Novel Synthetic oviductal fluid for Conventional Freezing 1 (SCF1) culture medium improves development and cryotolerance of in vitro produced Holstein embryos

Abstract

In vitro produced (IVP) embryos hold great promise in the cattle industry; however, suboptimal in vitro culture conditions induce metabolic dysfunction, resulting in poor development and low cryotolerance of IVP embryos. This limits the use of IVP embryos in the cattle industry for embryo transfer and commercial scale-up. Previous studies have reported the use of individual metabolic regulators in culture media to improve blastocyst development rates and cryopreservation. In this study, we hypothesized that using a combination of select regulators, chosen for their unique synergistic potential, would alleviate metabolic dysfunction and improve the development of in vitro produced embryos to make them more closely resemble in vivo derived embryos. To test this, we first compared lipid content between Holstein and Jersey embryos produced in vivo and in vitro, and then systematically determined the combination of metabolic regulators that led to the greatest improvements in embryonic development, lipid content, mitochondrial polarity, and cryotolerance. We also tested different slow freezing techniques to further improve cryotolerance and finally validated our results via a clinical trial. Overall, we found that the use of multiple metabolic regulators in one culture media, which we refer to as Synthetic oviductal fluid for Conventional Freezing 1 (SCF1), and an optimized slow freezing technique resulted in improved pregnancy rates for frozen IVP embryos compared to embryos cultured in a synthetic oviductal fluid media. Additionally, there was no difference in pregnancy rate between frozen and fresh IVP embryos cultured in SCF1. This suggests that optimizing culture conditions and slow freezing technique can produce cryotolerance IVP and should allow further dissemination of this assisted reproductive technology.

Lay Summary

In vitro produced (IVP) bovine embryos suffer from several physiological abnormalities that interfere with their ability to withstand the freezing process, a vital step in shipping and distribution of IVP embryos. To overcome these challenges, we performed a series of experiments to determine the optimal culture medium to best support the developing embryo. This new in vitro embryo culture medium is referred to as Synthetic oviductal fluid for Conventional Freezing 1 (SCF1). The medium is supplemented with various factors to more closely mimic the uterine environment, improve mitochondrial function, and decrease lipid accumulation. The results show that IVP embryos cultured in SCF1, slow frozen using an optimized technique, and transferred into recipients have a pregnancy rate that is similar to non-frozen IVP embryos. These findings suggest that SCF1 improves developmental competence of bovine IVP embryos and their ability to withstand cryopreservation, which can improve pregnancy rates and efficiency of assisted fertility operations within the dairy cattle industry.

This study shows that the use of a combination of metabolic regulators in in vitro bovine embryo production can enhance development and cryotolerance of embryos and result in higher pregnancy rates after transfer.

Introduction

Recent advances in assisted reproductive technologies have increased the production of bovine in vitro produced (IVP) embryos, with over 1 million IVP embryos transferred worldwide in 2019 (Viana, 2019). IVP embryos are favored over in vivo derived (IVD) embryos as oocytes can be retrieved from young and pregnant donors decreasing the generation interval for genetic gain (Hansen, 2006). Additionally, by cryopreserving IVP embryos, producers can transfer genetics throughout different herds and time embryo transfers to improve efficiency and increase their production capacity. Although promising, IVP embryos differ from IVD embryos and suffer from metabolic dysfunction, poor blastocyst development rates, and low cryotolerance (Seidel, 2006). Altogether, this decreases the success of IVP embryos and limits their potential in the cattle industry.

