A shorter equilibration period in the VitTrans in-straw bovine embryo vitri cation method improves post- warming outcomes

Iris Martínez-Rodero Universitat Autònoma de Barcelona Facultat de Veterinària: Universitat Autonoma de Barcelona Facultat de Veterinaria https://orcid.org/0000-0001-7057-2045 Tania García-Martínez Universitat Autònoma de Barcelona Facultat de Veterinària: Universitat Autonoma de Barcelona Facultat de Veterinaria Erika Alina Ordóñez-León Autonomous University of Barcelona Faculty of Veterinary: Universitat Autonoma de Barcelona Facultat de Veterinaria Meritxell Vendrell-Flotats Universitat Autònoma de Barcelona Facultat de Veterinària: Universitat Autonoma de Barcelona Facultat de Veterinaria Carlos Olegario-Hidalgo SERIDA: Servicio Regional de Investigacion y Desarrollo Agroalimentario Joseba Esmoris IK4-TEKNIKER Xabier Mendebil IK4-TEKNIKER Sabino Azcarate IK4-TEKNIKER Manel López-Béjar Universitat Autònoma de Barcelona Facultat de Veterinària: Universitat Autonoma de Barcelona Facultat de Veterinaria Marc Yeste Universitat de Girona Teresa Mogas (  teresa.mogas@uab.cat ) Universitat de Girona https://orcid.org/0000-0002-6733-1328

A shorter equilibration period in the VitTrans in-straw bovine embryo vitri cation method improves postwarming outcomes In beef and dairy cattle, in vitro embryo production (IVP) through assisted reproductive technologies is gaining popularity as an alternative to arti cial insemination and in vivo embryo transfer to improve genetic gains. This approach also helps circumvent breeding problems such as cows that may not ovulate or show compromised fertility during periods of heat stress (reviewed by [1]). Because of the large numbers of embryos generated through in vitro technologies, the cryopreservation of these embryos has become an important topic of research. Studies have shown that in vivo-derived transferable-stage embryos of many mammalian species can be successfully preserved through conventional slow freezing. In contrast, vitri cation seems the most effective method for embryos produced in vitro, as they are highly susceptible to cryoinjury [2]. While vitri cation is simpler, faster and cheaper than slow cryopreservation methods, it requires higher concentrations of cryoprotectant agents (CPAs) which could have deleterious effects on embryo development after their warming. To minimize this effect, warming is achieved via a complex dilution procedure along with the use of a stereomicroscope to completely remove the vitri cation solution. When working under farm conditions, this procedure is especially technically demanding.
When using vitri cation technology in veterinary practice, a practical approach is needed for the warming of vitri ed embryos so that embryos can be directly and easily transferred to the uterus. So far, there have been several attempts to replace successive dilution steps with one-step in-straw cryoprotectant dilution [3][4][5][6][7][8][9][10][11][12][13][14]. However, in some of these procedures, in-straw embryo warming requires more than one dilution step and proper handling of the carrier system, demanding more accuracy when these techniques are to be used in the eld by embryo-transfer practitioners [7,9,10,12]. Using the device VitTrans designed by our group, IVP embryos are easily warmed/diluted in-straw for their transfer to recipient females in eld conditions [15]. The performance of VitTrans assessed in terms of post-warming survival rates after 24 h of culture of IVP bovine embryos is comparable to that observed with our control vitri cationwarming method [15].
For the vitri cation of a solution, a radical increase in both the cooling rate and cryoprotectant concentration is required. Unfortunately, most cryoprotectants have some negative effects, including toxicity and osmotic injury.
Although there is no consensus regarding the toxicity of penetrating CPAs, it is widely accepted that the higher their concentration and higher the exposure temperature, the greater their toxicity. Hence, any variation in exposure time prior to cooling can cause dramatic differences in cellular hydration [16,17]. In any vitri cation protocol it is accordingly important to achieve an adequate balance between obtaining a high level of dehydration and high viscosity while also avoiding toxicity. The rst step is usually an equilibration stage in a solution containing a relatively low CPA concentration, followed by ultra-short (30-90 s) exposure to a vitri cation medium with a higher concentration of cryoprotectant (usually double the initial concentration) and dehydrating agent (usually, a disaccharide, such as sucrose). Exposure to the equilibration medium may be short (e.g., 1 min followed by 25 s during vitri cation) [12,18] or last for 3 min followed by vitri cation for 25 s [9,19,20] or even longer (e.g., 10-15 min followed by vitri cation for 60 s) [21]. These durations have provided adequate blastocyst survival, and hatching and pregnancy rates.
