Effect of embryo developmental kinetics and vitrification on pregnancy outcome, foaling rate and sex of the foal

Effect of embryo developmental kinetics and vitrification on pregnancy outcome, foaling rate and sex of the foal | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 13 November 2025 V1 Latest version Share on Effect of embryo developmental kinetics and vitrification on pregnancy outcome, foaling rate and sex of the foal Authors : Sofie Peere 0009-0001-2390-3435 [email protected] , M. Dewulf , Emma Van den Branden , M. Papas , K. Broothaers 0000-0001-7804-4693 , Tine De Coster , I. Briote , M. Hedia , D. Angel-Velez , M. Hoogewijs , Jan Govaere , and Katrien Smits Authors Info & Affiliations https://doi.org/10.22541/au.176304279.92865708/v1 296 views 88 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background: While fast embryo development has been associated with improved ICSI outcomes, the effects of slow embryo development (D9-11 post ICSI) and of vitrification vs. fresh IVP transfer on foaling rates and the foal’s sex are less explored. Objectives: To determine: 1) the effect of day of embryo development (D7-11) and 2) of vitrification and warming assessing two methods: direct warming in isotonic commercial embryo holding medium (one step) or serial dilution warming (three step) vs. fresh IVP embryos, on pregnancy, early pregnancy loss, foaling rates and the foal’s sex Study design: Retrospective clinical study Methods: Blastocysts (n=201) were collected on D7-11 after ICSI of in vitro matured (IVM) oocytes from Warmblood mares (n=22) and transferred either fresh or vitrified-warmed into recipient mares on D4 post-ovulation. Pregnancy, early pregnancy loss, foaling rates and the foal’s sex were compared by RStudio statistics. Results: The day of blastocyst development did not affect initial pregnancy rates, but transfer of D11 IVP embryos resulted in significantly higher early pregnancy loss (D7,-8,-9,-10 and -11; 0%, 5.7%, 11.1%, 20.0% and 83.3%, resp., p < 0.001) and lower foaling rates (D7,-8,-9,-10 and 11; 88.2%, 64%, 60.8%, 50% and 10%, resp., p < 0.0001). The foal’s sex showed a skewed sex ratio towards female in D10 (78.9%:21.1%) vs. D7 (35.7%:64.3%; P = 0.02), D8 (50%:50%; P = 0.05) and D9 (48.4%:51.6%) IVP embryos. Transfer of fresh vs. vitrified-warmed (one-and three step protocol) embryos did not significantly affect pregnancy nor foaling rates. Main limitations: Small sample size per day of blastocyst development Conclusions: Slow in vitro embryo development negatively affects foaling rates after transfer of D11 IVP embryos. Transfer of D10 IVP embryos yields acceptable foaling rates and favors female offspring. Vitrification provides a safe method to preserve equine embryos with the flexibility to use a one-step warming protocol. ORIGINAL ARTICLE Effect of embryo developmental kinetics and vitrification on pregnancy outcome, foaling rate and sex of the foal Summary Background: While fast embryo development has been associated with improved ICSI outcomes, the effects of slow embryo development (D9-11 post ICSI) and of vitrification vs. fresh IVP transfer on foaling rates and the foal’s sex are less explored. Objectives: To determine: 1) the effect of day of embryo development (D7-11) and 2) of vitrification and warming assessing two methods: direct warming in isotonic commercial embryo holding medium (one step) or serial dilution warming (three step) vs. fresh IVP embryos, on pregnancy, early pregnancy loss, foaling rates and the foal’s sex Study design: Retrospective clinical study Methods: Blastocysts (n=201) were collected on D7-11 after ICSI of in vitro matured (IVM) oocytes from Warmblood mares (n=22) and transferred either fresh or vitrified-warmed into recipient mares on D4 post-ovulation. Pregnancy, early pregnancy loss, foaling rates and the foal’s sex were compared by RStudio statistics. Results: The day of blastocyst development did not affect initial pregnancy rates, but transfer of D11 IVP embryos resulted in significantly higher early pregnancy loss (D7,-8,-9,-10 and -11; 