The decrease in IVP embryo quality is likely due to improper culture conditions, which are unable to mimic in vivo conditions and lead to altered morphology, gene expression, and metabolism of IVP embryos (Seidel, 2006). Throughout embryonic development, the embryo relies on different substrates for energy production. In the early 1-8 cell stage, pyruvate is the main substrate used for oxidative phosphorylation (Thompson, 2000); however, as the blastocoel cavity forms, glucose becomes the preferred substrate. During the progression from early (6) to late (7) stage blastocysts, the embryo uses free fatty acids as an energy source and lipid content depletes (Thompson, 2000), and thus, culture medium may have different effects during these transitional stages. Embryo culture media, such as synthetic oviductal fluid (SOF), do not adequately provide all the proper nutrients required for each stage of the developing embryo (Tervit et al., 1972). To address this shortcoming, sequential culture media were formulated to support the developing embryo’s energy requirements at different stages of development (Thompson and Peterson, 2000); however, there is insufficient evidence to suggest that it improves developmental outcomes (Reed et al., 2009; Sfontouris et al., 2016; Dieamant et al., 2017). Formulations of culture media have also been modified through the addition of amino acids, bovine serum albumin, and cytokines to improve development (Gandhi et al., 2000; Stoecklein et al., 2021). Despite these advances, culture medium is still unable to sufficiently mimic in vivo conditions and results in impaired embryonic development.

The most evident hallmark of IVP embryo development is altered metabolism, likely due to improper energy substrate utilization during development, and is marked by mitochondrial dysfunction and abnormal lipid accumulation (Barceló-Fimbres and Seidel, 2007; Paschoal et al., 2017). Dysfunctional mitochondria in the developing embryo leads to an accumulation of reactive oxygen species (ROS), which induces oxidative stress that damages DNA and organelles and increases lipid peroxidation (Li et al., 2016). IVP embryos are more lipid-laden than IVD embryos (Seidel, 2006; Barceló-Fimbres and Seidel, 2007). Increased lipid content is associated with poor cryopreservation outcomes perhaps through increased lipid peroxidation increasing oxidative stress post-thaw (Barceló-Fimbres and Seidel, 2007), or by altering the composition of cellular membranes and making them more sensitive to cryopreservation (Shehab-El-Deen et al., 2009). Decreasing lipid content through mechanical or chemical (Men et al., 2006; Barceló-Fimbres and Seidel, 2007) delipation improves cryotolerance, which suggests that decreasing lipid content directly impacts cryotolerance. However, mechanical delipation is too labor-intensive to incorporate into commercial production. Additionally, there are concerns regarding the toxicity of reagents used in chemical delipation. Thus, there is a need for a novel strategy to reduce embryonic lipid content.

Supplementing conventional culture media with additives and metabolic regulators during different stages of development to supplement embryo metabolism has been investigated as a method to reduce lipid accumulation and improve the development and cryotolerance of IVP embryos. The addition of conditioned medium from Vero cells (CM) during the initial stages of culture exposes embryos to signaling factors, hormones, and growth factors that would otherwise be lacking in a traditional in vitro culture system has also shown promising results in improving embryonic metabolism and development (Barcelo-Fimbres et al., 2015). Another promising mechanism is to add regulators that promote lipid metabolism. Adding l-carnitine facilitates the transport of fatty acids into the mitochondria, but also scavenges ROS to prevent oxidative stress that might occur from increased fatty acid metabolism (Sutton-McDowall et al., 2012, Baldoceda et al., 2015a, Takahashi et al., 2013). Thus, the addition at of L-carnitine at early stages of IVC, specifically at day 0, has previously shown to decrease lipid content and enhance cryosurvival (Baldoceda et al., 2015a, Takahashi et al., 2013). Another promising enhancer of lipolysis is forskolin, which activates lipase enzymes and has been shown to be beneficial for cryopreservation when added to early and late stage blastocysts when free fatty acids are the preferred energy source (Sanches et al., 2013, Paschoal et al., 2017). An alternative route to improving embryonic metabolism is to repair dysfunctional mitochondria, which may reduce lipid peroxidation, subsequently improving development and cryotolerance. Coenzyme Q (CoQ) and Vitamin K₂ (VitK) serve as electron carriers to improve mitochondrial function and reduce lipid peroxidation and increase bovine blastocyst rates when added to culture medium. This effect was shown when these substances were added after compaction, when glucose becomes the preferred energy substrate (Stojkovic et al., 1999; Baldoceda-Baldeon et al., 2014; Sefid et al., 2017). Although all additives have shown to be beneficial when used alone, combining all additives may work synergistically to improve the culture conditions and enhance IVP embryo development.