Given this background, we hypothesized that a shorter exposure time only in the rst step of the VitTrans protocol could be a safe approach to allow for delivery of the cryoprotectants to the blastocyst, thereby minimizing the likelihood of toxicity or osmotic damage. The objective of the present study was to determine whether a rst equilibration step of the VitTrans protocol shortened from 12 min to 3 min would serve to improve the post-warming quality of vitri ed day 7 and day 8 expanded blastocysts. Outcomes were assessed in terms of survival rates, differential cell counts, cell apoptosis and relative abundances of mRNAs of genes involved in apoptosis, oxidativestress pathways, water channels, implantation and gap junctions recorded in the warmed embryos.

Chemicals and suppliers
Unless stated otherwise, all chemicals and reagents were purchased from Sigma-Aldrich (Mo, USA).

In vitro production of bovine blastocysts
Embryos were produced according to our previously established procedures [22], with minor modi cations. Brie y, cow ovaries were collected at a local abattoir and transported to the laboratory in saline solution (0.9% NaCl) at 35-37 °C. Cumulus oocyte complexes (COCs) were obtained by aspiration from 3-to 8-mm follicles, and only COCs with three or more layers of cumulus cells and a homogeneous cytoplasm were selected for in vitro maturation (IVM).
After three washes in modi ed Dulbecco's PBS (phosphate-buffered saline) (PBS supplemented with 36 mg mL − 1 pyruvate, 50 mg mL − 1 gentamicin and 0.5 mg mL − 1 bovine serum albumin, (BSA)), groups of 40 to 50 COCs were transferred to 500 µL of maturation medium in four-well plates and cultured for 24 h at 38.5 °C in a 5% CO 2 humidi ed air atmosphere. The maturation medium consisted of TCM-199 supplemented with 10% (v/v) foetal calf serum (FCS), 10 ng mL − 1 epidermal growth factor and 50 mg mL − 1 gentamicin. For in vitro fertilization (IVF), motile spermatozoa were obtained by centrifuging frozen-thawed sperm from one Asturian bull (ASEAVA, Llanera, Asturias, Spain) of proven fertility at 300 x g for 10 min on a discontinuous gradient composed of 1 mL 40% Bovipure on 1 mL of 80% BoviPure (Nidacon Laboratories AB, Göthenborg, Sweden). The underlying sperm pellet was resuspended in 3 mL of BoviWash (Nidacon International, Gothenburg, Sweden) and pelleted by centrifugation at 300 x g for 5 min. Spermatozoa were counted in a Neubauer chamber and diluted in an appropriate volume of fertilization medium (Tyrode's medium supplemented with 25 mM bicarbonate, 22 mM Na-lactate, 1 mM Na-pyruvate, 6 mg mL − 1 fatty acid-free BSA and 10 mg mL − 1 heparin-sodium salt) to a nal concentration of 2 × 10 6 spermatozoa mL − 1 . Groups of 40 to 50 in vitro matured oocytes were transferred to 250 µL of IVF medium and co-incubated with 250 µL of sperm suspension ( nal concentration of 1 × 10 6 spermatozoa mL − 1 ) at 38.5 °C in a 5% CO 2 humidi ed air atmosphere.

Embryo vitri cation and warming
Blastocysts were vitri ed using the VitTrans device and vitri cation and warming solutions as previously described by Morató and Mogas [15]. The VitTrans consists of a carrier where the embryo is loaded, a hard plastic handle and a covering straw, which protects the device from mechanical damage during storage and serves as a 0.5-mL straw for sample dilution during warming and embryo transfer. The handle has an inner channel through which warming solutions are introduced to dilute the cryoprotectant and displace the embryo to the straw for transfer (Fig. 1). The holding medium (HM) used to formulate the vitri cation-warming solutions was TCM-199 HEPES with 20% (v/v) FCS. All steps were performed under a laminar ow hood heated to 38.5 °C using a stereomicroscope to visualize each step.
