- Email: [email protected]
stromal cell markers and steroid hormone receptors in the equine endometrium
Abstracts / Journal of Equine Veterinary Science 41 (2016) 51e84
53
media W1 (0.33 M sucrose), W2 (0.15 M sucrose) were used for both systems. In the closed system, the embryo was not in direct contact with liquid nitrogen. Twenty embryos were vitrified into 0.25-ml straws and sealed with a plug, using 2 steps: VS1 for 3 minutes and VS2 for 45 seconds. Vitrified embryos were stored into liquid nitrogen. Using the open system, the embryo was in direct contact with the liquid nitrogen. Fifty-seven embryos were placed in a 0.25 ml modified cut standard straw, using 5 steps: 3minute VS1 concentrating gradually (1:1, 1:2, 1:3), between 4 - 7 minutes in VS1 and 45 seconds in VS2. The time in VS1 was determined in a subjective manner by observing every minute, taking into account the degree of shrinkage dehydration. Three of the embryos vitrified with the open system and 15 using the closed system were warmed for embryo transfer. For both methods, the embryos were warmed and transferred to recipient mares in the following 30-60 minutes, always carried out by the same technician. No pregnancies were obtained after warming and transfer of embryos vitrified with the closed system (0/15). In this method the time of exposure to the average VS1 is probably too short to allow a complete dehydration, as observed by the degree of shrinkage. Three pregnancies were obtained with the open system (3/3). Pregnancy diagnoses were made at day 14, Parturitions are expected to occur between March and April 2016. In the open system exposure time in VS1 is greater, and during this time, for a greater effectiveness in the process, the embryo is continuously pipetted in the last phase equilibrium in VS1. This facilitates the internalization of the cryoprotectants, favoring dehydration and preventing the formation of ice crystals during cryopreservation and warming. No pregnancies were obtained using the closed system. With the open system, the advantage is that it allows us to hold the embryo in a micro-volume, increasing the cooling rate and minimizing the toxicity of the cryoprotectants in order to reduce its concentration.
D7 after ovulation. After collection, 18 embryos were cryopreserved immediately (group 0h) while 10 embryos were refrigerated in an Equitainer at 4 C during 24h prior cryopreservation (group 24h). Just before embryo vitrification, a glass micropipette was positioned above the embryos under a stereomicroscope, introduced into the blastocoel cavity and quickly retrieved. This step was performed manually without the help of a micromanipulator. Then embryos were deposited in medium with increasing sucrose concentration (0.1 - 0.2 - 0.4M) for 3 min each. Embryos collapsed and were immediately vitrified using the OPS procedure. Briefly, embryos were vitrified in 2 steps: 1.5M ethylene glycol (EG) for 5 min and 7M EG supplemented with 0.6M galactose for 30 sec. One embryo was then loaded per straw. Two base media were tested for collapsing and vitrification in group 0h: culture medium (CM: modified synthetic fluid + 20% foetal calf serum + 19mM glucose) (N¼10) and embryo holding medium (EHM: PBS + 4g/L BSA) (N¼8). In group 24h, only EHM was tested. At thawing, all embryos were deposited in EHM with decreasing sucrose concentration (0.2 - 0.1 - 0.0M) for 3 min each and placed in CM for 24h in a humidified atmosphere of 5% CO2, 5% O2 and 90% N2 at 38.5 C. Embryo survival rate was noted at the end of the in vitro culture. In both groups, one embryo lost its capsule at thawing and was discarded. After 24h of in vitro culture, 9/9 (100%) 7/7 (100%) and 8/9 (89%) embryos had survived for group 0h-CM, 0h-EHM and 24h-EHM, respectively. This technique is easy, quick to perform and inexpensive. Moreover the results are very exciting, particularly with the 24h refrigerated embryos.
Key Words: vitrification, collapse, blastocyst, open device.