Improving the slow freezing technique could be an additional way to enhance IVP embryo cryotolerance. Slow-freezing is the preferred method of cryopreservation in the cattle industry due to the ease of transfer in the field; however, ice crystal formation, osmotic shock, lipid peroxidation, and other causes lead to low success rates (Seidel, 2006; Ferré et al., 2020). Standard slow freezing procedure involves equilibrating embryos in ethylene glycol (EG) for 10 min before being loaded into straws (Hasler, 2001). For slow freezing to be successful, water must be drawn out of the cells by cryoprotectants to prevent intracellular ice crystal formation (Inaba et al., 2016). Multiple studies have investigated ways to improve embryo survival by collapsing the blastocoel cavity before freezing to reduce water content inside the embryos and prevent ice crystal formation. However, those studies show that pregnancy rates are still lower than fresh embryos (Min et al., 2014). Alternatively, studies have used a lower concentration of EG surrounding the embryo to decrease osmotic shock initiated during thawing (Seidel, 2006; Sanches et al., 2016), but the resulting pregnancy rates are still lower than IVD embryos. Other possible mechanisms to increase dehydration before slow freezing are the addition of an additional extracellular cryoprotectant such as sucrose (Chaytor et al., 2012), or by increasing the time at which embryos are exposed to cryoprotectants to further dehydrate them.

Our objectives in this study were to 1) compare the lipid content of in vivo and in vitro produced Holstein and Jersey embryos, 2) determine the optimal culture conditions to improve embryonic development and metabolism, 3) optimize slow-freezing techniques to improve cryosurvival, and 4) validate these results in vivo with clinical pregnancy data. We hypothesized that a select combination of metabolic regulators, used at concentrations that have been shown to be beneficial to embryonic development when used alone, would improve embryonic development and metabolism to be more similar to IVD embryos and, in combination with optimized slow freezing techniques, would lead to higher pregnancy rates compared to standard IVP frozen embryos.

Materials and Methods

Animals and handling

All procedures involving animals were approved by the Animal Care and Use Committee of California Polytechnic State University, San Luis Obispo (San Luis Obispo, CA).

Experiment 1: Comparison of lipid levels of in vitro produced and in vivo derived Holstein and Jersey embryos

Experimental design

The first experiment was designed as a 2 × 3 factorial model to compare lipid content between two breeds (Jersey and Holstein) and three embryo production methods (IVD, IVP, and IVP with the addition of forskolin and L-carnitine). Data were analyzed by ANOVA, and differences between groups were compared using Tukey’s HSD for multiple comparisons.

IVD embryo production

Holstein and Jersey cows were superovulated according to the MoFA Pluset protocol. Cows were fitted with a controlled internal drug release device (EAZI-BREED CIDR Cattle insert, Zoetis USA) on stimulation day 0. On stimulation day 2, 86 µg/mL GnRH (Fertagyl; Intervet Inc, Summit, NJ) was administered intramuscularly. Cows then received 700 IU or 575 IU follicle stimulating hormone (FSH) (Pluset Flex H; MoFA, Verona, WI), respectively, in 8 decreasing doses on days 4 to 8 via intramuscular injection. On injections 6 and 7, 250 mg/mL prostaglandin (Estrumate, Intervet Inc, Summit, NJ) was also administered. Cows were monitored for signs of heat and artificially inseminated with frozen-thawed Holstein or Jersey semen twice on day 9, approximately 12 h apart.

On day 17 of the cycle, a non-surgical embryo flush was performed according to standard procedures (Seidel and Seidel, 1991) to collect day 8 IVD embryos. Briefly, cows were rectally palpated and a two-way foley catheter connected to an embryo collection cup was inserted into the vagina, through the cervix, into the uterus. The uterine body was flushed with a continuous flow method using 1 to 2 liters of flush solution. The flush solution was emptied through a mesh filter into an embryo collection cup and recovered embryos were immersed into a pH stable holding solution.

