Collection and evaluation of mare s oocytes for in vitro fertilization

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1 PhD THESIS Collection and evaluation of mare s oocytes for in vitro fertilization (SUMMARY OF THE Ph.D THESIS) PhD student Aryan Hussam Scientific coordinator Prof. Dr. Ioan Ştefan Groza

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3 The research was conducted on several priorities: evaluation and identification of the best recovery techniques of mare oocytes, in season and out of season after slaughter; improvement of morphological and selection methods for mare oocytes for in vitro maturation; assessment degree of maturation after in vitro cultivation by morphological and morphocitometric aspects; semen preparation used in the in vitro fertilization protocol of mare oocytes; evaluation of in vitro fertilization rate of mare oocytes. FACTORS AFFECTING THE RECOVERY RATE OF MARE OOCYTES The purpose of this investigation was to verify the average number of preovulatory follicles, their average diameter in a large group of mares and investigate if their size is affected by the age of the animals. The research was conducted during on a number of 240 ovaries collected from 120 slaughtered mares. Ovaries were collected two times/month, 10 ovaries every time, at an interval of two weeks. The animals were from households being brought to the abattoir for slaughter. Follicles were masured using a roller and were classified in 4 categories depending on their size: category 1: follicule < 1 cm; category 2: follicule between 1-2 cm; category 3: follicule between 2-3 cm; category 4: follicule > 3 cm. Influence of season on ovarian weight, follicular diameter, recovery rate and the average number of follicles existing per ovary On right ovary were recorded the following average values for each category of follicles: category 1 (follicles <1 cm): the smallest diameter was recorded (0.32 cm) in December, and the largest diameter was recorded (0.83 cm) in February; category 2 (follicles between 1-2 cm): the smallest diameter was recorded (1.2 cm) in November and the largest diameter was recorded (1.87 cm) in April; category 3 (follicles 2-3 cm): the smallest diameter was recorded (2.1 cm) in November and the largest diameter was recorded (2.87 cm) in May; category 4 (follicles> 3 cm): the smallest diameter was recorded (3.0 cm) in November and the largest diameter was recorded (4.92 cm) in April. The mean values obtained at the level of left ovary follicles in terms of size for each category are: category 1 (follicles <1 cm): the smallest diameter was recorded (0.27 cm) in December and the largest diameter was recorded (0.85 cm) in April; category 2 (follicles between 1-2 cm): the smallest diameter was recorded (1.1 cm) in November and the largest diameter was recorded (1.84 cm) in May; category 3 (follicles 2-3 cm): the smallest diameter was recorded (2.2 cm) in December and the largest diameter was recorded (2.63 cm) in June; category 4 (follicles> 3 cm): the smallest diameter was recorded (3.1 cm) in December and the largest diameter was recorded (4.63 cm) in July. Concerning the average number of follicles presented on the ovary data revealed by the experiment are: right ovary: the lowest number (2.4 follicles/ovary) was recorded in December and the highest number (5.6 follicles/ovary) was recorded in April; left ovary: the lowest number (2.7 follicles/ovary) was recorded in November and the highest number (4.7 follicles/ovary) was recorded in May. In terms of the number of oocytes collected/month the average values were: right ovary: the lowest number of oocytes collected (0.2 oocytes/month) was registered in December and the highest number (1.5 oocytes/month) in May; left ovary: lowest number of ovoicte harvested (0.1 oocytes/month) was registered in November and the highest number (1.1 oocytes/month) in March. III