Vitri cation protocol Day 7 (D7) and Day 8 (D8) blastocysts were randomly transferred to an equilibration solution (ES) consisting of 7.5% (v/v) ethylene glycol (EG) and 7.5% (v/v) dimethyl sulfoxide (DMSO) in HM for 3 min (short equilibration: SE) or 12 min (long equilibration: LE). The blastocysts were then transferred to vitri cation solution (VS) containing 15% (v/v) EG + 15% (v/v) DMSO + 0.5 M sucrose dissolved in HM. After incubating for 30-40 s, embryos (up to 2) were loaded onto the embryo attachment piece of the VitTrans device, almost all the solution removed to leave only a thin layer covering the blastocysts and the sample quickly plunged in liquid nitrogen. Subsequently, the VitTrans device was covered with the 0.5-mL plastic straw. The entire process from immersion in VS to plunging in liquid nitrogen was completed within 1 min. The loaded devices were stored in liquid nitrogen.

Warming protocol
For warming, the cover of the VitTrans inside liquid nitrogen was twisted open for 10 s to release pressure. Then, the whole VitTrans device (with its cover) was removed from the liquid nitrogen, held for 1 s in air and submerged in a water bath at 45 °C for 3 s, leaving the hard handle above the water surface. While in the water bath, a syringe containing the diluting solution (0.5 M sucrose in HM) at 45 °C was connected to the hard handle using the Luer connector. Next, the whole VitTrans device (with its cover) was removed from the water bath as the diluting solution was injected through the lumen of the device. Once the diluting solution entered the straw, the outside was dried to remove any remaining water, and the VitTrans removed from the straw. At this point, the straw containing the warmed embryos is ready for transfer.
To determine embryo survival in subsequent experiments, the cotton plug end of the straw was cut and the contents of the straw expelled into a Petri dish. Blastocysts were then transferred to the culture medium and incubated at 38 °C in a 5% CO 2

Differential staining and TUNEL
At 24 h post-warming, expanded and hatched blastocysts surviving vitri cation in each group underwent immunostaining plus the TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) assay to quantify TCN, ICM cell number, TE cell number and AR. Fresh non-vitri ed D8 blastocysts served as controls. The protocol for embryo staining was based on Vendrell-Flotats et al. [23] with some modi cations. All steps were done at 38.5 °C unless otherwise stated. Blastocysts were xed in 2% (v/v) paraformaldehyde in PBS for 15 min at room temperature (RT). After xation, embryos were washed at least three times in PBS and permeabilised in 0.01% Triton X-100 in PBS supplemented with 5% normal donkey serum (PBS-NDS) for 1 h at RT. The embryos were washed in PBS (3×) and incubated at 4 °C overnight with mouse anti-SOX2 primary antibody (1:100; Invitrogen, CA, USA) in a humidi ed chamber. Next, the embryos were washed in PBS (3×) for 20 min and permeabilised again with 0.005% Triton X-100 in PBS-NDS for 20 min. The embryos were then incubated with the secondary antibody goat anti-mouse IgG Alexa Fluor 568 (1:500; ThermoFisher, Ma, USA) diluted for 1 h in a humidi ed chamber. They were then transferred to PBS-NDS-0.005% Triton X-100 for 20 min, washed in PBS (3×) and incubated in the TUNEL () reaction mixture dilution following the manufacturer's instructions (in situ Cell Death Detection Kit, Fluorescein) for 1 h in the dark. Positive and negative control samples were included in each assay. Blastocysts exposed to DNase I for 15 min at RT served as positive controls and blastocysts not exposed to the terminal TdT enzyme served as negative controls. Embryos were then washed thoroughly in 0.005% Triton X-100 in PBS-NDS for 5 min, mounted on poly-l-lysine treated coverslips tted with a self-adhesive reinforcement ring in a 3-µL drop of Vectashield containing 125 ng mL − 1 4′,6-diamidino-2phenylindole (DAPI) (Vectorlabs, Burlingame, CA), and attened with a slide. The preparation was sealed with nail varnish and stored at 4 °C protected from light until observation within the following 3 days. Confocal images in serial sections separated by 0.5 µm were captured with a confocal laser scanning microscope (Leica TCS SP5, Leica Microsystems CMS GmbH, Mannheim, Germany) to examine the ICM cell nuclei (SOX2-Alexa Fluor 568; excitation 561 nm), cell nucleus (DAPI; excitation 405 nm) and DNA fragmentation ( uorescein isothiocyanate-conjugated TUNEL label; excitation 488 nm). TCN, ICM cell number, and apoptotic cells were analysed using Imaris 9.2 software (Oxford Instruments, UK). Individual nuclei were counted as intact (TUNEL(−); blue/red stain) or fragmented (TUNEL(+), green stain) DNA, TE cells (SOX2(−); blue stain) or ICM cells (SOX2(+); red stain) (Fig. 2). The total number of cells was calculated as the sum of the TE and ICM cells. The AR was calculated as the ratio TUNEL(+) cells/total number of cells.