IVP embryo production

Holstein and Jersey ovaries were collected from a local abattoir and transported to the laboratory 2 h away at room temperature in 0.15 M saline. Cumulus oocyte complexes were aspirated from 2 to 8 mm follicles using an 18 g needle via vacuum pump aspiration. Oocytes with at least three layers of cumulus cells and an even cytoplasm were matured in maturation media (TCM-199, 10% fetal calf serum [FCS], 4.03 mM pyruvate, 25 µg/mL gentamicin, 5 mIU FSH, and 0.5 mM l-carnitine) and cultured for 22 to 24 h at 38.5 °C and 5% CO₂ in air.

Sperm from two fertility-proven bulls per breed were processed through a 40/80 Sperm Talp Percoll (Sigma, P1644) gradient and co-incubated with mature oocytes for 16 to 24 h in fertilization medium (FCDM) (Mendes et al., 2003). Presumptive zygotes were vortexed to removed cumulus cells and placed in SOF-based commercially available culture media (BBH7, BoviPro, MOFA Global, WI) supplemented with 1.5 mM l-carnitine at 38.5 °C in 5% O₂, 6% CO₂ until day 7. Forskolin (10 µM) was added to the culture media on day 5 post-insemination.

Lipid quantification

IVD embryos and day 7 IVP blastocysts were fixed in 10% formalin in PBS + 0.01% PVA solution or 100% ethanol as a negative control for at least 16 h in the dark at room temperature. Embryos were washed to remove the fixative before being stained with 1 µg/mL Nile red dye in PBS for 30 min in the dark at room temperature. Embryos were washed once more to remove excess dye. Images were acquired along the equatorial plane of the embryo at 40× magnification using a widefield fluorescent microscope equipped with a FITC filter (Zeiss Axioskop 40, Oberkochen, Germany). Images were analyzed by determining the mean fluorescence of the embryo and subtracting the background to calculate the corrected mean total fluorescence in 10 representative images in IMAGE J software (NIH).

Experiment 2: Effects of sequential media, embryonic stage, and individual metabolic regulators on embryonic metabolism

Experimental design

This experiment was designed in two parts. For part 1, a 2 × 2 × 2 factorial model was used to compare culture systems (continuous vs. 3-step sequential), blastocyst stage (stage 6 [early] vs. stage 7 [late]), and cumulative additives (no additives vs. L-carnitine [0.5 mM at day 1], plus CoQ [30 μM at day 3], plus VitK [0.5 mM at day 3], plus forskolin [10 μM at day 5]). Total lipid content was evaluated in grade 1–2 embryos from each treatment.

For part 2, a 2 × 2 × 2 × 2 factorial model was used to compare each additive (0 vs. 0.5 mM L-carnitine at day 1, 0 vs. 30 μM CoQ [Homogenous liposomal ubiquinol, Tishcon Corp, Westbury, NY]) at day 3, 0 vs. 0.5 mM VitK (V9378; Sigma) at day 3, and 0 vs. 10 μM forskolin at day 5) as well as all combinations of these additives. Mitochondrial polarity was used to evaluate late-stage (stage 7), grade 1–2 blastocysts from each treatment group.

IVP embryo production

IVP embryos were produced as described above, with the following modifications. Holstein ovaries were collected from an abattoir (Cargill Meat Solutions., Inc, Fresno, CA) and cooled to 22–25 °C in 0.15 M saline during transportation. Oocytes were then fertilized in FCDM for 16 to 24 h at 38.5 °C in 5% CO₂ in the air, and then vortexed to remove cumulus cells. Presumptive zygotes were randomly assigned to either continuous medium (SOFaaci [Holm et al., 1999] supplemented with 1 mM glucose for 7–8 d) or sequential medium (SOFaaci supplemented with non-essential amino acids, 0.2 mM glucose, 0.33 mM pyruvate, and 6 mM lactate, pH 7.34 +/− 0.02 for days 1–4, SOFaaci supplemented with essential and nonessential amino acids, 0.5 mM glucose, 0.10 mM pyruvate, and 1 mM lactate, pH 7.26 +/– 0.02 for days 4–6, and SOFaaci supplemented with essential and nonessential amino acids, 1 mM glucose, 0.10 mM pyruvate, and 1 mM lactate, pH 7.17 +/– 0.02 for days 6–8). Both media were made in house, and preliminary studies showed no difference in blastocyst rate or re-expansion rate between BBH7 (used in experiment 1) and continuous SOF medium used in this experiment (unpublished results). Both treatments were cultured in 5% O₂ and 6% CO₂ and 90% N₂ at 38.5 °C.