4 Regarding the average number of follicles per ovary existing experimental data revealed are the following: right ovary: the smallest number (2.4 follicles/ovary) was recorded in December and the highest number (5.6 follicles/ovary) was recorded in April; left ovary: the smallest number (2.7 follicles/ovary) was recorded in November and the highest number (4.7 follicles/ovary) was recorded in May. In terms of the number of oocytes recovered/month averages were: right ovary: the lowest number of recovered oocytes (0.2 oocytes/month) was recorded in December and the highest number (1.5 oocytes / month) in May; left ovary: the lowest number of recovered oocytes (0.1 oocytes / month) was recorded in November and the highest number (1.1 oocytes / month) in March. Regarding the average weight of ovaries were observed the following values: for the ovary the lowest weight was recorded (62.13 g) in January and the highest weight was recorded (105.5 g) in April. For the left ovary values are: lowest weight was recorded (57.3 g) in November and the highest weight was recorded ( g) in April. The effect of age on mare preovulatory follicle diameter The average values during the research reported in the diameter of the hair follicle, depending on the age of the animals, are the following: category 1 (follicles <1 cm): the largest average diameter (0.8 cm) was recorded in mares in Group 3 (8-10 years) and the lowest average diameter (0.2 cm) of was recorded in mares in group 7 (19 years); category 2 (follicles between 1-2 cm): the largest diameter (1.7 cm) was reported in mares belonging to Group 2 (5-7 years) and the smallest diameter (1.2 cm) was referred to the mares in group 7 (19 years); category 3 (follicles between 2-3 cm): the largest diameter (2.8 cm) was observed in mares in the first group (4 years) and the smallest diameter (2 cm) was recorded in mares from group 4 (11-13 years); category 4 (follicles > 3 cm): the largest diameter (4.78 cm) was obtained from mares in group 1 (4 years) and the smallest diameter (3.12 cm) was recorded in mares from group 6 (17-19 years). COLLECTION OF MARE OOCYTES FROM SLAUGHTERED ANIMALS The purpose of this chapter was to assess and identify the best techniques of mare oocytes recovery for in vitro fertilization and performing a morphological assessment of oocytes and their classification according to structural aspects. Research has been conducted during on 752 ovaries collected from 376 mares aged 4 to 19 years and 150 ovaries from 75 mares 2 years of aged and less than 2 years, sexually immature. Collection of mare oocytes by aspiration follicles method - Were used needles with a diameter of 18G attached to 20 ml syringe. Sterile tube sedimentation are required to maintain follicular fluid at a constant temperature of 22 0 C. Collection of mare oocytes by scraping follicles - Using sterile scalpel blades, cutting follicles was done with a diameter greater than 1 cm and superficial scraping inside the follicle. Results of mare oocytes collection by follicular aspiration method From the 752 ovaries studied, 424 ovaries were collected in season and 328 ovaries were collected in out of season. Thus were formed two experimental groups: group I: consists of 424 mare's ovaries collected in reproduction season; were collected 720 oocytes from 1688 follicles, the recovery rate was 42.61%, with an average of 1.69 oocyte/ovary; group II: consisting of 328 mare's ovaries collected in out of season; 224 oocytes were collected from 1136 follicles, the recovery rate was 19.7% with an average of 0.70 oocyte/ovary. IV

5 After morphological examination oocytes were classified as follows: group I: from 720 collected oocytes, 576 oocytes (80%) were cultivable; group II: from 224 collected oocytes 112 oocytes (50%) were cultivable. Results of mare oocytes collection by follicles scraping method From the total of 752 ovaries studied, 424 ovaries were collected in season and 328 ovaries were collected in out of season. Thus were formed two experimental groups: group I: consists of 424 mare's ovaries collected in reproduction season; 432 oocytes were collected from 1688 follicles, the recovery rate was 25.5%, with an average of 1.01 oocyte / ovary; group II: consists of 328 mare's ovaries collected in out of season; 176 oocytes were collected from 1136 mare follicles, the recovery rate was 15.4% with an average of 0.53 oocytes / ovary. The classification of oocytes after performing morphological exam is the following: group I: from 432 collected oocytes 256 oocytes (59.25%) were cultivable; group II: from 176 collected oocytes 120 oocytes (68.18%) were cultivable. COLLECTION OF MARE OOCYTES FROM OVIDUCTAL LEVEL This aim of study was to collect mare oocytes from oviduct level of slaughtered mares and to mrealised their assessment