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Home / Equine Research Bank / Equine Vet J / The efficacy of embryo recovery on consecutive days from Day...
Equine veterinary journal2025; doi: 10.1111/evj.70098

The efficacy of embryo recovery on consecutive days from Day 6.5 to obtain small embryos for vitrification.

Abstract: Vitrified embryos ≤300 μm give better pregnancy rates following warming and transfer than larger ones. Embryo recovery undertaken close to when the embryo enters the uterus (Day 6-6.5) helps in the recovery of embryos ≤300 μm. However, flushing early can mean missing an embryo not yet in the uterus, whereas later can result in embryos >300 μm. Objective: To evaluate if repeated embryo flushing on consecutive days from Day 6.5 would increase the number of embryos ≤300μm recovered. Methods: Retrospective case series. Methods: Four hundred and ninety-six inseminations with cooled (n = 339) or frozen (n = 130) semen were undertaken in 91 mares over a three-year period; pre-ovulatory for cooled and post-ovulatory for frozen semen, with mares checked for ovulation daily or every 6 h, respectively. At a presumed Day 6.5 (cooled semen) or 7 (frozen semen), mares' uteri were flushed, with flushing repeated 24 and 48 h later if no embryo was recovered. Linear mixed-effects models and likelihood ratio tests were used to investigate factors potentially influencing embryo diameter, embryo grade, and oviduct transport time (e.g., semen type, ovulatory drug, stallion age, mare age, season and flush number). Results: Using repeated flushing, 98.8% (243/246) of embryos recovered were ≤300 μm. Second and third flushes yielded 61 embryos (24.8% of recovered embryos). The only variable that influenced embryo diameter was flush number (p = 0.003); for embryo grade, no variable was significant, whereas oviduct transport time was affected by semen type (p = 0.003), ovulatory drug (p = 0.029) and stallion age (p = 0.005). Conclusions: Noncontrolled clinical study. Conclusions: Embryo flushing on consecutive days from the time of expected entry of the embryo into the uterus was effective in collecting embryos ≤300 μm and resulted in additional embryos being retrieved compared with a single flush. Oviductal transport time in some mares is >156 h from ovulation.
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Publication Date: 2025-09-09 PubMed ID: 40923566DOI: 10.1111/evj.70098Google Scholar: Lookup
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Summary

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Overview

  • This study investigated whether flushing the uterus of mares on consecutive days starting around Day 6.5 after ovulation increases the recovery of small embryos (≤300 μm), which are known to have better pregnancy success after vitrification (freezing).

Background

  • Vitrification is a freezing method used to preserve embryos for later transfer.
  • Embryos that are ≤300 μm in diameter tend to survive vitrification better and give improved pregnancy rates after warming and transfer compared to larger embryos.
  • It is important to recover embryos when they first enter the uterus, typically around Day 6 to 6.5 post-ovulation.
  • If the uterus is flushed too early, the embryo might not yet be present, causing a missed recovery.
  • If flushed too late, the embryo may have grown beyond the preferred ≤300 μm size.

Objective

  • The study aimed to evaluate if performing repeated uterine flushing on consecutive days starting around Day 6.5 would increase the recovery of small (≤300 μm) embryos.

Methods

  • Design: A retrospective case series analyzing data collected over three years.
  • Subjects: 91 mares undergoing a total of 496 inseminations using either cooled (339) or frozen (130) semen.
  • Timing:
    • For cooled semen, inseminations were done pre-ovulation; for frozen semen, post-ovulation.
    • Mares were checked for ovulation either daily or every 6 hours.
  • Embryo Recovery:
    • Uterine flushing started at presumed Day 6.5 (cooled semen) or Day 7 (frozen semen).
    • If no embryo was recovered on the first flush, flushing was repeated 24 hours and then again 48 hours later (up to 3 flushes).
  • Data Analysis:
    • Linear mixed-effects models and likelihood ratio tests were conducted.
    • Factors studied included semen type, ovulatory drugs, stallion and mare age, season, and flush number.
    • Outcomes measured were embryo diameter, embryo quality grade, and oviduct transport time (time taken for embryo to travel from ovary to uterus).