RNA extraction, reverse transcription and quantitative real-time PCR Gene expression analysis was performed 24 h after warming following the procedures used for RNA extraction and real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) previously described by [24]. Blastocysts were washed three times in Dulbecco's PBS containing 0.3% polyvinyl alcohol (PVA) at 38.5 °C and then pipetted in pools of 5 embryos into 0.5 mL microtubes. Tubes were then immediately plunged in liquid nitrogen and stored at − 80 °C until further processing.
Poly-(A)-RNA was extracted using the Dynabeads mRNA Direct Extraction Kit (Dynal Biotech, Oslo, Norway) following the manufacturer's instructions with minor modi cations. For poly-(A)-RNA extraction, each pool of blastocysts was lysed in 50 µL of lysis buffer at RT for 5 min by gently pipetting, and the uid lysate was then hybridized with 10 µL of prewashed beads for 5 min at RT by gently shaking. After hybridization, poly-(A)-RNA-bead complexes were washed at RT twice in 50 µL of Washing Buffer A and two more times in 50 µL of Washing Buffer B. Next, the samples were eluted in 16 µL of Elution Buffer (Tris HCl) and heated to 70 °C for 5 min. Immediately after extraction, 4 µL of qScript cDNAsupermix (Quanta Biosciences; Gaithersburg, MD, USA) was added and the Reverse Transcription (RT) reaction carried out using oligo-dT primers, random primers, dNTPs and qScript reverse transcriptase. The RT reaction was run for 5 min at 25 °C, followed by 1 h at 42 °C to allow the RT-qPCR of mRNA, and 10 min at 70 °C to denature the reverse transcriptase enzyme. After RT, the resulting cDNA was diluted in 25 µL of Tris HCl (elution solution).
The relative abundance of mRNA transcripts was quanti ed by the qPCR method using a 7500 Real Time PCR System (Applied Biosystems, Foster City, California, USA). The qPCR reaction mix contained 10 µL of Fast SYBR Green Master Mix (Applied Biosystems, Foster City, California, USA), 1.2 µL of each primer (500 nM; Life Techno Life Technologies, Madrid, Spain) and 2 µL of cDNA template. Nuclease-free water was added to a nal volume of 20 µL.
PCR ampli cation consisted of one cycle of denaturation at 95 °C for 10 min, 45 cycles of ampli cation with a denaturation step at 95 °C for 15 seconds, an annealing step for 1 min at 60 °C (the appropriate annealing temperature of the primers) and a nal extension step at 72 °C for 40 s. Fluorescence data were acquired during the nal extension step. The identity of the ampli ed PCR product was veri ed by melting curve analysis and gel electrophoresis (on a 2% agarose gel containing 0.1 µg/mL SafeView; Applied Biological Materials, Vancouver, Canada). The melting protocol consisted of heating the samples from 50 to 95 °C and holding at each temperature for 5 s while monitoring uorescence. In each run there were three technical replicates from each of the four biological replicates per individual gene. Further, negative controls were also included for the template and reverse transcription and ampli ed by PCR to ensure no cross-contamination.