Addition of metabolic regulators

After insemination, presumptive zygotes were randomly assigned to a culture medium that contained either 0 or 0.5 mM l-carnitine. CoQ was added at day 3 after insemination at a final concentration of 30 μM. VitK powder was dissolved in 95.5% ethanol and added to embryo culture groups on day 3 after insemination at a final concentration of 0.5 mM. Forskolin (344270; Sigma) was dissolved into dimethyl sulfoxide solution and added at a final concentration of 10 μM on day 5 after insemination.

Lipid quantification

In part 1, embryos were stained with Nile Red dye and imaged as described in experiment 1. For part 2, images were acquired by confocal microscopy at 40× magnification (Olympus Fluoview, Tokyo, Japan). Stage 7, grade 1-2 blastocysts were fixed in 3.9% paraformaldehyde a minimum of 16 h in the dark at 4 °C. Following fixation, blastocysts were washed three times in PBS-PVP and counter-stained with 1 μg/mL DAPI for 15 min in the dark. Embryos were then stained with 1 µg/mL Nile Red for 30 min. Finally, embryos were washed 3 times in PBS-PVP and then placed in PBS-PVP for image acquisition to preserve the 3-dimensional structure. Twenty images were taken at a 5 µm step size from the equator of the embryo to the bottom, such that only half of the embryo was imaged at 40× magnification on a confocal microscope (Olympus Fluoview, Tokyo, Japan). Images were analyzed by determining the mean fluorescence of the embryo and subtracting the background to calculate the corrected mean total fluorescence in 10 representative images using IMAGE J software (NIH).

Mitochondrial polarity

Stage 7, grade 1–2 blastocysts were stained for mitochondrial polarity using 300 nM Mitotracker Red CMX-Rosamine (ThermoFischer, Waltham, MA) for 50 min in culture conditions. Blastocysts were washed through PBS-PVP, and then fixed in 3.9% paraformaldehyde a minimum of 16 h in the dark at 4 °C. Following fixation, blastocysts were counter-stained with 1 μg/mL DAPI for 15 min in the dark and then transferred to the confocal microscope in PBS-PVP. Twenty images were taken at ×40 magnification with a 5 μm step size from the equator of the embryo to the bottom. Images were analyzed by determining the mean fluorescence of the embryo and subtracting the background to calculate the corrected mean total fluorescence in 10 representative images using IMAGE J software (NIH).

Experiment 3: Effects of metabolic regulators on embryonic development and metabolism

Experimental design

This experiment was separated into two parts. In part 1, a 2 × 2 factorial model was used to compare the effects of CM (0% vs. 5% on day 1 after IVF) and l-carnitine (0 mM vs. 0.5 mM on day 1 after IVF). Blastocyst rate, lipid content, mitochondrial polarity, cryopreservation survival rate, and percentage of apoptotic cells after re-expansion were used to evaluate embryos from each treatment group. The experiment was performed in 8 replicates with cryopreservation performed in 6.

For part 2, a 2 × 2 × 2 factorial model was used to compare the effects of CM (0% vs. 5% on day 1), VitK (0 mM vs. 0.5 mM on day 3), and forskolin (0 μM vs. 10 μM on day 5). l-carnitine was added at a concentration of 0.5 mM on day 1 after IVF in all treatment groups. Embryos were evaluated based on blastocyst rate, lipid content, mitochondrial polarity, cryopreservation survival rate, and percentage of apoptotic cells after re-expansion for each treatment group. The experiment was performed in 8 replicates with cryopreservation performed in 4.