0%, 5.7%, 11.1%, 20.0% and 83.3%, resp., p < 0.001) and lower foaling rates (D7,-8,-9,-10 and 11; 88.2%, 64%, 60.8%, 50% and 10%, resp., p < 0.0001). The foal’s sex showed a skewed sex ratio towards female in D10 (78.9%:21.1%) vs. D7 (35.7%:64.3%; P = 0.02), D8 (50%:50%; P = 0.05) and D9 (48.4%:51.6%) IVP embryos. Transfer of fresh vs. vitrified-warmed (one-and three step protocol) embryos did not significantly affect pregnancy nor foaling rates. Main limitations: Small sample size per day of blastocyst development Conclusions: Slow in vitro embryo development negatively affects foaling rates after transfer of D11 IVP embryos. Transfer of D10 IVP embryos yields acceptable foaling rates and favors female offspring. Vitrification provides a safe method to preserve equine embryos with the flexibility to use a one-step warming protocol. Keywords: Embryo kinetics; In vitro production; Horse; Foal’s sex; Pregnancy; Foaling rate; Vitrification Introduction In vitro production (IVP) of equine embryos by intracytoplasmatic sperm injection (ICSI) became increasingly popular. The possibility of performing ovum pick-up (OPU) year-round, obtaining multiple embryos per year from both fertile and subfertile animals created a high interest among horse breeders. Moreover, cryopreservation of equine embryos, via conventional slow freezing or vitrification, enables worldwide distribution of valuable genetics and enhances efficiency in mare management [1–3]. Producing a healthy foal is crucial and compels to address all factors influencing pregnancy- and foaling rates following transfer of an IVP embryo. As shown previously, the ‘speed of in vitro embryo development’ (i.e. the interval between ICSI and blastocyst formation) affects pregnancy rate, embryonic loss, foaling rate and the foal’s sex [1,2,4–7]. Early blastocyst formation on days 6 to 8 post ICSI (D6-8), results in higher pregnancy and foaling rates compared to slow embryo development (D9-11) [1,2,4,5]. Transfer of slow developing embryos (D9 and beyond) lead to disappointing pregnancy rates and higher losses [1,5]. Only one study reported foaling rates after transfer of slow IVP embryos with D9 generating 38.5% and D10 only 25% [2]. Fast in vitro embryo development has been shown to favor male offspring. Claes et al. (2022) showed a skewed sex ratio towards male after transfer of D7 IVP equine embryos compared to D8, suggesting that male embryos develop more rapidly in vitro . While slow development has been suggested to favor female offspring [4,7], conclusive data for IVP embryos developing beyond D9 are lacking. Vitrification of IVP embryos is a well-established technique, resulting in similar pregnancy rates, when compared to transfer of vitrified small (<480 µm) in vivo recovered embryos [8,9]. Development of a simplified method using a commercial vitrification kit followed by direct warming in isotonic embryo holding medium (EHM) further supported embryo survival and pregnancy rates [10,11]. However, large scale foaling rates directly comparing transfer of vitrified-warmed (one- and three step protocol) vs. fresh IVP embryos in clinical settings are limited. The aims of this study were to evaluate the effects on pregnancy outcomes and the foal’s sex of: (1) slow in vitro embryo development, and (2) fresh transfer versus vitrification and warming by two methods in our IVP system. Material and methods Equine embryos ( n =201) were produced in vitro from privately owned Warmblood mares ( n =22) presented for commercial OPU-ICSI at the Faculty of Veterinary Medicine, {masked for review}. The oocyte donor mares were housed on the same breeding farm under similar conditions. They were all aged between 3-23 yrs old and were selected based on their follicular count (>10 follicles; sizes: 5-25mm) by transrectal ultrasonography (7.5-MHz transducer, transrectal linear probe, MyLabOne, Essaote, Genoa, Italy). The IVP year (2021-2024) was recorded. 