The comparative threshold cycle (Ct) method was used to quantify the relative expression of six candidate genes (BAX, BCL2L1, AQP3, SOD1, CX43 and IFNτ) in vitri ed/warmed viable blastocysts at 24 h post-warming, normalized to the endogenous control housekeeping (HK) genes PPIA and H3F3A. Fluorescence data were acquired after each elongation step to determine the threshold cycle for each sample. The threshold cycle, which is set in the log-linear phase, re ects the PCR cycle number at which the uorescence generated within a given reaction is just above background uorescence. Within this region of the ampli cation curve, a difference of one cycle is equivalent to doubling of the ampli ed PCR product. According to the comparative Ct method, the ΔCt value was determined by subtracting the mean between PPIA and H3F3A Ct values for each sample from the Ct value of each target gene of the sample for each replicate separately. Calculation of ΔΔCt involved the subtraction of the ΔCt value for the average ΔCt across all embryos per target. Fold differences in relative transcript abundances were calculated for target genes assuming an ampli cation e ciency of 100% and using the formula 2 −(ΔΔCt) . Primer sequences, amplicon size and GenBank accession numbers for each gene are provided in Table 1. The e ciency of primer ampli cation was 100%. Non-template controls were not ampli ed or returned a Ct value 10 points higher than the average Ct value for the genes. The experiment was repeated independently four times. Chicago, Illinois, USA). The data were rst checked for normality using the Shapiro-Wilk test, and for homogeneity of variances using the Levene test.
Survival rates were compared by two-way analysis of variance (ANOVA) followed by Sidak's test for pair-wise comparisons. Total cell counts, number of ICM cells, and apoptosis rate were analyzed by the three-factor general linear model. Relative transcript abundances were assessed by two-factor ANOVA followed by the post-hoc Sidak's test. When data were not normally distributed or variances were not homogenous, data were linearly transformed into arcsine square roots, square roots or logarithms. When transformed data did not ful l parametric assumptions, Kruskal-Wallis and Mann-Whitney tests were used as non-parametric alternatives. Data are expressed as means ± standard error of the mean (SEM). Signi cance was set at P ≤ 0.05.

Results
A shorter time of exposure of embryos to the equilibrium solution leads to improved embryo development (Experiment 1) Post-warming survival and hatching rates of D7 and D8 expanded blastocysts vitri ed after a short (3 min) or long (12 min) period of exposure to the equilibration solution are shown in Table 2. Vitri cation led to signi cant reductions in D7 and D8 embryo survival rates recorded at 3 h or 24 h post-warming when compared to fresh control blastocysts. While no effects of the equilibration time were observed on embryo survival assessed at 3 h postwarming, both D7 and D8 vitri ed blastocysts subjected to SE showed signi cantly higher survival and hatching rates (P < 0.05) than those blastocysts vitri ed after a longer equilibration period. Hatching rates of D7 blastocysts in the SE group did not differ from those observed for the fresh non-vitri ed blastocysts (31.4 ± 3.7 vs. 35.9 ± 4.0, respectively). However, vitri ed D8 blastocysts showed signi cantly lower hatching rates than those derived from fresh non-vitri ed embryos, regardless of SE or LE.
In addition, survival at 24 h post-warming was signi cantly higher for the vitri ed D7 blastocysts than D8 blastocysts, regardless of the equilibration period. The SE treatment signi cantly increased the hatching capacity of vitri ed D7 blastocysts when compared to vitri ed D8 blastocysts. However, no differences in hatching rates were observed between D7 and D8 blastocysts vitri ed after the LE treatment.  The outcomes TCN, ICM and TE cell numbers and AR determined 24 h post-warming of D7 and D8 expanded bovine blastocysts vitri ed after the short and long equilibration times are shown in Table 3. TCN and TE cell numbers were signi cantly lower in expanded blastocysts derived from vitri ed/warmed D7 blastocysts compared to those derived from fresh control blastocysts, regardless of the length of exposure to the equilibration solution. However, both outcome measures were similar in non-vitri ed fresh and SE-vitri ed D7 blastocysts reaching the hatching stage at 24 h post-warming, while they were signi cantly lower in LE-vitri ed D7 blastocysts. The rate of apoptotic cells was signi cantly higher in both vitri cation groups when compared to fresh controls, although vitri cation using the SE protocol produced less apoptosis than when the LE protocol was used.