and classification. Morphological study was correlated with the degree of maturation for in vitro fertilization selection. Research was conducted during on a total of 60 mare oviducts collected from slaughtered animals. From 30 studied genital apparatus, 30 oviducts were collected in season (February, March, April) and 30 oviducts were collected in out of season (September, October, November) and two experimental groups were formed : group I: 30 oviducts collected in reproduction season - 22 oocytes were collected, recovery rate was 0.73 oocyte/oviduct; group II: 30 oviducts collected in out of season - oocytes were not obtained, recovery rate was 0 oocyte/oviduct. Morphological study allowed to establish the developmental stage of collected oocyte and their classification into the following categories: cultivable oocytes - 12 oocytes (54.55%); non-cultivable or degenerated oocytes: 8 oocytes (36.36%) ; mature oocytes: two oocytes (9%). IN VITRO CULTIVATION OF MARE OOCYTES The aims of this study was to evaluate media and solutions used for in vitro maturation of equine oocytes for medium term and correlating morphological study with maturation degree of those suitable for in vitro fertilization. The study was conducted during on a total of 930 mare oocytes collected from ovaries of slaughtered animals in slaughterhouses Vinţu de Jos (Alba Iulia) and Cetina (Baia Mare). Medium-term cultivation of mare oocyte For in vitro maturation of mare oocytes we used sterile plates with 12 compartments. In each of them was added 1 ml of culture medium, technique carried out under sterile hood. With the aid of a fine pipette with a diameter of 50 µl, we placed 5-6 oocytes in the culture medium. The prepared plates were placed in the incubator for 24/28 hours at a temperature of C and 5.3% CO 2. A total of 765 oocytes (82.25%) were classified as "cultivable" and 165 oocytes (17.75%) were classified in "non-cultivable" category being excluded from the protocol. Medium-term cultivation was conducted on a total of 765 oocytes in five culture media as follows: V

6 175 oocytes were cultured in DMEM-F12 medium supplemented with 10% serum replacement; 150 oocytes were cultured TCM-199 medium supplemented with 10% FCS, 25 mm HEPES, 1 mm sodium pyruvate, 1% glutamine, 1% AA, 10 mg / ml of FSH, 2 mg / ml of LH, 500 µl antibiotics; 137 oocytes were cultured in TCM-199 medium supplemented with 5% SOF, 5% FCS, 1% glutamine, 1% AA, 500 µl antibiotics; 145 oocytes were cultured in SOF medium supplemented 10% FCS, 1% glutamine, 1% AA, 500 µl antibiotics; 158 oocytes were cultured in pure follicular fluid. MORPHOCYTOMETRICAL EVALUATION AND CLASSIFICATION OF MARE OOCYTES AFTER CULTIVATION The aim of this chapter was to improve the morphological assessment of mare oocytes by introducing into current practice the morphocytometrical assessments. These evaluations help determine morphological and morphocytometrical parameters after maturation. We wanted to also development a new classification of oocytes evaluated according to the morphocytometrical criteria. The research was conducted during on the ovaries collected from slaughtered mares in abattoirs from Vinţu de Jos, Alba county and Baia Mare, Maramures County. The collection of the mare oocytes was achieved by three methods described above, were collected 930 oocytes, a number of 765 oocytes (82.25%) were classified as "cultivable" and subjected to in vitro maturation. Morphological examination - In vitro cultivation of mare oocytes was performed for a total number of 765 oocytes collected and classified as "cultivable". Culture media were prepared after original formulas. Morphocytometrical examination - Morphocytometrial analysis was performed with the software Motic Image Plus of inverted microscope, taking into studio thickness of the zona pellucida and cumulus expansion after maturation, oocyte diameter, presence and size of the first polar body. Based on morphological aspects, oocytes were classified into two quality classes: mature oocytes (expanded cumulus cells, homogeneous or slightly granular cytoplasm, the presence of the first polar body, perivitelin space uniform, zona pellucida integrity); degenerate oocytes (total or partial loss of cumulus cells, strongly granular cytoplasm, vacuolated, perivitelin space ununiform, broken zona pellucida). Morphocytometrical analysis allowed us to complete morphological examination done before, which led to the reclassification of cultured oocytes in four quality classes. From 765 cultured oocytes 197 oocytes (25.75%) were classified as excellent mature" class, 108 oocytes (14.11%) were classified as mature good class, 203 oocytes (26.53%) in immature class and the remaining 257 oocytes (33.59%) were considered degenerated. VI