1) Equine IVP embryos 1.1) Transvaginal Ultrasound-Guided follicle aspiration Immature oocytes were collected by OPU, as previously described [12,13]. Briefly, prior to the OPU, the mares were premedicated with benzylpenicillin (IM; Penikel, Kela, Sint Niklaas, Belgium, 20,000 IU/kg), flunixin meglumine (IV; Wellicox, Ceva Santé Animale, Naaldwijk, The Netherlands, 1.1 mg/kg), detomidine hydrochloride (IV; Domidine, Eurovet Animal Health BV, Bladel, The Netherlands, 0.01 mg/kg) and butorphanol tartrate (IV; Dolorex, MSD Animal Health, Sint-Lambrechts-Woluwe, Belgium, 0.01 mg/kg). Additionally, to enhance rectal relaxation, N-butylscopolammonium bromide (IV; Buscopan, Boehringer Ingelheim, Brussel, Belgium, 0.3 mg/kg), was administered. Antral follicles (5-25mm) were punctured using a 12G double-lumen needle, aspirated and flushed 10 times with pre-warmed commercial flushing medium (Equiplus, Minitube, Tiefenbach, Germany), while rotating the needle. Recovered follicular and flushing fluids were filtered (70 µm oocyte filter; Cell strainer, BD Biosciences, Falcon, Erembodegem, Belgium) and the cumulus oocyte complexes (COCs) were identified using a stereomicroscope (Olympus SZX7, Olympus Corp., Japan). The COCs were collected in Medium 199 with Hank’s salts (Gibco, Life technologies, Merelbeke, Belgium) containing 10% fetal bovine serum (FBS, Gibco) and 0,1% gentamycin (Sigma-Aldrich, Bornen, Belgium). 1.2) In-vitro maturation (IVM) of oocytes, ICSI and in-vitro culture (IVC) The recovered oocytes were either placed in commercial EHM (Emcare, Spervital, The Netherlands) for 20-24h at room temperature (22°C) prior to IVM or immediately transferred to maturation medium (Medium 199 with Earl’s salts (Gibco) containing 10% (v/v) FBS (Gibco), 10 ug/mL follicle stimulating hormone, 2 ug/mL luteinizing hormone (Stimufol, Reprobiol, Ouffet, Belgium) and 0,1% gentamycin). In vitro maturation was conducted in groups of 2-20 COCs in 100-500 µL maturation medium under oil (CooperSurgical, Venlo, The Netherlands) for 28-34h at 38.2 °C in 5% CO2 in air. Oocytes were denuded in Medium 199 with Hank’s salts containing 10% FBS and 0.1% Hyaluronidase (Sigma-Aldrich, Bornen, Belgium) and those with an extruded first polar body were selected for ICSI. ICSI was performed using frozen-thawed semen from a warmblood stallion ( n =19) selected by the mare owner. A small piece of a 0.5 ml straw was cut-off and thawed in 1mL of preheated (38.2 °C) G-MOPS (Vitrolife, Londerzeel, Belgium). Subsequently, a double centrifugation at room temperature (400 g for 3 min at 22°C) was performed. The supernatant was removed and the sperm suspension was dissolved in 200 µL of G-MOPS. A small quantity of the semen suspension was added to a 5uL droplet of 7% polyvinylpyrrolidone (Coopersurgical, Venlo, The Netherlands) and Piezo-driven ICSI was conducted as described previously [12]. The injected oocytes were cultured in groups of 1-16 in 20 µL droplets of DMEM/F-12 (Gibco) with 10% FBS and 0.1% Gentamycin under oil at 38.2 °C in a humified atmosphere of 5% 02, 5% C02 and 90% N2. Blastocyst formation was assessed daily between D7 and D11 post ICSI. 1.4) Vitrification and warming of equine IVP blastocysts Equine IVP blastocysts were transferred fresh ( n =37) or after vitrification ( n =164) in a minimal volume on a Cryolock (Irvine Scientific) and warmed following two protocols: A one step protocol: vitrification, using a commercial vitrification kit (VitKit; Minitube, Germany), followed by direct warming in EHM ( n = 100): vitrification at 23°C in VS1 (5min), VS2 (5 min) and VS3 (40 s), containing increasing concentrations of glycerol and/or ethylene glycol [14], followed by direct warming at 38.2°C for 5 min in 4 ml commercial EHM (ABT holding, NIFA, The Netherlands) [10,11]. A three step protocol, consisting of vitrification, followed by a stepwise serial dilution warming ( n = 64) [8]: holding of embryos in base solution (BS; DMEM-F12 with 20% FBS), equilibration for 5 min in vitrification solution 1 (VS1; BS + 1.5M ethylene glycol) followed by 40 s in vitrification solution 2 (VS2; BS + 7M ethylene glycol and 0.6M galactose); Subsequent warming was performed in a three-step protocol: warming medium (WM; DPBS with 0.1% glucose, 36 mg/L pyruvate, and 0.4% BSA) with 0.3M sucrose (1 min), followed by WM with 0.15M sucrose (5 min) and holding in WM (5 min), all performed at 38.2°C [8,11]. Both fresh and vitrified-warmed IVP embryos were washed four times in commercial EHM (ABT holding, NIFA, The Netherlands) and loaded into a 0.25-mL straw, which was placed into a Cassou gun (IVM technologies) for immediate embryo-transfer (ET). 