No differences were observed when TCN and TE cell number were assessed at 24 h post-warming in expanded blastocysts derived from fresh D8 blastocysts or D8 blastocysts vitri ed using the SE protocol. However, both counts were signi cantly lower in expanded blastocysts derived from D8 blastocysts vitri ed using the LE protocol. ICM cell numbers in expanded blastocysts derived from vitri ed/warmed D8 blastocysts were signi cantly lower compared to control fresh blastocysts, regardless of the vitri cation protocol. A similar trend was observed for TCN, and ICM and TE cell numbers assessed in hatched blastocysts derived from vitri ed/warmed D8 blastocysts. Apoptosis rates were signi cantly higher for vitri ed/warmed D8 blastocysts when compared to non-vitri ed embryos, although the SE protocol yielded a signi cantly lower apoptosis rate than the LE protocol. Control: fresh non-vitri ed expanded blastocysts; SE: expanded blastocysts vitri ed after a short equilibration period (3 min); LE: expanded blastocysts vitri ed after a long equilibration period (12 min).

Different times of exposure to the equilibration solution modify gene expression patterns in warmed expanded blastocysts vitri ed using the VitTrans as the cryodevice (Experiment 3)
Given the effects on embryo development and embryo quality observed in our initial experiments, the effects of the shorter and longer equilibration times on the relative abundance of genes was only assessed in post-warmed expanded and hatched blastocysts derived from vitri ed/warmed D7 expanded embryos (Fig. 3). While no signi cant differences in BAX expression was observed between the two treatments, the BCL2L1 gene was overexpressed in both expanded and hatched blastocysts derived from SE-vitri ed blastocysts compared to blastocysts derived from fresh or LE-vitri ed blastocysts. mRNA transcript abundances of the SOD1 gene were signi cantly higher in blastocysts derived from SE-than LE-vitri cation although SOD1 mRNA abundances in both vitri cation groups did not differ from those detected in blastocysts derived from fresh non-vitri ed blastocysts. No differences in AQP3, CX43 and IFNτ transcript abundances were observed between treatments. However, expanded and hatched blastocysts derived from blastocyst vitri ed using the SE protocol showed a clear trend (P = 0.07) towards higher CX43 and AQP3 gene expression levels compared to expanded and hatched blastocysts vitri ed using the LE protocol. When gene expression was compared between blastocyst stages, hatched blastocysts derived from vitri ed blastocysts had higher CX43 expression and lower IFNτ expression than their expanded counterparts, while no differences between the two stages were observed for the other genes.

Discussion
To effectively transfer vitri cation technology to the eld, the procedures used for the warming and transfer of cryopreserved bovine embryos should be kept as simple as possible. The VitTrans device was designed to facilitate the vitri cation/warming technique by including an easy one-step in-straw dilution method followed by direct embryo transfer to the uterus [15]. While we have reported post-warming survival rates of around 60% for D7 expanded blastocysts vitri ed using VitTrans, here we modi ed the two-step vitri cation protocol to improve post-warming outcomes. The objective of this study was to investigate the effects of different equilibration times on several postwarming outcome measures in bovine D7 and D8 expanded blastocysts vitri ed using the VitTrans procedure. Our results indicate that a short equilibration time (3 min) during vitri cation improves post-warming survival and the hatching ability of both D7 and D8 expanded blastocysts, whereas lengthening the equilibration time to 12 min does not seem to offer any further bene ts. In addition, the hatching rates of D7-blastocysts vitri ed by the SE protocol were similar to those recorded for fresh non-vitri ed embryos. Several studies have compared equilibration times used in the vitri cation of in vitro produced blastocysts of different species [25][26][27][28][29]. In cattle, Do et al. [27] found similar re-expansion (24 h post-warming) and hatching rates (48 h post-warming) when bovine expanded blastocysts were vitri ed after a short (3 min) or long equilibration (8 min) time possibly explained by differences in temperature and equilibration times. Thus, while the short equilibration time tested by Do et al. [27] was similar to ours (3 min at 37 °C), their long equilibration protocol consisted of 8 min at room temperature, which may have resulted in reduced cytotoxicity and osmotic stress [16] and thus minimized differences between the use of their short or long protocol. Consistently, in a study carried out in the dromedary camel, loading of CPAs at 37 °C for a short exposure time (3 min) led to an outcome comparable to that of original processing at room temperature with a longer exposure time (15 min) [26]. When working at room temperature in humans and mice, different equilibration times did not affect post-warming embryo survival [25,28]. However, lengthening the exposure time to the equilibration solution from 4 to 8 min was found to improve the DNA integrity index after the vitri cation of murine blastocysts [25,28]. Prior to the vitri cation of human blastocysts, 9-10 min of exposure to the equilibration solution improved the outcomes clinical pregnancy, embryo implantation and live birth rates compared to shorter exposure times [25].