Results

  • Total embryos recovered: 246.
  • Percentage of embryos ≤300 μm: 98.8% (243 out of 246 embryos).
  • Contribution of second and third flushes: they recovered 61 embryos, which is 24.8% of all embryos recovered, indicating substantial additional yield from repeated flushes.
  • Statistically significant findings:
    • Flush number influenced embryo diameter (p = 0.003), suggesting embryo size may change depending on day of flushing.
    • No factor significantly affected embryo grade (quality).
    • Oviduct transport time was affected by:
      • Semen type (p = 0.003)
      • Use of ovulatory drugs (p = 0.029)
      • Stallion age (p = 0.005)
    • Some mares had oviduct transport times longer than 156 hours (~6.5 days) from ovulation.

Conclusions

  • Repeated embryo flushing on consecutive days starting around the time embryos enter the uterus is effective for recovering small (≤300 μm) embryos suitable for vitrification.
  • This approach yields more embryos than a single flush procedure.
  • There is considerable variability between mares in transit time of embryos through the oviduct, with some taking longer than expected to reach the uterus.
  • Understanding and accounting for this variability with repeated flushing can improve embryo recovery and thus potentially enhance pregnancy outcomes after transfer.
  • The study emphasizes the practical benefit of timed repeated embryo recovery in equine breeding clinics.

Cite This Article

APA
Couto GR, Vigano DWA, Santos GDC, Allen WRT, Wilsher S. (2025). The efficacy of embryo recovery on consecutive days from Day 6.5 to obtain small embryos for vitrification. Equine Vet J. https://doi.org/10.1111/evj.70098

Publication

Equine veterinary journal
ISSN: 2042-3306
NlmUniqueID: 0173320
Country: United States
Language: English

Researcher Affiliations

Couto, Guillerme R
  • Al Wathba Stables, Abu Dhabi, UAE.
Vigano, Debraym W A
  • Al Wathba Stables, Abu Dhabi, UAE.
Santos, Gabriel D C
  • Al Wathba Stables, Abu Dhabi, UAE.
Allen, W R Twink
  • Sharjah Equine Hospital, Sharjah, UAE.
Wilsher, Sandra
  • Sharjah Equine Hospital, Sharjah, UAE.
  • The Paul Mellon Laboratory, Suffolk, UK.