Vero cell conditioned media

Vero cells (African Green Monkey kidney cells, Cercopithecus aethiops) were seeded at a concentration of 1 × 10⁵/mL using Tissue Culture Medium 199 (Life Technologies, Inc., Grand Island, NY) supplemented with 5% chemically defined FBS replacement (MB Biomedicals), which is free of hormones, cytokines, and fatty acids. Cells were incubated at 37 °C in humidified air containing 5% CO₂. The cells had a total of 2 passages and were cultured for 48 h after the last passage. Conditioned media was aspirated from the wells, filtered, and stored at −80 °C until use.

IVP embryo production

IVP embryos were produced in the continuous medium as described above, with the following modifications. Metabolic regulators (5% CM and 0.5 mM L-carnitine were added at day 1, 0.5 mM VitK on day 3_, and 10 µM forskolin on day 5) were added as indicated throughout the experiments. Blastocyst rate was assessed on days 7 and 8 post-insemination, and stage 7, grade 1-2 blastocysts were used for subsequent analysis.

Lipid quantification and mitochondrial polarity assessment

Stage 7, grade 1-2 blastocysts were assessed for lipid content and mitochondrial polarity using confocal microscopy as described in experiment 2.

Slow freezing and thawing

Stage 7–9 (late blastocysts, hatching blastocysts, or expanded hatched blastocysts), grade 1–2 blastocysts were slow frozen following a 10-min equilibration in 1.5 M EG with 0.5 M sucrose. Blastocysts were loaded into 0.25 cc straws and then placed into the cryo-chamber (Crysalys C2346S, Napa Valley, CA) at −6 °C. Embryos were held for one minute at −6 °C, then seeded, and held for another 9 min before they were cooled at a rate of −0.6 °C per minute to a final temperature of −33 °C before being plunged in liquid nitrogen. Straws were stored in liquid nitrogen until thawing. Straws were thawed in the air for 10 s followed by 30 s in 32–35 °C water. Straw contents were expelled, and blastocysts were placed into culture media and assessed for re-expansion at 24 and 48 h post-thaw.

TUNEL assay

Re-expanded blastocysts were assessed for apoptosis using a TUNEL assay (ThermoFisher, Waltham, MA). Blastocysts were washed three times in PBS-PVP and fixed in 3.9% paraformaldehyde for 3 to 7 d. Following fixation, blastocysts were washed three times in PBS-PVP and permeabilized in 0.5% Triton X for 30 min at room temperature. Blastocysts were washed three more times in PBS-PVP and then stained with TUNEL in the dark at 38.5 °C and 85% humidity. Blastocysts were counter-stained with 1 μg/mL DAPI for 15 min in the dark, then transferred to the confocal microscope in PBS-PVP, and imaged at 40× with 20 images per embryo taken at a 5 μm step size from the equator to the bottom of the embryo. Ten images per embryo were analyzed by counting total and apoptotic cells and calculating the percent of apoptotic cells.

Experiment 4: Effects of novel media and improved slow freezing techniques on embryo cryotolerance

Formulation of novel culture medium

Based on the previous experiments, we determined the components of an optimum culture medium and call it Synthetic oviductal fluid for Conventional Freezing 1 (SCF1). The continuous culture medium is supplemented with 5% CM, 0.5 mM l-carnitine and 0.5 mM VitK on day 1, and 10 µM forskolin on day 5. In contrast to previous results (Baldoceda-Baldeon et al., 2014), there was no difference in embryonic development when VitK was added on day 1 or 3 (results not shown), so it was therefore added on day 1 to avoid unnecessary embryo handling.