Different vitri cation outcomes have been recently reported after vitri cation of expanded blastocysts using various one-step warming devices and short equilibration times. As we have also observed, one-step in-straw warming/dilution of expanded blastocysts vitri ed in berplugs returned similar [9] or higher survival rates [20] for D7 than D8 blastocysts. However, either lower [9] or higher [20] hatching rates were observed at 24 h post-warming when D7 or D8 expanded blastocysts were vitri ed in berplugs compared to our results. Further, one-step warming of bovine D7 expanded blastocysts vitri ed on hand-pulled glass micropipettes as the cryodevice led to higher hatching rates assessed at 72 h post-warming rather than 24 h post-warming [12,19].
When 24 h post-warming outcomes were compared after the vitri cation of blastocysts produced after different times of in vitro culture, our results are consistent with those of others. Thus, signi cantly higher survival, re-expansion and hatching rates have been described after the vitri cation of D7 compared to D8 IVP bovine blastocysts [20,21,30,31] such that cryotolerance diminishes as the length of embryo culture increases. In the present study, although the hatching ability of D7 blastocysts vitri ed/warmed within the SE protocol was comparable that of fresh non-vitri ed D7 blastocysts, Day 8 vitri ed/warmed blastocyst gave rise to under half of the hatching yield observed in the fresh control group. Early developing embryos are better at surviving than later embryos. This has been highlighted in prior work in which vitri ed/warmed earlier cryopreserved IVP bovine blastocysts returned higher survival, hatching and pregnancy rates [20,21].
The correct distribution of cells in the ICM and outer TE layer of the blastocyst is crucial for embryo development. However, while it is accepted that a minimal number of embryonic cells is needed to establish pregnancy [32], optimal ICM and TE cell numbers and distributions in the blastocyst remain unclear. Thus, higher ICM cell counts may lead to increased pregnancy rates [33] and an excessive number of cells allocated to the TE may lead to pregnancy abnormalities [34,35]. Here, the TUNEL assay combined with differential staining for ICM and TE cells revealed signi cantly lower TCN and TE-cell numbers and a higher apoptosis rate in vitri ed/warmed D7 re-expanded blastocysts compared to fresh ones while no differences emerged in ICM cell numbers, suggesting that the main site of cryopreservation-related membrane damage was the trophectoderm. Similar [36] or reduced total cell counts have been reported after bovine embryo vitri cation [37,38], mainly due to a low cell count in the TE. This effect is consistent with a greater accumulation of lipids in the TE than ICM [39], as cytoplasmic lipid contents appear strongly related to survival of cryopreservation [2]. In contrast, Gomez et al. [40] described that vitri cation seemed to exert a detrimental effect on the ICM, while TE cells survived cryopreservation in numbers comparable to those counted in embryos before vitri cation. However, we detected no differences in TCN, or in ICM and TE cell numbers between D7 blastocysts vitri ed after SE and fresh blastocysts, while D7 blastocysts vitri ed after LE showed signi cantly lower TCN and numbers of TE cells. This suggests that D7 expanded blastocysts vitri ed using our SE protocol suffered less cryodamage or were able to recover from any damage at 24 h post-warming, showing similar hatching rates and embryo quality as those of fresh ones. However, among the D8 embryos subjected to vitri cation/warming, TCN, and ICM and TE cell numbers were signi cantly lower in hatching blastocysts when compared to fresh blastocysts, regardless of the equilibration time. The timing of blastocyst formation is a good marker of embryo quality determining that early-cavitating embryos are of better quality than later cavitating embryos in terms of total cell numbers, inner cell mass and trophectoderm cell distributions, and cryosurvival [30,34]. While we still lack reliable blastocyst stage morphological predictors of competence after embryo transfer, it is accepted by many research groups and commercial companies that best pregnancy rates are achieved after the transfer of day 7 expanded bovine blastocysts whether fresh or cryopreserved (reviewed by [41,42]).