References

This article includes 65 references
  1. Eldridge‐Panuska WD, di Brienza VC, Seidel GE Jr, Squires EL, Carnevale EM. Establishment of pregnancies after serial dilution or direct transfer by vitrified equine embryos.. Theriogenology 2005;63:1308–1319.
  2. Sanchez R, Blanco M, Weiss J, Rosati I, Herrera C, Bollwein H. Influence of embryonic size and manipulation on pregnancy rates of mares after transfer of cryopreserved equine embryos.. J Equine Vet Sci 2017;49:54–59.
  3. Wilsher S, Rigali F, Couto G, Camargo S, Allen WR. Vitrification of equine expanded blastocysts following puncture with or without aspiration of the blastocoele fluid.. Equine Vet J 2019;51:500–505.
  4. Wilsher S, Rigali F, Kovacsy S, Allen WT. Successful vitrification of manually punctured equine embryos.. Equine Vet J 2021;53:1227–1233.
  5. Kovacsy S, Ismer A, Hoogewijs M, Funes J, Wilsher S. Successful vitrification of equine embryos >300 μm without puncture or aspiration.. Equine Vet J 2024;56(4):815–822.
    doi: 10.1111/evj.14081google scholar: lookup
  6. Choi YH, Velez IC, Riera FL, Roldán JE, Hartman DL, Bliss SB. Successful cryopreservation of expanded equine blastocysts.. Theriogenology 2011;76(1):143–152.
  7. Diaz F, Bondiolli K, Paccamonti D, Gentry GT. Cryopreservation of Day 8 equine embryos after blastocyst micromanipulation and vitrification.. Theriogenology 2016;85(5):894–903.
  8. Freeman DA, Weber JA, Geary RT, Woods GL. Time of embryo transport through the mare oviduct.. Theriogenology 1991;35:823–830.
  9. Battut I, Colchen S, Fieni F, Tainturier D, Bruyas J‐F. Success rates when attempting to nonsurgically collect equine embryos at 144, 156 or 168 hours after ovulation.. Equine Vet J 1998;29(S25):60–62.
  10. Betteridge KJ. The structure and function of the equine capsule in relation to embryo manipulation and transfer.. Equine Vet J 1989;21(S8):92–100.
  11. Oriol JG, Betteridge KJ, Clarke AJ, Sharom FJ. Mucin‐like glycoproteins in the equine embryonic capsule.. Mol Reprod Dev 1993;34:255–265.
  12. Betteridge KJ. Equine embryology: an inventory of unanswered questions.. Theriogenology 2007;68(S):S9–S21.
  13. Enders AC, Schlafke S, Kantz KC, Liu IKM. Endodermal cells of the equine yolk sac from Day 7 until formation of the definitive yolk sac placenta.. Equine Vet J 1993;25(S15):3–9.
  14. Squires EL, Garcia RH, Ginther OJ. Factors affecting success of equine embryo transfer.. Equine Vet J 1985;17(S3):92–95.
  15. Boyle MS, Sanderson MW, Skidmore JA, Allen WR. Use of serial progesterone measurements to assess cycle length, time of ovulation and timing of uterine flushes in order to recover equine morulae.. Equine Vet J 1989;21(S8):10–13.
  16. McKinnon AO, Squires EL. Morphological assessment of the equine embryo. J Am Vet Med Assoc 1988;192:401–406.
  17. Colchen S, Battut I, Fieni F, Tainturier D, Siliart S, Bruyas JF. Quantitative histological analysis of equine embryos at exactly 156 and 168h after ovulation. J Reprod Fertil Suppl 2000;56:527–537.
  18. McKinnon AO, Perriam WJ, Lescun TB, Walker J, Vasey JR. Effect of a GNRH analogue (Ovuplant), hCG and dexamethasone on the time to ovulation in cycling mares. World Equine Vet Res 1997;2:16–18.
  19. Carnevale EM. Vitrification of equine embryos. Vet Clin North Am Equine Pract 2006;22:831–841.
  20. Lascombes FA, Pashen RL. Results from embryo freezing and post ovulation breeding in a commercial embryo transfer programme. Proceedings 5th International Symposium on Equine Embryo Transfer, Havemeyer Foundation Monograph series 3, Saari, Finland Newmarket: R&W Publishing; 2000. p. 95–96.
  21. Cuervo‐Arango J, Claes AN, Stout TAE. Horse embryo diameter is influenced by the embryonic age but not by the type of semen used to inseminate donor mares. Theriogenology 2018;115:90–93.
  22. Carnevale EM, Griffin PG, Ginther OJ. Age‐associated subfertility before entry of embryos into the uterus in mares. Equine Vet J 1993;25(S15):31–35.