7 Expression of mesenchymal stem/stromal cell markers and steroid hormone receptors in the equine endometrium
6 Easy, quick and cheap technique to cryopreserve Welsh B pony blastocyst
Acknowledgements A grant from IFCE was received to perform this experiment.
re 2, Thierry Florence Guignot 1, *, Thierry Blard 2, Philippe Barrie 2 2 2 Gasgogne , Yvan Gaude , Jean-Marie Yvon , Pascal Mermillod 1, Fabrice Reigner 2 1 UMR INRA-CNRS-Universit e de Tours-IFCE, Physiologie de la Reproduction et des Comportements, F-37380 Nouzilly, France; 2 UE1297, Unit e Exp erimentale de Physiologie Animale de l’Orfrasi ere, INRA, F-37380 Nouzilly, France *Corresponding author: fl[email protected]
E. Rink 1, 2, 3,*, J. Kuhl 3, C. Aurich 3, H. French 1, R. Nino-Fong 1, E. Watson 4, F.X. Donadeu 2 1 Ross University School of Veterinary Medicine, P.O. Box 334, Basseterre, St. Kitts, West Indies; 2 The Roslin Institute, The University of Edinburgh, Easter Bush, Midlothian, Scotland, UK; 3 €rmedizinische Universita €t Wien, Veterina €rplatz 1, Vienna, Veterina Austria; 4 The University of Edinburgh, Easter Bush, Midlothian, Scotland *Corresponding author: [email protected]
In equine embryos, cryopreservation of early embryos <300 mm in diameter leads to high pregnancy rates (70%-80%) but blastocysts >300 mm in diameter, results in lower pregnancy rates after transfer. It was hypothesized that the combination of the presence of the capsule and the large amount of fluid within the blastocoel could be responsible of pregnancy failures with frozen/ thawed embryos. It was demonstrated that blastocoel collapse could circumvent this difficulty and allow the cryopreservation of large equine embryos [Choi YH, et al. Theriogenology 2011;76:143e152]. We have validated this technique on pony embryos [Guignot F et al. Journal of American Science, 2015,93: e de la Recherche Equine, 5222e31; Guignot F, et al. 41eme Journe 12 mars 2015,78-83]. However, this technique requires skillfulness and expensive equipment. The aim of this study was to develop an easier and cheaper technique to reduce blastocoelic fluid volume before cryopreservation of Welsh pony embryos. Twenty eight expanded blastocysts, 166 - 777 mm in diameter (mean of the diameter ± SEM ¼ 370 ± 30 mm), were collected at
The equine endometrium is a dynamic tissue undergoing alterations during the estrous cycle. Mesenchymal stem/stromal cells (MSCs) are multipotent precursor cells that have been isolated from many tissues, including endometrium in other species. These cells are necessary for tissue homeostasis, which in the cycling endometrium is regulated in part by changes in concentration of steroid hormones. The expression of estrogen and progesterone receptors during the equine estrous cycle has been studied before, but information regarding MSC gene expression is lacking. This study was designed to examine mRNA expression of MSC markers (CD29, CD44, CD73, CD90, CD105), perivascular markers (CD146 and NG2) and steroid hormone receptors (ESRa, ESRb and PGR) in the equine endometrium. The aim was to describe estrous cycle-related differences in healthy mares. Results might contribute to the understanding of endometrial proliferation and differentiation. Endometrial biopsies were taken from healthy mares (n¼5) during the early and late luteal phase (day 5 (d5) and day 13 (d13) post ovulation, respectively)
54
Abstracts / Journal of Equine Veterinary Science 41 (2016) 51e84
and during estrus (one follicle > 3.5cm, pronounced uterine edema). RNA was extracted and cDNA reverse transcribed for analysis via RT-qPCR. Data was normalized to the geometric means of the housekeeping genes 18S and GAPDH. DCt values were used for statistical analysis using IBM SPSS Statistics 22. Data for qPCR are presented as gene expression relative to the mean of 18S and GAPDH. Expression of ESRa (11.1 ±1.1) and PGR (6.6 ±1.0) was significantly higher during estrus (p<0.01) compared to the early (5.0±0.2 and 2.2±0.1 respectively) and late luteal phase (3.8±0.6 and 1.4±0.2). In addition, ESRb mRNA expression tended to be higher during estrus than early diestrus (p¼0.07) but no difference was found for steroid receptor mRNA expression during diestrus, apart from a tendency for PGR to be higher at d5 than d13 (p¼0.08). Detectable levels of mRNA for all 5 MSC markers analyzed were present throughout the estrous cycle. While the levels of CD73 were consistent, the expression of three MSC markers (CD29, CD44, CD105) was elevated at d13. This difference was substantial between d13 and estrus for CD29 (37.6±6.2 and 12.2±3.4; p<0.01) and CD105 (8.3±0.9 and 4.5±0.6; p<0.05) and