1.5) Embryo transfer, pregnancy outcome, foaling rate and sex of the foal All selected standardbred recipient mares had a good BCS (4-6/9) and were aged between 3 and 12 years old. They were housed in group at the same breeding farm under similar conditions. Cycle follow-up was performed, including daily control during late estrus to determine the day of ovulation. Fresh ( n =37) and vitrified-warmed (one step ( n =100) vs. three step ( n =64) protocol) IVP embryos were transferred into sedated recipient mares (detomidine hydrochloride and butorphanol tartrate) by an experienced practitioner using the Wilsher technique [15,16] on D4 after ovulation. Pregnancy checks were performed 7 to 10 days and 35 to 38 days after ET, defined as D14 and D42 of pregnancy. Pregnancy loss was categorized as early pregnancy loss (D42). Date of birth, foaling rate and the foal’s sex were recorded. 1.7) Statistical analysis Statistical analyses were performed using RStudio (version 2023.06.0+421). Descriptive statistics were presented as mean ± standard deviation (SD). Data distribution was assessed using boxplots and bar plots. All variables (embryo age, preservation method, survival, sex, and embryonic loss) were categorical. Pearson’s Chi-squared test, Fisher’s Exact Test, and pairwise comparisons with Bonferroni correction for multiple comparisons were used to analyze categorical variables. The P-values presented in this study are the adjusted P-values. Bar plots were employed to visually inspect associations between categorical variables. Differences were considered significant when P < 0.05 with tendencies noted at P < 0.1. Results A total of 201 IVP embryos were transferred. Two vitrified-warmed (one step) IVP embryos that resulted in monozygotic twin pregnancies were excluded prior to analysis. Of the 199 embryos, 60 were transferred in 2022, 80 in 2023, and 59 in 2024. Overall, pregnancy rates at D14 and D42, the foaling rate and sex ratio (colt:filly) were: 73.9% ( n = 147), 62.8% ( n = 125), 57.8% ( n = 115) and 52.2%:47.8% (n=60:55). Slow in vitro embryo development is associated with reduced foaling rates Speed of in vitro embryo development ranged from D7 to D11 (D7 (n=17), -8 (n=73), -9 (n=51), -10 (n=38), -11 (n=20)) with a mean day of blastocyst formation of 8.7 ± 1.13 days. Pregnancy rates at D14 were not significantly affected by the day of blastocyst formation (P = 0.10) (88.2%, 81.3%, 70.6%, 65.8% and 60% for D7, -8, -9, -10 and -11; resp.) (Figure 1) . However, a significant association was found between the day of blastocyst formation and both pregnancy rate on D42 (p < 0.001) and foaling rate (p < 0.001). Post-hoc analyses with Fisher’s exact test showed a significantly higher chance of early pregnancy loss (83.3%) and reduced foaling rate (10.0%) after transfer of D11 IVP embryos compared to D7, -8, -9 and -10 (0.0%, 5.7%, 11.1%, 20.0% and 88.2%, 64.0%, 60.8%, 50.0%, respectively). Since only two D11 IVP embryos resulted in live born foals (2/20; 10%), they were excluded from subsequent analyses. Figure 1: Bars represent the mean pregnancy rate at 14 days of gestation (blue), pregnancy rate at 42 days of gestation (red), and foaling rate (green) per embryo age (D7-D11). Different lowercase letters (a, b, x, y) indicate significant differences between days of blastocyst development within each outcome measure (P < 0.05; Fisher’s exact test with Bonferroni correction). Capital letters (A) indicate no significant differences between groups for pregnancy rate at 14 days. Slow in vitro embryo development favors the birth of female offspring A significant association between embryo kinetics