Apoptosis has been frequently used as a marker for embryo quality as high rates of apoptotic cells have been linked to the reduced developmental competence of both in vivo or in vitro produced embryos [43][44][45]. Vitri cation requires adequate dehydration and a high viscosity across all blastomeres and blastocele which is di cult given the characteristics of the blastocyst (multicellularity, presence of blastocele with high water content). This determines that vitri cation leads to a post-warming increase in apoptosis [46]. Our results revealed that both equilibration solution exposure times induced apoptosis in surviving blastocysts by the time of their re-expansion and hatching. However, while the apoptosis rate for D7 expanded blastocysts vitri ed via the VitTrans LE protocol was similar to that reported previously by Morató and Mogas [15], the apoptotic cell rate was signi cantly higher for the LE than SE protocol or control embryos. This nding suggests that the high toxicity effect of CPAs produced at high temperature can be avoided to some extent by reducing the time of exposure to the cryoprotectant [16]. Moreover, D8 embryos induced higher percentages of apoptotic cells that D7 embryos, in agreement with results observed when expanded blastocysts were vitri ed/warned using a one-step direct transfer procedure [20].
When genes related to apoptosis were analysed, a signi cantly higher abundance of BCL2L1 transcripts was observed in both expanded and hatched blastocysts derived from the SE protocol when compared to fresh embryos or those vitri ed after the long equilibration period, while there were no differences in BAX gene expression among treatments. Yang and Rajamahendran [47] related a higher expression level of Bcl-2 to better quality embryos less prone to apoptosis. However, the levels of BCL2L1 gene expression observed in our study were inconsistent with apoptosis levels assessed by TUNEL in fresh or vitri ed D7 blastocysts, suggesting that apoptosis detected by TUNEL is independent of the expression of BCL2L1 or BAX genes, as observed previously [48]. Similarly, mRNA levels of SOD1 were upregulated after SE treatment, indicating that a shorter exposure time may reduce oxidative stress by improving the activity of antioxidant enzymes and improving the quality of vitri ed/warmed embryos [49]. In addition, a trend although not signi cant (P = 0.07) was observed towards greater CX43 and AQP3 gene expression in blastocysts subjected to SE compared to LE. In effect, high expression of CX43, a gene related to cell compaction and adhesion [50], has been linked to better quality and more cryotolerant embryos [51]. AQP3 plays an important role in the transport of cryoprotectants and uids during the cryopreservation of bovine embryos [52]. The presence of mRNA encoding this protein has been also related to better embryo cryotolerance [53]. While not always signi cant, the differences in gene expression observed in surviving blastocysts derived from D7 blastocysts vitri ed after the SE treatment could be indicative of better embryo quality. In effect, these blastocysts showed an improved hatching ability together with higher TCN, and TE cell numbers and a lower apoptosis rate.

Conclusions
In conclusion, vitri cation of IVP D7 bovine embryos using the VitTrans device with a brief 3 min exposure to the equilibration solution gave rise to post-warming outcomes comparable to those of fresh non-vitri ed blastocysts. In addition, our gene expression analysis indicated that the SE treatment could lead to the production of more highquality blastocysts, promoting the e ciency of embryo transfer. This strategy of shortening the exposure time to the equilibration medium within the VitTrans procedure could have important implications for commercial in vitro embryo transfer programs simplifying the use of this technique in eld conditions. Future experiments are underway to establish the full survival potential of these cryopreserved embryos after their transfer to recipient cows.

Availability of data and material
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.  (1) showing the out ow of the inner channel (5) and embryo attachment piece (6). Scale bar: 1 cm.