between d13 and d5 for CD29 (37.6±6.2 and 7.4±2.3) and CD44 (12.9±1.8 and 4.1±0.3; p<0.01). In contrast, CD90 expression was higher at estrus (27.8±3.8) than at d5 (6.7±0.9) or d13 (12.0±2.1; p<0.01). The expression of perivascular markers, CD146 and NG2, tended to be highest on d13 compared to d5 (p¼0.07). Elevated quantities of MSC and perivascular marker transcripts during late diestrus might be linked to the preparation of the equine endometrium for the proliferation phase associated with estrus. Interestingly, this increase in marker expression appears before the estrogen peak during estrus, but still under the influence of elevated levels of progesterone. In summary, this study shows that the equine endometrium expresses MSC markers and it does so at variable levels throughout the estrous cycle. This information will be useful for future studies aiming to derive endometrial MSCs from mares. Key Words: mesenchymal stem cell, equine, endometrium, steroid hormone receptor
8 Dynamics of in vitro maturation of equine oocytes recovered by transvaginal follicular aspiration n Rodríguez 1, 2, *, Andre s Gambini 1, 2, Amada Eugenia María Bele n Largel 4, Daniel Felipe Ynsaurralde 1, 3, Olinda Briski 1, Herna Salamone 1, 2 1 Laboratorio de Biotecnología Animal, Facultad de Agronomía, noma de Buenos Aires, Universidad de Buenos Aires, Ciudad Auto Argentina; 2 Consejo Nacional de Investigaciones Científicas y noma de Buenos Aires, Argentina; T ecnicas (CONICET), Ciudad Auto 3 n Instituto Nacional de Tecnologías Agropecuarias (INTA), Estacio Experimental Agropecuaria, Mercedes, Corrientes, Argentina; 4 Centro de Transferencia Embrionaria Equina El Palenque, 25 de Mayo, Buenos Aires, Argentina *Corresponding author: [email protected] Assisted reproduction techniques are acquiring more interest in the current equine breeding industry. However, those clinical procedures and researches on horse oocyte maturation were limited by the difficulty in obtaining a meaningful number of mature oocytes. Transvaginal follicular aspiration (TVA) in the mare is more complex, expensive and time-consuming than in cows, and the amount of recovered oocytes is typically low. These reasons justify the need of taking the major advantage of recovered oocytes. In vitro maturation (IVM) is one of the essential aspects for oocyte quality, but the best timing for IVM in the horse has not been established. In our study, we aim to describe the in vitro maturation dynamics of equine oocytes recovered by TVA at
different time schedules. Forty six mares were used as donors during spring transitional period (from July to September, southern hemisphere, Argentina). Recovered cumuluseoocyte complexes (COCs) were held in TALP-H medium at room temperature until the last mare of the day was aspirated (up to 8 h). Then, COCs were transferred to equilibrated IVM medium (Gambini et al, 2012) to allow the initiation of IVM, and transported to the lab (2.5 h of transportation) in a portable incubator at 38.5 C. Finally, COCs were placed in 50 ml drops of IVM medium under mineral oil in a 6.5% CO2 humidified atmosphere at 38.5 C. Mechanical cell cumulus removal of all COCs and the evaluation of first polar body extrusion started at 20 h of IVM. Oocytes with intact membrane and an extruded polar body were considered mature. Oocytes without a visible polar body were consider immature, and they were kept in IVM medium and were reevaluated every 2 hours up to 40 h of IVM. A total of 217 COCs were recovered from 46 aspirated mares, with a recovery rate of 4.9 COCs per mare. Oocyte classification from a total of 217 COCs at 37 h of IVM was (mean % ± SEM): matured, 67.1 ± 4.6; immature, 11.5 ± 3.02; degenerated, 19.3 ± 4.3. None oocyte was found mature after 35 h of IVM. From a total of 99 COCs, accumulative percentage of matured oocytes at different time schedules were (mean % ± SEM): 20-22 h, 42.3 ± 4.3; 23-25h, 62.3 ± 7.0; 26-28 h, 80 ± 5.7; 29-31 h, 93.2 ± 5.1; 32-34 h, 96.52 ± 3.5; 35-37 h, 100. No statistical differences in maturation rates were found among groups after the interval of 23-25 h of maturation. These results indicate that at 20 h of IVM 42.3% of oocytes are already matured, but 64.5% of immature oocytes at this time schedule will mature in the following 16 hours of IVM. Oocytes that are found immature after 37 h of IVM are more likely to degenerate. For clinical purposes, immature equine oocytes can be held in IVM medium up to 37 hours to allow the extrusion of the first polar body. Current studies are addressing oocyte developmental competence at different time schedules by ICSI embryo production.
