 58% (7/12) and untreated cycle 50% (6/12). Based these results and following the protocol of the present study, we can concluded that the NSAID firocoxib can be safely used during the estrus cycles of mares in embryo transfer programmes without interfer on ovulation and embryo recovery rates. Key Words: Mare, embryo, ovulation, firocoxib
Acknowledgements Haras LUB Breeding Unesp-FMVZ
52 Successful in vitro production of mammalian embryos: a strict quality management approach C. Herrera 1, 2, E. Jeannerat 2, S. Wyck 1, L. Bittner 1, M. Van den Bergh 3, F. Janett 1, D. Burger 2, H. Bollwein 1 1 Clinic for Animal Reproduction Medicine, Vetsuisse - Faculty, University of Zürich, Switzerland; 2 Swiss Institute of Equine Medicine, Agroscope and University of Berne, Avenches, Switzerland; 3 Quartec GmbH Switzerland Continuous successful in vitro production of mammalian embryos depends largely on a stable cell-culture system, characterized by minimal variations in evident physical and chemical conditions (CO2, O2, pH, Temperature, Osmolality) and the less evident prevention of infiltration of embryo toxic pollutants (Volatile Organic Compounds VOCs, Residual Disinfectants, cleaning agents etc.). We report here about an efficient quality management (QM) of
those parameters in newly established equine and bovine in vitro embryo production laboratories. The QM consisted in a preliminary control and tuning of the basic parameters, temperature, % CO2 % O2% of all incubators and heating stages and an assessment of passive absorption and gas-chromatographic analysis of VOCs in the laboratory environment. Where needed, data loggers were installed to estimate the fluctuations in temperature and gas atmosphere in the incubators during intensive working periods. Infra-Red photography was applied to control homogenous heating of microscope heating stages. Based on the collected data, all parameters were adjusted to obtain the ideal culture conditions for every step during the embryo manipulations. The second series consisted in a weekly monitoring of the performances of the “In House Made” culture media by pH measurement after overnight equilibration and the % blastocyst formation in a bovine In Vitro Production System using oocytes collected from ovaries obtained from the slaughterhouse. When the pH was out of the range 7.42 -7.44 and/or the blastocyst formation was low, corrective measures were taken. The third surveillance consisted in a quarterly monitoring by an external company of the VOCs together, with a control of the culture media by means of a blood gas analyser. Once a stable system for in vitro production of bovine embryos was produced, we were also able to produce equine blastocyst after ICSI on oocytes obtained from the slaughterhouse or after direct oocyte collection from mares by OPU. The collected data of 72 pH measurements during the first year led to 17 adjustments of the % CO2 settings of the incubators to achieve the correct pH for our media with 2.1 mg/m NaHCO3. The temperature surveillance revealed a difference of -2 C between the indicated temperature on the display of the heating stages and the real temperature in the culture dishes, obliging to adjust the settings to 40.1 C. A constant difference of +0.8 C exists between the upper and lower shelves of the incubators, but without affecting embryo development. VOCs surveillance resulted in the replacement of ethanol as a surface disinfectant by a quaternary ammonium compound free from VOCs. The Total VOCs levels in the laboratory could be reduced below the alarm level of 0.5 ppm. A strict control of all culture conditions and the exclusion of any negative environmental factors is essential for the establishment of successful laboratories producing in vitro bovine and equine embryos.
53 In vitro growth of small equine embryos after vitrification Celina M. Checura*, Alexis Powers School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA, 53706 *Corresponding author: [email protected] Some laboratory procedures, like the derivation of embryonic stem cells (ESC), benefit from the availability of embryos in groups to optimize the use of time and supplies. Due to the low efficiency of superovulation in mares, cryopreservation of embryos becomes an indispensable tool for grouping embryos for these type of procedures. However, cryopreservation of equine embryos is challenging due to embryo size and permeability properties, resulting in low survival of the embryonic inner cell mass. This study was designed to compare two vitrification procedures for small equine embryos (<300 mm) for post-warming viability under in vitro conditions, measured as diameter growth. Fourteen embryos were recovered by uterine lavage from light-horse mares at days 6 and 6.5 after ovulation. Embryos were randomly assigned to one of two vitrification/warming protocols: A) a commercial vitrification kit (SYNGRO® Equine Vitrification Kit, Bioniche Animal Health) was
Abstracts / Journal of Equine Veterinary Science 41 (2016) 51e84
used for straw vitrification as recommended by the manufacturer: equilibration in vitrification solution 1 (VS1) for 5 min; VS2 for 5 min; and VS3 45-60 sec; cooling in liquid-nitrogen vapor for 1 min and then plunged into liquid nitrogen. For warming, straws were held in the air for 10 sec, placed into a 37 C water bath for 20 sec; contents of the straw were mixed by flicking the straw and then emptied into 400ml of 0.5 M Galactose in DPBS+20% FCS for 6 min, moved to DPBS+20% FCS (Holding) for 6 min; and placed in culture. B) Embryos were incubated in VS1 (1.5 M Ethylene Glycol -EG-) for 3 min; and in VS2 (7 M EG and 0.6 M galactose) for 45-60 seconds; cooled in liquid-nitrogen vapor for 1 minute, and then plunged into liquid nitrogen. For warming, straws were held in the air for 10 sec, placed into a 37 C water bath for 20 sec; flicked, and then emptied into 400ml of 0.5 M Galactose in Holding for 3 min; moved to 0.25; and 0.125 M Galactose in Holding for 3 min each, to Holding for 6 min; and placed in culture. Culture was in DMEM/ Ham's F12 (1:1) with 10% FCS at 38.5ºC, in 5% O2, 5% CO2 and 90% N2. Embryo diameter was calculated by averaging two perpendicular diameters on saved images taken before vitrification and at 24 and 48 hours post-warming. Data was log transformed for normality, analyzed as repeated measures, and compared by LSM. The main effect of treatment was significant (p<0.05), with larger diameters for treatment A, but there was no effect of time or treatment by time interaction. Means and SEM for the non-transformed data were 193.8±15.0; 239.3±45.1; and 266.6±68.3 mm, and 173.5±5.8; 158.2±8.1; 159.4±8.7 mm for 0; 24; and 48 hours for groups A and B respectively. In conclusion, treatment A was better than treatment B in maintaining embryo growth in vitro after vitrification/warming. Further studies are needed to determine the integrity of the inner cell mass of group-A embryos and to improve culture conditions.