and the foal’s sex was observed (Fisher’s exact test, P = 0.01; Figure 2 ). Pairwise comparisons between groups revealed significant differences in the male-to-female ratio for D7 versus D10 embryos (P = 0.02) and for D8 versus D10 (P = 0.05). No other comparisons were statistically significant (P > 0.05). Figure 2: Distribution of male (blue) and female (red) offspring according to day of blastocyst formation (D7 to D10). A significant association between day of blastocyst development and sex was observed (Fisher’s exact test, P = 0.01). Pairwise comparisons showed more females than males on D10 compared to D7 (P = 0.02) and a trend toward significance compared to D8 (P = 0.05). Embryo vitrification does not affect pregnancy and foaling rate Day 7-10 IVP embryos ( n = 179) were transferred using one of the three methods: fresh ( n = 35), one step ( n = 95) and three step ( n = 49) vitrification/warming. No significant differences were observed between preservation methods for pregnancy rate at D14 ( P = 0.70) (80.6%, 72.9% and 75.5%, resp.), D42 ( P = 0.85) (68.6%, 68.4% and 69.4%), or foaling rate ( P = 0.50) (65.7%, 63.2% and 61.2%) as shown in Figure 3. Figure 3.: Pregnancy rates at D14 (blue) and D42 (red), and foaling rate (green) for transferred IVP embryos preserved using three different methods: fresh and vitrification/warming in a one and three step protocol. No significant differences were observed between preservation methods for pregnancy rate at D14 (P = 0.70), pregnancy rate at D42 (P = 0.85), or foaling rate (P = 0.50) Discussion This study reports the effect of slow in vitro embryo development (D9 and beyond) on pregnancy and foaling outcomes and the sex of the foal after transfer of fresh vs. vitrified-warmed (one- and three step protocol) IVP embryos. In line with Lewis et al. (2023), competitive initial pregnancy rates were demonstrated, which were not significantly affected by the day of blastocyst formation (ranging from 88.2% for D7 to 60% for D11 IVP embryos). This contrasts with previous studies reporting significantly lower initial pregnancy rates for slower embryos (69% for D6-9 vs. 27% after D9 IVP embryos [5] and 68% for D7 vs. 59% for D8 IVP embryos [4]), which is probably due to the smaller sample size in our study. Similar to previous studies [1,2,4], a significant decline in foaling rate with delayed blastocyst formation was observed (ranging from 88.24% for D7 to 10% for D11 IVP embryos). Claes et al. (2020) reported a 10% higher likelihood of foaling after transfer of D7 vs. D8 embryos, but foaling rates on D9 and beyond were not published. While Lewis et al. (2023) reported low foaling rates after transfer of D9 and D10 fresh IVP embryos (38.5% and 25%, resp.), our study still showed acceptable results (60.8% and 50%, resp.). These findings may be influenced by differences between IVP systems, since laboratory protocols are not standardized across centers. For example, the timing of ICSI and vitrification (morning vs. evening) determines the developmental age of the embryo, meaning that an embryo classified as “D9” in one laboratory may not correspond to the same developmental stage in another. Differences in lab scheduling and culture conditions can therefore complicate direct comparison between studies. This offers a cut-off time for IVP embryos to reach blastocyst stage specific to the production system and subsequently, complicates comparison of data among varying studies. Since only two foals were born after transfer of 20 D11 IVP embryos (10%), vitrification and transfer of D11 IVP embryos and beyond are no longer performed in our IVP system. In the present study, slow in vitro embryo development favored the birth of female offspring with significantly more fillies than colts born after transfer of D10 compared to D7 IVP embryos (P = 0.02) and a trend toward significance compared to D8 (P = 0.05). As observed in cattle [17], the speed of in vitro embryo development has been described to affect the foal’s sex too, with a higher colt:filly ratio in D7 vs. D8 equine IVP embryos [4,6]. Here, this was not significant, most likely due to our smaller sample size in the D7 IVP embryo age group. No IVP embryos beyond D8 were included in the study of Claes et al. (2020), however an increased proportion of female embryos at advanced embryonic age was suggested. Similarly, a higher incidence of female embryos was reported in Arabian and Quarter horses compared to Warmblood horses [7]. This breed difference could be attributed to the slower developmental pace of in vitro embryos in Arabian and Quarter horses. The present study further corroborates these data, with slow in vitro embryo development being associated with a significantly higher incidence of fillies. Both fresh and vitrified-warmed IVP embryos, using either a one- or three step warming protocol, were transferred with no significant differences observed in pregnancy- and foaling rates. These comparable success rates provide flexibility in the client’s choice to vitrify or immediately transfer the IVP embryos. Consistent with previous reports, vitrification of IVP embryos proved to be a reliable technique achieving high outcomes without posing the challenges intrinsic to the cryopreservation of in vivo flushed embryos [3,8,9,18]. Warming of vitrified embryos has typically been performed by serial dilutions with decreasing sugar concentrations (three step) to limit osmotic damage [8]. However, since direct warming in EHM, as first described by Canesin et al. (2020), showed high success rates and key advantages, such as eliminating the need for media preparation and allowing efficient mare-side performance, followed by immediate transfer by the veterinary practitioner, this warming method was favored. In our study, the one step protocol achieved high pregnancy (D14, D42)- and foaling rates (72.9%, 68.4%, and 63.2%, resp.), supporting its routine application within our IVP system. In contrast to Canesin et al. 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Supplementary Material File (list of figures evj.docx) Download 50.76 KB Information & Authors Information Version history V1 Version 1 13 November 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Authors Affiliations Sofie Peere 0009-0001-2390-3435 [email protected] Universiteit Gent Vakgroep Interne Geneeskunde Voortplanting en Populatiegeneeskunde View all articles by this author M. Dewulf Universiteit Gent Vakgroep Interne Geneeskunde Voortplanting en Populatiegeneeskunde View all articles by this author Emma Van den Branden Universiteit Gent Vakgroep Interne Geneeskunde Voortplanting en Populatiegeneeskunde View all articles by this author M. Papas Universiteit Utrecht Faculteit Diergeneeskunde View all articles by this author K. Broothaers 0000-0001-7804-4693 Universiteit Gent Vakgroep Interne Geneeskunde Voortplanting en Populatiegeneeskunde View all articles by this author Tine De Coster Universiteit Gent Vakgroep Interne Geneeskunde Voortplanting en Populatiegeneeskunde View all articles by this author I. Briote Universiteit Gent Vakgroep Interne Geneeskunde Voortplanting en Populatiegeneeskunde View all articles by this author M. Hedia Cairo University Faculty of Veterinary Medicine View all articles by this author D. Angel-Velez Universidad Cooperativa de Colombia - Medellin View all articles by this author M. Hoogewijs BV ReproVets View all articles by this author Jan Govaere Universiteit Gent Vakgroep Interne Geneeskunde Voortplanting en Populatiegeneeskunde View all articles by this author Katrien Smits Universiteit Gent Vakgroep Interne Geneeskunde Voortplanting en Populatiegeneeskunde View all articles by this author Metrics & Citations Metrics Article Usage 296 views 88 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Sofie Peere, M. Dewulf, Emma Van den Branden, et al. Effect of embryo developmental kinetics and vitrification on pregnancy outcome, foaling rate and sex of the foal. Authorea . 13 November 2025. DOI: https://doi.org/10.22541/au.176304279.92865708/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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