Experimental design and statistical model

Experiment 3 was designed as a 2 × 2 × 2 factorial model to compare effects of culture media (SOF vs. SCF1), sucrose dehydration (0 vs. 0.6 M), and equilibration time of embryos in EG (10 vs. 20 min). Statistical analysis was performed in SAS (SAS Institute, Cary, NC). Blastocyst rate and re-expansion data were analyzed via ANOVA, and groups were compared with Tukey’s HSD correction for multiple comparisons. Mitotracker, Nile Red, and TUNEL analysis data were analyzed for normality using the Shapiro Wilk test, log transformed as appropriate, and analyzed via ANOVA and groups were compared with Tukey’s HSD correction for multiple comparisons.

IVP embryo production, lipid quantitation, and mitochondrial polarity assessment

Embryos were produced and lipid content and mitochondrial polarity were assessed using confocal microscopy as previously described in experiment 3.

Slow freezing

Stage 7–9 blastocysts were dehydrated in 0 or 0.6 M sucrose for 2 min and then equilibrated in 1.5 M EG with 0.5 M sucrose for 10 or 20 min before slow freezing. Following treatment, blastocysts were slow frozen, thawed, assessed for re-expansion, and post-thaw apoptosis as described in experiment 3.

Experiment 5: Effect of novel culture media and slow freezing technique on pregnancy rates

Experimental design

The clinical trial was designed as a 2 × 2 factorial model to compare the effects of two culture media (SOF vs. SCF1) and cryopreservation (fresh vs. slow-freezing) on pregnancy rates after embryo transfer. Data were analyzed with ANOVA and Tukey’s HSD.

Embryo transfer

Client-owned Holstein dry cow donors (n = 55) were selected for follicular wave synchronization, FSH stimulation, and follicular aspiration as described previously (Barceló-Fimbres et al., 2015). Briefly, donor cows received 86 µg GnRH IM (Fertagyl) and a CIDR intravaginal insert on stimulation day 0 am. On stimulation day 2 am, 300 IU FSH (Folltropin; Vetoquinol USA, Inc. Ft. Worth, TX) was administered intramuscularly in five 60 IU injections every 12 h. Forty-eight to 50 h after the last FSH injection, the CIDR was removed and the donor cows underwent follicular aspiration (142 cycles). The resulting oocytes were matured, fertilized, and cultured in SOF or SCF1 and cryopreserved (20-min equilibration in EG) as in experiment 4 using 37 different bulls selected by the clients. The resulting embryos were either transferred fresh or frozen for direct transfer as in experiment 4. Embryos were transferred by one experienced technician into virgin Holstein heifers 13 to 15 mo of age in two adjacent locations. Estrus synchronization of recipients took place weekly using the 5-d CIDR-synch protocol [day 0: 86 µg GnRH IM (Fertagyl) and a CIDR insert; days 5 and 6: 250 µg prostaglandin IM (Estrumate)] and CIDR was removed on days 5 and 7: 86 µg GnRH IM (Fertagyl) (Reproductive Management Strategies for Dairy Heifers, 2018). Transvaginal embryo transfer took place on day 8 after the last GnRH injection. Recipient heifers were moved with clean-up bulls 1 wk after embryo transfer and pregnancy determination using transrectal ultrasonography was performed by a skilled veterinarian 60 d after embryo transfer.

Results

Experiment 1: Comparison of lipid levels of in vitro produced and in vivo derived Holstein and Jersey embryos

We first investigated differences in lipid content between IVP and IVD Jersey and Holstein embryos (Rhodes-Long et al., 2016). IVP embryos had higher lipid content than IVD embryos (56.9 ± 3.1 vs. 49.2 ± 7.9 AFU, respectively, in Jersey embryos, and 55.7 ± 2.7 vs. 43.2 ± 3.3 AFU, respectively, in Holsteins, P < 0.05, Figure 1, Table 1). The addition of forskolin and L-carnitine to the culture medium decreased lipid content to levels comparable to IVD embryos in both Jersey (46.1 ± 1.6 AFU) and Holstein embryos (44.5 ± 1.6 AFU, P > 0.05, Figure 1, Table 1).

Figure 1.