75
was done in duplicate, using a male-specific and male-female, autosomal primer in separate tubes. DNA amplification was performed in 500 ml micro centrifuge tubes with 25 ml reaction mixture containing: 1.6 mM inner primers, 0.2 mM outer primers, 0.8 mM loop primers, 2.5 ml of 10X Bst Isothermal Amplification Buffer, 1.6 mM dNTPs and 4 ml of sample. Reaction tubes were placed in dry bath incubator at 95 C for 5 min then 2U Bst 2.0 of WarmStart DNA polymerase was added to each tube. DNA amplification proceeded in a dry bath incubator at 66 C for 40 min. Subsequently, 0.5 ml of 1000X SYBR green was added to the amplification tube and color change was recorded within 5 min (Picture 1). For each test, two tubes containing no DNA template had been used as a negative control. Serial dilution (1 ng/ml to 1 pg/ml) of genomic DNA extracted from stallions and mares confirmed that as little as 40 pg total volume of genomic DNA could be detected using LAMP. Blastocoel fluid (containing some loose cells) from Day 8, in vivo produced equine embryos were aspirated and discharged in a 4 mL microdroplet of DNAse-free water then diluted up to 10 ml. Harvested specimens were divided equally into two tubes, one for male-specific reaction and one for male-female common reaction and analyzed using LAMP. Results were compared to conventional PCR using extracted DNA from the lysed whole embryo. In 5 out of 6 embryos the same gender was detected with the LAMP and PCR techniques and in one embryo the blastocoel fluid sample no amplification was obtained with the LAMP method.
Key Words: equine embryo, vitrification
54 Preimplantation Gender Determination on Equine Embryos using LAMP P. Dini 1, *, M. Van Poucke 2, C. Herrera 3, 4, L. Peelman 2, Peter Daels 1 1 Department of Reproduction, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Belgium; 2 Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Belgium; 3 Clinic of Reproductive Medicine, Vetsuisse Faculty, Zürich, Switzerland; 4 Swiss Institute of Equine Medicine, Agroscope and University of Berne, Avenches, Switzerland *Corresponding author: [email protected] Prediction and selection of gender before transferring embryos to recipient mares is gaining interest in the equine embryo transfer industry. The availability of a simple, same-day test for gender determination may allow transferring fresh embryos with the desired gender and is expected to have a high economic impact on commercial programs. Gender determination based on PCR has been reported in horses and uses either embryonic cells or, more recently, cell-free genomic DNA present in blastocoel fluid (Choi et al., 2010; Herrera et al., 2015). However, these techniques remain time consuming and require sophisticated equipment and skilled personnel. LAMP is a DNA amplification method that can amplify a specific DNA sequence at constant temperature (60-65 C), in a short period of time (<1 hr), without the need for agarose gel electrophoresis (Notomi et al., 2000). In the present study, we have successfully designed a set of primers (Inner, Outer and Loop primers) for the ETY-1 gene on the male specific region of the equine Y-chromosome and autosomal specific primers as a housekeeping gene. Gender determination
Picture 1. LAMP assay on male and female genomic DNA. Top row: male, female and 2 negative controls (MQ-Water) with male specific primers. Bottom row male, female and 2 negative controls (MQ-Water) with autosomal specific primers. Green-yellow color indicates positive reaction.
55 Comparison of pregnancy rates between acyclic mules and cyclic mares as recipients for embryo transfer C.E. Camargo 1, *, R.C. Macan 2, S.F. Rechsteiner 1, 3, E.L. Gastal 4 1 Postgraduate Program in Animal Medicine: Equine, UFRGS, Porto Alegre, RS, Brazil; 2 Veterinary Medicine Faculty, PUCPR, Curitiba, PR, Brazil; 3 HISTOREP, Biology Institute, UFPel, Pelotas, RS, Brazil; 4 Department of Animal Science, Food and Nutrition, Southern Illinois University, Carbondale, IL, USA *Corresponding author: [email protected] Shortages in recipient mares are a real problem in countries where the equine embryo transfer technique has become very common. The objectives of this study were to compare pregnancy rates between acyclic mules and cyclic mares used as recipients for embryo transfer, and evaluate the number of embryo transfer attempts to achieve a pregnancy. This study was conducted at the Experimental Farm Gralha Azul of the Pontifical Catholic University of Paran a. Four embryo donor mares aged 4 to 10 years were used. To test the efficacy of the embryo transfer technique in mules and to compare it with mares, two groups of recipients