7 IN VITRO FERTILIZATION OF MARE OOCYTES Chapter aims was to study the influence of morphological assessment of oocytes on in vitro fertilization protocol. The research aimed to improve techniques for preparing semen, oocytes used for IVF protocol and evaluation of media and solutions used in the embryo culture. We also assessed the importance of morphological study correlated with the stage of development in determining the viability of the embryo. The research was conducted from 2009 to 2015 in the Laboratory of Biotechnology of Reproduction, Obstetrics and Veterinary Gynecology Department at Faculty of Veterinary Medicine Cluj-Napoca. Semen preparation for in vitro fertilization In order of sperm capacitation, semen straws were thawed by placing them in hot water at 37 ºC for 40 seconds. The sperm selection was made through swim-up method described by (Choi et al., 2003). using 2 ml of medium standard - the Sperm TALP medium (minitube, Germany) which was previously equilibrated by incubating at 39 C and 5% CO 2 for 2 hours. The pellet was resuspended in 800 µl Sperm TALP, preheated, and the prepared tubes were incubated for 1 hour in order to activate the sperm. Preparing media for in vitro fertilization Based on the composition of a known culture medium for mare oocytes fertilization- the TALP (Tyrode's Albumine Lactate Pyruvate Medium), in the laboratory were prepared culture media prepared in original way. Protocol for in vitro fertilization of mare oocytes We used microdroplets fertilization system: in the culture plates were placed oocytes/drop and 0.5 µl semen (concentration of 1.3 x 10 6 sperm/ml). Cultivation parameters were: temperature 39 C, 5% CO 2, 90% humidity for 24 hours. ICSI fertilization protocol of mare oocytes Sperm injection was performed under strict control of micromanipulation and inverted microscope. The sperm suspension was diluted with 5% PVP in 0.9% saline solution for preparation and dispose in three drops of 2 round and one elongated as follows: first drop (10 μl) is used to wash the pipette (saline PVP (polyvinylpyrrolidone)); second drop (10 μl) contains sperm suspension in saline PVP; third elongated drop (150 μl) was with TCM 199 buffered with HEPES with 1mg/ml BSA for oocytes. After covering the droplets with mineral oil, we proceed to placing inside them 6 denuded oocytes. One sperm was taken from the sperm drop by aspiration tail and deposited in droplet with oocytes to be injected. In vitro culture of mare oocytes fertilized After vortexing, the fertilization media has been replaced by three successive washes with the necessary culture media of embryos, two culture media supplemented and enriched in the original way with different materials to ensure proper development of in vitro fertilized oocytes to the young blastocyst stage. Plates were introduced to the incubator for 24 hours at 39 ºC, 5% CO2, 5% O2, 90% humidity. The results of in vitro fertilization of mare oocytes are showed in table 1: Table 1 The results of in vitro fertilization of mature oocytes IVF Non-fertilized Degenerated Oocytes/medium Fertilized oocytes medium oocytes oocytes F I ,72% 21,90% 32,38% F II 85 F III 80 37,64% 24 30% 30,58% 22 27,5% 31,76% 34 42,5% Results of mare oocytes fertilization with ICSI method are presented in table 2. VII