Acknowledgments The authors want to thank Alejandra Jacobsen, Mariano Arnaude and the team of El Palenque embriones. Key Words: Oocyte; Transvaginal aspiration; Mare; Maturation; Equine; Ovum Pick-Up.
9 Variability in efficiency in a commercial oocyte transfer program n 1, Katrin Hinrichs 2, 3 Fernando L. Riera 1, Jaime E. Rolda 1 ~a Laboratorio de Reproduccion Equina Prof. Robert M. Kenney, Don Pilar Embriones, Lincoln (B), Argentina; 2 Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA Oocyte transfer (OT), that is, the surgical transfer of a mature oocyte into oviduct of a synchronized, inseminated recipient mare, represents an effective method for foal production. If efficient ICSI laboratories are not available, OT becomes the next option to produce offspring from mares with infertility due to abnormalities of the tubular reproductive tract. We have completed five consecutive breeding seasons using OT in a commercial embryo transfer center in Argentina. Notably, the first three breeding seasons produced acceptable results compared to those reported in the literature, but the last two seasons yielded poor results which could not be explained. These seasons are analysed
53
media W1 (0.33 M sucrose), W2 (0.15 M sucrose) were used for both systems. In the closed system, the embryo was not in direct contact with liquid nitrogen. Twenty embryos were vitrified into 0.25-ml straws and sealed with a plug, using 2 steps: VS1 for 3 minutes and VS2 for 45 seconds. Vitrified embryos were stored into liquid nitrogen. Using the open system, the embryo was in direct contact with the liquid nitrogen. Fifty-seven embryos were placed in a 0.25 ml modified cut standard straw, using 5 steps: 3minute VS1 concentrating gradually (1:1, 1:2, 1:3), between 4 - 7 minutes in VS1 and 45 seconds in VS2. The time in VS1 was determined in a subjective manner by observing every minute, taking into account the degree of shrinkage dehydration. Three of the embryos vitrified with the open system and 15 using the closed system were warmed for embryo transfer. For both methods, the embryos were warmed and transferred to recipient mares in the following 30-60 minutes, always carried out by the same technician. No pregnancies were obtained after warming and transfer of embryos vitrified with the closed system (0/15). In this method the time of exposure to the average VS1 is probably too short to allow a complete dehydration, as observed by the degree of shrinkage. Three pregnancies were obtained with the open system (3/3). Pregnancy diagnoses were made at day 14, Parturitions are expected to occur between March and April 2016. In the open system exposure time in VS1 is greater, and during this time, for a greater effectiveness in the process, the embryo is continuously pipetted in the last phase equilibrium in VS1. This facilitates the internalization of the cryoprotectants, favoring dehydration and preventing the formation of ice crystals during cryopreservation and warming. No pregnancies were obtained using the closed system. With the open system, the advantage is that it allows us to hold the embryo in a micro-volume, increasing the cooling rate and minimizing the toxicity of the cryoprotectants in order to reduce its concentration.