  23. Battut I, Grandchamp des Raux A, Nicaise JL, Fieni F, Tainturier D, Bruyas JF. When do equine embryos enter the uterine cavity: An attempt to answer?. Proc 5th Int Symp Equine Embryo Transfer, Havemeyer Monograph No. 3 Newmarket: R&W Publications; 2000. p. 66–68.
  24. McCue PM, DeLuca CA, Ferris RA, Wall JJ. How to evaluate equine embryos. Proc Am Assoc Equine Pract 2009;55:252–256.
  25. Zuur AF, Ieno EN, Walker N, Saveliev AA, Smith GM. Mixed effects models and extensions in ecology with R. New York, NY: Springer; 2009.
    doi: 10.1007/978-0-387-87458-6google scholar: lookup
  26. R Core Team. R: a language and environment for statistical computing. Vienna: R Foundation for Statistical Computing; 2025.
  27. Hartig F. DHARMa: residual diagnostics for hierarchical (multi‐level/mixed) regression models. R package version 0.4.7. 2024.
  28. Lindblom B, Hamberger L, Wiqvist N. Differentiated contractile effects of prostaglandins E and F on the isolated circular and longitudinal smooth muscle of the human oviduct. Fertil Steril 1978;30:553–559.
  29. Wånggren K, Stavreus‐Evers A, Olsson C, Andersson E, Gemzell‐Danielsson K. Regulation of muscular contractions in the human Fallopian tube through prostaglandins and progestagens. Hum Reprod 2008;23:2359–2368.
  30. Van Niekerk CH, Gerneke WH. Persistence and parthenogenetic cleavage of tubal ova in the mare. Onderstepoort J Vet Res 1966;31:195–232.
  31. Onuma H, Ohnami Y. Retention of tubal eggs in mares. J Reprod Fertil 1975;(Suppl 23):507–511.
  32. Flood PF, Jong A, Betteridge KJ. The location of eggs retained in the oviducts of mares. J Reprod Fertil 1979;57:291–294.
    doi: 10.1530/jrf.0.0570291google scholar: lookup
  33. Betteridge KJ, Eaglesome MD, Mitchell D, Flood PF, Beriault R. Development of horse embryos up to twenty‐two days after ovulation: observations on fresh specimens. J Anat 1982;135:191–209.
  34. Aguilar JJ, Woods GL, Miragaya MH, Olsen LM. Living fibroblasts cells in the oviductal masses of mares. Equine Vet J 1997;29(S25):103–108.
  35. Weber JA, Woods GL, Aguilar JJ. Location of equine oviductal embryos on Day 5 post ovulation and oviductal transport time of Day 5 embryos autotransferred to the contralateral oviduct. Theriogenology 1996;46:1477–1483.
    doi: 10.1016/s0093-691x(96)00325-1google scholar: lookup
  36. Sullivan JJ, Parker WG, Larson LL. Duration of estrus and ovulation time in non‐lactating mares given hCG during three successive estrous periods. J Am Vet Med Assoc 1973;163:895–898.
  37. McKinnon AO, Nobelius AM, Figueroa STD, Skidmore J, Vasey JR, Trigg TE. Predictable ovulation in mares treated with an implant of the GnRH analogue Deslorelin. Equine Vet J 1993;25:321–323.
  38. Samper JC, Jensen S, Sergeant J, Estrada A. Timing of induction of ovulation in mares treated with Ovuplant or Chorolun. J Equine Vet Sci 2002;22:320–323.
  39. Morrissey J, Ferris R, McCue P. Comparison of deslorelin and histrelin for induction of ovulation in mares. Clin Theriogenol 2019;11:123–126.
  40. Cazales N, Pereyra F, Icatt S, Colmán NJ, Camacha CA, Estradé MJ. hCG versus histrelin acetate: ovulation inducing agents in mares (Equus caballas) in a commercial breeding program in Uruguay. J Equine Vet Sci 2023;125:104698.
  41. Segabinazzi LGTM, Oba E, Alvarenga MA. The combination of hCG and GnRH analog to hasten ovulation in mares does not change luteal function and pregnancy outcome in embryo recipient mares. J Equine Vet Sci 2021;105:103691.
  42. Weber JA, Freeman DA, Vanderwall DK, Woods GL. Prostaglandin E2 secretion by oviductal transport stage equine embryos. Biol Reprod 1991;45:540–543.
  43. Ball BA, Little TV, Hillman RB, Woods GL. Pregnancy rates at Days 2 and 14 and estimated embryonic loss rates prior to Day 14 in normal and subfertile mares. Theriogenology 1986;26:611–619.
  44. Ball BA, Little TV, Weber JA, Woods GL. Survival of Day‐4 embryos from young, normal mares and aged, subfertile mares after transfer to normal recipient mares. Reproduction 1989;85:187–194.