8 35 oocytes from mature class were used in the protocol of fertilization, based on morphological examination and completed with morphocitometrical examination. Table 2,,ICSI medium Results obtained after ICSI fertilization of mature oocytes Oocytes/medium Fertilized oocytes Non-fertilized Degenerated oocytes oocytes ,71% 8,57% 25,72% F 35 The results of in vitro cultivation of mare embryo For in vitro cultivation 127 fertilized oocytes were selected with homogeneous cytoplasm, integrated pellucida zone, two global polar bodies, and the two pronuclear (male and female). The results obtained after in vitro fertilization and ICSI method of mature mare oocytes is the following: in C l culture medium: were cultured 62 fertilized oocytes, 38 integrated embryos were obtained (61.29%) and 24 degenerated embryos (38.71%); in C II culture medium: 65 fertilized oocytes were cultured, 48 integrated embryos were obtained (73.48%) and 17 degenerated embryos (26.52%). GENERAL CONCLUSIONS AND PRACTICAL RECOMMENDATIONS After research conducted in and the interpretation of results, the following conclusions were formulated: 1. the results obtained after the follicle measurements allowed classification into 4 categories as: Category 1 (follicles <1 cm), Category 2 (follicles between 1-2 cm), Category 3 (follicles between 2-3 cm), category 4 (follicles> 3 cm); 2 were applied two methods oocytes collection of from the ovaries of slaughtered animals: follicles aspiration method and scraping method of the follicles to hair; 3. was applied a new method for oocytes collection from the oviduct of animals slaughtered by washing the oviduct using PBS; 4. the number of cultured oocytes recovered by aspiration method was 688 from 944 collected oocytes, the collecting rate being 72.88%; 5. the number of cultured oocytes recovered by scraping method was 376 of 608 collected oocytes, the collecting rate being 61.84%; 6. the number of oocytes recovered from the slaughtered animals oviduct is reduced (22 oocytes); 7. morphological characters determined after cultivation allowed classification into two quality categories: 305 mature oocytes, (39.86%), 460 degenerated oocytes (60.14%); 8. morphocytometric measurements made after cultivation, complemented by morphological examination, allowed us reinstatement oocytes into four quality classes: 197 excellent mature oocytes, 25.75%, 108 good mature oocytes, 14.11%, 203 immature oocytes, 26.53%, 257 degenerate oocytes, 33.59%; 9. from 305 cultivated oocytes, 270 oocytes were used for in vitro fertilization and the remaining 35 oocytes were used for ICSI fertilization method; 10. comparing the results of in vitro fertilization, stand out those obtained in the F I, where fertilization percentage was 45.72% (48 oocytes ), followed by the M II and M III media, where fertilization percentages were 37.64% (32 oocytes) and 30% (24 oocytes); 11. comparing the results of ICSI fertilization, fertilization percentage was 65.71% (23 oocytes), the lowest percentage of unfertilized oocytes was 8,57% (three oocytes), and the percentage of degenerated oocytes after fertilization was 25.72% (9 oocytes); 12. the results obtained by in vitro fertilization of oocytes ICSI method mare mature embryos classification is as follows: in the IC composed of SOF, BSA, pyruvate, lactic acid and glutamine, in which fertilized oocytes have grown 62, and They've won 38 embryos upright VIII

9 (61.29%) and 24 embryos degenerate (38.71%) in the CII medium (DMEM-F12, 10% FCS, antibiotics), which were cultured fertilized oocytes 65 results in 48 integrity embryos (73.48%) and 17 degenerated embryos (26.52%); 13. the percentage of embryos at the stage of 2-8 cells was higher in the CI, 28.95% (11 embryos) than in the CII % (13 embryos); PRACTICAL RECOMMENDATIONS Following the results obtained in this study may state the following practical recommendations: 1. Our research regarding collection of mare oocytes through two methods, aspiration and scraping method, allows us to recommend into the research use of the second method because of the advantages it brings: superior quality of oocytes; 2. We recommend the use for various biotechnologies (IVF CIV, cryopreservation) of only mature mare oocytes with the following morphometric characteristics: oocyte diameter of 110 µm compaction and expansion of cumulus cells over 40 µm, pellucid membrane integrity with a thickness of 13 µm, homogeneous cytoplasm, uniform perivitelin space; 3. We recommend for embryos cultivation derived from in vitro fertilization oocytes the use of DMEM-F12 medium supplemented with 10% FCS and antibiotics because it providesto ensure proper embryonic development; 4. For a better efficiency of in vitro fertilization protocol in order to obtain superior results, we recommend the use of oocytes whose maturation was based on morphological examination in conjunction with morphocytometry evaluation. REFERENCES 1. ABDOON, A.S.S., 2001 Factors affecting in vitro production of bovine embryos, Department. Of Animal Reproduction A.J. National Research Dokki. 