D7 after ovulation. After collection, 18 embryos were cryopreserved immediately (group 0h) while 10 embryos were refrigerated in an Equitainer at 4 C during 24h prior cryopreservation (group 24h). Just before embryo vitrification, a glass micropipette was positioned above the embryos under a stereomicroscope, introduced into the blastocoel cavity and quickly retrieved. This step was performed manually without the help of a micromanipulator. Then embryos were deposited in medium with increasing sucrose concentration (0.1 - 0.2 - 0.4M) for 3 min each. Embryos collapsed and were immediately vitrified using the OPS procedure. Briefly, embryos were vitrified in 2 steps: 1.5M ethylene glycol (EG) for 5 min and 7M EG supplemented with 0.6M galactose for 30 sec. One embryo was then loaded per straw. Two base media were tested for collapsing and vitrification in group 0h: culture medium (CM: modified synthetic fluid + 20% foetal calf serum + 19mM glucose) (N¼10) and embryo holding medium (EHM: PBS + 4g/L BSA) (N¼8). In group 24h, only EHM was tested. At thawing, all embryos were deposited in EHM with decreasing sucrose concentration (0.2 - 0.1 - 0.0M) for 3 min each and placed in CM for 24h in a humidified atmosphere of 5% CO2, 5% O2 and 90% N2 at 38.5 C. Embryo survival rate was noted at the end of the in vitro culture. In both groups, one embryo lost its capsule at thawing and was discarded. After 24h of in vitro culture, 9/9 (100%) 7/7 (100%) and 8/9 (89%) embryos had survived for group 0h-CM, 0h-EHM and 24h-EHM, respectively. This technique is easy, quick to perform and inexpensive. Moreover the results are very exciting, particularly with the 24h refrigerated embryos.
Key Words: vitrification, collapse, blastocyst, open device.
7 Expression of mesenchymal stem/stromal cell markers and steroid hormone receptors in the equine endometrium
6 Easy, quick and cheap technique to cryopreserve Welsh B pony blastocyst
Acknowledgements A grant from IFCE was received to perform this experiment.
re 2, Thierry Florence Guignot 1, *, Thierry Blard 2, Philippe Barrie 2 2 2 Gasgogne , Yvan Gaude , Jean-Marie Yvon , Pascal Mermillod 1, Fabrice Reigner 2 1 UMR INRA-CNRS-Universit e de Tours-IFCE, Physiologie de la Reproduction et des Comportements, F-37380 Nouzilly, France; 2 UE1297, Unit e Exp erimentale de Physiologie Animale de l’Orfrasi ere, INRA, F-37380 Nouzilly, France *Corresponding author: fl[email protected]
E. Rink 1, 2, 3,*, J. Kuhl 3, C. Aurich 3, H. French 1, R. Nino-Fong 1, E. Watson 4, F.X. Donadeu 2 1 Ross University School of Veterinary Medicine, P.O. Box 334, Basseterre, St. Kitts, West Indies; 2 The Roslin Institute, The University of Edinburgh, Easter Bush, Midlothian, Scotland, UK; 3 €rmedizinische Universita €t Wien, Veterina €rplatz 1, Vienna, Veterina Austria; 4 The University of Edinburgh, Easter Bush, Midlothian, Scotland *Corresponding author: [email protected]
In equine embryos, cryopreservation of early embryos <300 mm in diameter leads to high pregnancy rates (70%-80%) but blastocysts >300 mm in diameter, results in lower pregnancy rates after transfer. It was hypothesized that the combination of the presence of the capsule and the large amount of fluid within the blastocoel could be responsible of pregnancy failures with frozen/ thawed embryos. It was demonstrated that blastocoel collapse could circumvent this difficulty and allow the cryopreservation of large equine embryos [Choi YH, et al. Theriogenology 2011;76:143e152]. We have validated this technique on pony embryos [Guignot F et al. Journal of American Science, 2015,93: e de la Recherche Equine, 5222e31; Guignot F, et al. 41eme Journe 12 mars 2015,78-83]. However, this technique requires skillfulness and expensive equipment. The aim of this study was to develop an easier and cheaper technique to reduce blastocoelic fluid volume before cryopreservation of Welsh pony embryos. Twenty eight expanded blastocysts, 166 - 777 mm in diameter (mean of the diameter ± SEM ¼ 370 ± 30 mm), were collected at