  45. Grondahl C, Hyttel P. Nucleogenesis and ribonucleic acid synthesis in preimplantation equine embryos. Biol Reprod 1996;55:769–774.
  46. Goszczynski DE, Tinetti PS, Choi YH, Hinrichs K, Ross PJ. Genome activation in equine in‐vitro produced embryos. Biol Reprod 2022;106:66–82.
  47. Hoshi K, Aita T, Yanagida K, Yoshimatsu N, Sato A. Variation in the cholesterol/phospholipid ratio in human spermatozoa and its relationship with capacitation. Hum Reprod 1990;5:71–74.
  48. Ostermeier GC, Cardona C, Moody MA, Simpson AJ, Mendoza R, Seaman E. Timing of sperm capacitation varies reproducibly among men. Mol Reprod Dev 2018;85:387–396.
    doi: 10.1002/mrd.22972google scholar: lookup
  49. McCue PM, Ferris RA, Lindholm AR, Deluca CA. Embryo recovery procedures and collection success: results from 492 embryo‐flush attempts. Proc Am Assoc Equine Pract 2010;56:318–321.
  50. Panzani D, Rota A, Marmorini P, Vannozzi I, Camillo F. Retrospective study of factors affecting multiple ovulations, embryo recovery, quality and diameter in a commercial embryo transfer program. Theriogenology 2014;82:807–814.
  51. Bedford SJ, Meyers SA, Varner DD. Acrosomal status of fresh, cooled and cryopreserved stallion spermatozoa. J Reprod Fert 2000;Suppl 56:133–140.
  52. Schembri MA, Major DA, Suttie JJ, Maxwell WMC, Evans G. Capacitation‐like changes in equine spermatozoa throughout the cryopreservation process. Reprod Fertil Dev 2002;14:225–233.
  53. Thomas AD, Meyers SA, Ball BA. Capacitation‐like changes in equine spermatozoa following cryopreservation. Theriogenology 2006;65:1531–1550.
  54. DePamphilis ML. Genome duplication at the beginning of mammalian development. In: DePamphilis ML, editor. Current topics in developmental biology. Volume 120. Cambridge, MA: Academic Press; 2016. p. 55–102.
  55. Hartman DL. Embryo transfer: donor mare management after the flush. In: McKinnon AO, Squires EL, Vaala WE, Varner DD, editors. Equine reproduction. 2nd ed. Oxford: Wiley‐Blackwell; 2011. p. 2878–2879.
  56. Martínez‐Boví R, Sala‐Ayala L, Querol‐Paajanen A, Plaza‐Davila M, Cuervo‐Arango J. Effects of repeated embryo flushing without PGF2α administration on luteal function, percentage of unwanted pregnancy and subsequent fertility in mares. Equine Vet J 2024;56:796–805.
  57. Carnevale EM, Ramirez EL, Squires EL, Alvarenga MA, Vanderwall DK, McCue PM. Factors affecting pregnancy rates and early embryonic death after embryo transfer. Theriogenology 2000;54:965–979.
  58. Balbuena G, Garzarón A, Cuervo‐Arango J. The effect of embryo diameter and developmental stage on embryo transfer rates. 7th International Symposium on Equine Embryo Transfer. Cambridge: The Havemeyer Foundation; 2008. p. 88–90.
  59. Carmago CE, Weiss RR, Kozicki LE, Pastorello Duarte M, Garcia Duarte MC, Lunelli D. Some factors affecting the rate of pregnancy after embryo transfer derived from the Brazilian jumper horse breed. J Equine Vet Sci 2013;33:924–929.
  60. Cuervo‐Arango J, Claes AN, Stout TAE. Small Day 8 equine embryos cannot be rescued by a less advanced recipient mare uterus. Theriogenology 2019;126:36–40.
    doi: 10.1016/j.theriogenology.2018.11.026google scholar: lookup
  61. Chevalier F, Palmer E. Ultraosonic echography in the mare. J Reprod Fertil Suppl 1982;32:423–430.
  62. Newcombe JR. Embryonic loss and abnormalities of early pregnancy. Equine Vet Educ 2000;12:88–101.
  63. Vanderwall D, Squires EL, Brinsko SP, McCue PM. Diagnosis and management of abnormal embryonic development characterised by formation of an embryonic vesicle without an embryo in the mare. J Am Vet Med Assoc 2000;217:58–63.
  64. Handler J, Königshofer M, Kindahl H, Schams D, Aurich C. Secretion patterns of oxytocin and PGF2alpha‐metabolite in response to cervical dilation in cyclic mares. Theriogenology 2003;59:1381–1391.
  65. Rubio MS, Allen WR, Wilsher S, Mouguelar H. Effects of embryo flushing on serum progesterone levels and the inter‐ovulatory period in Arabian donor mares. J Equine Vet Sci 2023;125:103.

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