12: BAVISTER, B.D., 1982 Evidence for a role of post-ovulatory cumulus contribution components in supporting fertilizing ability of hamster spermatozoa, J. Andrology, 3: BOITOR, I., CIUPERCESCU, D., GROZA, I., MOISE, D., SOCACIU, C., DAMIAN, A., 1987 Progrese în transferul de embrioni la animalele de fermă. Filiala ICVB Pasteur Cluj-Napoca. 4. BRACKETT,. B.D., EVANS, J.F., 1980 Fertilization and early development of cow ova, Biol. Reprod., 23: CARNEVALE, EM, MACLELLAN, LJ, COUTINHO DE SILVA, MA, SQUIRES, EL., Pregnancies attained after collection and transfer of oocytes from ovaries of five euthanatized mares. J Am Vet Med Assoc;222: CELESTINOS, M., Evaluación de la sobrevivencia in vitro de embriones de coneja bipartidos antes y después de la vitrificación. Tesis de Magister en Ciencias Mención Reproducción Animal. Universidad Austral de Chile. Valdivia, Chile. 7. CHOI, Y.T., TAKAGI, M., KAMISHITA, H., ACOSTA, T.J., 1997 Effects of follicular fluid on fertilization and embryonic development of bovine oocytes in vitro, Theriogenology 49: DAVIS, I.C., J.E. CORREA., Inducción de superovulación y transferencia de embriones en oveja. Agro Sur. 12: DELLA AQUILA, ME, CHO, YS, MINOIA, P, TRAINA, V, LACALANDRA, GM, MARITATO, F., Effects of follicular fluid supplementation of in-vitro maturation medium on the fertilization and development of equine oocytes after in-vitro fertilization or intracytoplasmic sperm injection. Hum Reprod;12: DUCHAMP, G., BEZARD, J., PALMER, E., 1995 oocyte yield and the consequences of puncture of all follicles larger than 8 millimeters in mares. In: Sharp, D.C.; Bazer, F.W. (Eds.), Equine reproduction VI. Society for the study of reproduction, Madison, WI, pp DURAN, H., 2000 Technical aspect of in vitro embryo production, Phil. Jour. Vet. Anim. Sci. 24, 1-2: EVANS, J.P., 2002 The molecular basis of sperm-oocyte membrane interactions during mammalian fertilization, Human Reproduction Update, 8: IX

10 X 13. GALLI, C, CROTTI, G, TURINI, R, DUCHI, G, MARI, G, ZAVAGLIA, G, et al., Frozen-thawed embryos produced by ovum pickup of immature oocytes and ICSI are capable to establish pregnancies in the horse. Theriogenology ;58:705 8 [abstract]. 14. GARDNERR, D.K., LANE, H., BATT, P., Uptake and metabolism of pyruvate and glucose by individual sheep preattachement embryos developed in vivo. Mol. Reprod. Dev., 36: GREVE T., MADISON V., 1991 In vitro fertilization in cattle: to review, Reprod. Nutr. Dev. 31: GROZA I. şi col., 2006 Ginecologie, Andrologie şi Obstetrică Veterinară Compendiu, Ed. Academiei Române, Bucureşti. 17. GROZA, I., 1996 Actualităţi şi perspective în biotehnologia transferului de embrioni la specia ovină. Ed.Ceres. 18. HASHIMOTO, S., MINAMI, N., TAKAKURA, R., 2000a Low oxygen tension during in vitro maturation is beneficial for supporting the subsequent development of bovine cumulus oocyte complexes, Mol. Reprod. Dev., 57: 353/ HINRICHS, K, BESTCHART, RW, McCUE, PM, SQUIRES, EL., Effect of timing of follicle aspiration on pregnancy rate after oocyte transfer in the mares. J Reprod Fertil ;56(Suppl): HINRICHS K., SCHMIDT, AL., SELGRATH, JP., 1993b - Atlas of chromatin configuration of germinal vesicle-stage and maturing horse oocytes. Equine Veterinary Journal Supplement. 15, HUNTER, R.F.F. and POLGE., C Maturation of follicular oocytes in the pig after injection of human chorionic gonadotropin. Jour. Reprod. Fertil. 12: KRISHER, R.L., BAVISTER, B.D., 1998 Responses of oocytes and embryos to the culture environment, Theriogenology 49: RIEGER, D. LOSKUTOFF, N.M., BETTERIDGE,K.J., Developmentally related changes in the metabolism of glucose and glutamine by cattle embryos produced and cocultured in vitro. J. Reprod. Fertil. 195: SIRARD, M.A., RICHARD, F., BLONDIN, P., ROBERT, C., 2006 Contribution of the oocyte to embryo quality. Theriogenology, 65: WALES, R.G., Measurement of metabolic turnover in single mouse embryos. J. Reprod. Fertil., 76: YANG, N.S., LU, K.H., GORDON, I., 1990 In vitro fertilization (FIV) and culture (IVC) of bovine oocytes from stored ovaries. Theriogenology 33: ZHANG, J.J., BOYLE, M.S., ALLEN, W.R., GALLI, C., Recent studies on in vivo fertilization of in vitro matured horse oocytes. Equine Vet. J. Suppl. 8,

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