The equine endometrium is a dynamic tissue undergoing alterations during the estrous cycle. Mesenchymal stem/stromal cells (MSCs) are multipotent precursor cells that have been isolated from many tissues, including endometrium in other species. These cells are necessary for tissue homeostasis, which in the cycling endometrium is regulated in part by changes in concentration of steroid hormones. The expression of estrogen and progesterone receptors during the equine estrous cycle has been studied before, but information regarding MSC gene expression is lacking. This study was designed to examine mRNA expression of MSC markers (CD29, CD44, CD73, CD90, CD105), perivascular markers (CD146 and NG2) and steroid hormone receptors (ESRa, ESRb and PGR) in the equine endometrium. The aim was to describe estrous cycle-related differences in healthy mares. Results might contribute to the understanding of endometrial proliferation and differentiation. Endometrial biopsies were taken from healthy mares (n¼5) during the early and late luteal phase (day 5 (d5) and day 13 (d13) post ovulation, respectively)
54
Abstracts / Journal of Equine Veterinary Science 41 (2016) 51e84
and during estrus (one follicle > 3.5cm, pronounced uterine edema). RNA was extracted and cDNA reverse transcribed for analysis via RT-qPCR. Data was normalized to the geometric means of the housekeeping genes 18S and GAPDH. DCt values were used for statistical analysis using IBM SPSS Statistics 22. Data for qPCR are presented as gene expression relative to the mean of 18S and GAPDH. Expression of ESRa (11.1 ±1.1) and PGR (6.6 ±1.0) was significantly higher during estrus (p<0.01) compared to the early (5.0±0.2 and 2.2±0.1 respectively) and late luteal phase (3.8±0.6 and 1.4±0.2). In addition, ESRb mRNA expression tended to be higher during estrus than early diestrus (p¼0.07) but no difference was found for steroid receptor mRNA expression during diestrus, apart from a tendency for PGR to be higher at d5 than d13 (p¼0.08). Detectable levels of mRNA for all 5 MSC markers analyzed were present throughout the estrous cycle. While the levels of CD73 were consistent, the expression of three MSC markers (CD29, CD44, CD105) was elevated at d13. This difference was substantial between d13 and estrus for CD29 (37.6±6.2 and 12.2±3.4; p<0.01) and CD105 (8.3±0.9 and 4.5±0.6; p<0.05) and between d13 and d5 for CD29 (37.6±6.2 and 7.4±2.3) and CD44 (12.9±1.8 and 4.1±0.3; p<0.01). In contrast, CD90 expression was higher at estrus (27.8±3.8) than at d5 (6.7±0.9) or d13 (12.0±2.1; p<0.01). The expression of perivascular markers, CD146 and NG2, tended to be highest on d13 compared to d5 (p¼0.07). Elevated quantities of MSC and perivascular marker transcripts during late diestrus might be linked to the preparation of the equine endometrium for the proliferation phase associated with estrus. Interestingly, this increase in marker expression appears before the estrogen peak during estrus, but still under the influence of elevated levels of progesterone. In summary, this study shows that the equine endometrium expresses MSC markers and it does so at variable levels throughout the estrous cycle. This information will be useful for future studies aiming to derive endometrial MSCs from mares. Key Words: mesenchymal stem cell, equine, endometrium, steroid hormone receptor
8 Dynamics of in vitro maturation of equine oocytes recovered by transvaginal follicular aspiration n Rodríguez 1, 2, *, Andre s Gambini 1, 2, Amada Eugenia María Bele n Largel 4, Daniel Felipe Ynsaurralde 1, 3, Olinda Briski 1, Herna Salamone 1, 2 1 Laboratorio de Biotecnología Animal, Facultad de Agronomía, noma de Buenos Aires, Universidad de Buenos Aires, Ciudad Auto Argentina; 2 Consejo Nacional de Investigaciones Científicas y noma de Buenos Aires, Argentina; T ecnicas (CONICET), Ciudad Auto 3 n Instituto Nacional de Tecnologías Agropecuarias (INTA), Estacio Experimental Agropecuaria, Mercedes, Corrientes, Argentina; 4 Centro de Transferencia Embrionaria Equina El Palenque, 25 de Mayo, Buenos Aires, Argentina *Corresponding author: [email protected] Assisted reproduction techniques are acquiring more interest in the current equine breeding industry. However, those clinical procedures and researches on horse oocyte maturation were limited by the difficulty in obtaining a meaningful number of mature oocytes. Transvaginal follicular aspiration (TVA) in the mare is more complex, expensive and time-consuming than in cows, and the amount of recovered oocytes is typically low. These reasons justify the need of taking the major advantage of recovered oocytes. In vitro maturation (IVM) is one of the essential aspects for oocyte quality, but the best timing for IVM in the horse has not been established. In our study, we aim to describe the in vitro maturation dynamics of equine oocytes recovered by TVA at
different time schedules. Forty six mares were used as donors during spring transitional period (from July to September, southern hemisphere, Argentina). Recovered cumuluseoocyte complexes (COCs) were held in TALP-H medium at room temperature until the last mare of the day was aspirated (up to 8 h). Then, COCs were transferred to equilibrated IVM medium (Gambini et al, 2012) to allow the initiation of IVM, and transported to the lab (2.5 h of transportation) in a portable incubator at 38.5 C. Finally, COCs were placed in 50 ml drops of IVM medium under mineral oil in a 6.5% CO2 humidified atmosphere at 38.5 C. Mechanical cell cumulus removal of all COCs and the evaluation of first polar body extrusion started at 20 h of IVM. Oocytes with intact membrane and an extruded polar body were considered mature. Oocytes without a visible polar body were consider immature, and they were kept in IVM medium and were reevaluated every 2 hours up to 40 h of IVM. A total of 217 COCs were recovered from 46 aspirated mares, with a recovery rate of 4.9 COCs per mare. Oocyte classification from a total of 217 COCs at 37 h of IVM was (mean % ± SEM): matured, 67.1 ± 4.6; immature, 11.5 ± 3.02; degenerated, 19.3 ± 4.3. None oocyte was found mature after 35 h of IVM. From a total of 99 COCs, accumulative percentage of matured oocytes at different time schedules were (mean % ± SEM): 20-22 h, 42.3 ± 4.3; 23-25h, 62.3 ± 7.0; 26-28 h, 80 ± 5.7; 29-31 h, 93.2 ± 5.1; 32-34 h, 96.52 ± 3.5; 35-37 h, 100. No statistical differences in maturation rates were found among groups after the interval of 23-25 h of maturation. These results indicate that at 20 h of IVM 42.3% of oocytes are already matured, but 64.5% of immature oocytes at this time schedule will mature in the following 16 hours of IVM. Oocytes that are found immature after 37 h of IVM are more likely to degenerate. For clinical purposes, immature equine oocytes can be held in IVM medium up to 37 hours to allow the extrusion of the first polar body. Current studies are addressing oocyte developmental competence at different time schedules by ICSI embryo production.
Acknowledgments The authors want to thank Alejandra Jacobsen, Mariano Arnaude and the team of El Palenque embriones. Key Words: Oocyte; Transvaginal aspiration; Mare; Maturation; Equine; Ovum Pick-Up.
9 Variability in efficiency in a commercial oocyte transfer program n 1, Katrin Hinrichs 2, 3 Fernando L. Riera 1, Jaime E. Rolda 1 ~a Laboratorio de Reproduccion Equina Prof. Robert M. Kenney, Don Pilar Embriones, Lincoln (B), Argentina; 2 Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA Oocyte transfer (OT), that is, the surgical transfer of a mature oocyte into oviduct of a synchronized, inseminated recipient mare, represents an effective method for foal production. If efficient ICSI laboratories are not available, OT becomes the next option to produce offspring from mares with infertility due to abnormalities of the tubular reproductive tract. We have completed five consecutive breeding seasons using OT in a commercial embryo transfer center in Argentina. Notably, the first three breeding seasons produced acceptable results compared to those reported in the literature, but the last two seasons yielded poor results which could not be explained. These seasons are analysed