Representative images of lipid content of in vivo derived (IVD, n = 27), in vitro produced (IVP, n = 60), and IVP with forskolin and l-carnitine (IVP+forskolin+l-carnitine, n = 199). Lipid content was determined by Nile Red (green) staining, and images were taken at the equatorial plane of each embryo using an epifluorescent microscope.

Table 1.

Lipid levels of in vivo derived (IVD, n = 27), in vitro produced (IVP, n = 60), and in vitro produced with forskolin (IVP+forskolin, n = 199) in Holstein and Jersey cattle

Breed	Embryo production methods
	IVD (n = 27)	IVP (n = 60)	IVP+Forskolin+ L-carnitine (n = 199)
Jersey	49.2 ± 7.9ab	56.9 ± 3.1b	46.1 ± 1.6a
Holstein	43.2 ± 3.3a	55.7 ± 2.7b	44.5 ± 1.6a

Quantification of lipid levels determined using Nile Red dye and epifluorescent microscopy and quantified by mean arbitrary fluorescent units (mean ± SEM, ^a,bP < 0.01).

Experiment 2: Effects of sequential media, embryonic stage, and individual metabolic regulators on embryonic metabolism

We next sought to determine the media type, embryonic stage, and metabolic regulators that would have the greatest impact on embryonic metabolism (Table 2) (Roberts et al., 2016a). Since there were similar results between Jersey and Holstein embryos in experiment 1, we chose to continue the following experiments with only Holstein embryos due to availability at the abattoir. Lipid content was not affected by continuous or 3-step sequential media (33.78 ± 0.90 AFU vs. 32.40 ± 1.00 AFU, respectively), and this was consistent between stage and additives (Table 2). Thus, media type was eliminated as a factor in the model for further analyses and continuous media was used for subsequent studies to reduce embryo handling. Stage 7 embryos had less intracellular lipid accumulation (P < 0.05) than early-stage embryos when cultured with additives (23.79 ± 0.85 vs. 33.41 ± 0.80 AFU, respectively) or without additives (31.33 ± 1.80 vs. 23.79 ± 0.85, respectively, Figure 2). We also found that culturing embryos with l-carnitine, CoQ, and forskolin decreased lipid content in both embryo stages tested (P < 0.05, Figure 2).

Table 2.

Effect of different culture conditions on lipid accumulation

Media type	Interaction	n	Nile Red AFU	SEM
Continuous	None	149	33.78	± 0.90
3-Step Sequential	None	189	32.40	± 1.00
Continuous	Early (6)	80	39.34	± 1.17
3-Step Sequential	Early (6)	101	37.88	± 1.27
Continuous	Late (7)	69	28.21	± 1.30
3-Step Sequential	Late (7)	88	26.91	± 1.35
Continuous	Additive	112	29.38	± 0.90
3-Step Sequential	Additive	163	27.82	± 0.74
Continuous	No Additive	37	38.18	± 1.56
3-Step Sequential	No Additive	26	36.97	± 1.86

Embryos were cultured in designated treatment groups, then assessed for lipid content using Nile Red dye and quantified by mean arbitrary fluorescent units (AFU, mean ± SEM). There were no differences in the mean lipid content between the culture groups (P < 0.05).

Figure 2.

Lipid content of early or late-stage embryos with or without additives. (A) Flowchart depicting experimental design and generation of IVP embryos in experiment 2 part 1. (B) Quantification of Nile Red staining expressed by mean arbitrary fluorescent units (AFU, mean ± SEM). Lipid content was evaluated in early-stage embryos cultured with no additives (n = 37), early-stage embryos cultured with additives (n = 15), late-stage embryos with no additives (n = 26), and late-stage embryos with additives (n = 13). Values without common labels differ (P < 0.05).

The effects of individual or combinations of additives on mitochondrial polarity were assessed in part 2 of experiment 2. l-carnitine did not increase mitochondrial polarity compared to the no additive treatment group (Figure 3). CoQ, vitamin K₂ (VitK), and forskolin all increased mitochondrial polarity individually compared to the no additive treatment (5442 ± 340 AFU, 6288.6 ± 299 AFU, 6075.1 ± 326 AFU, respectively, vs. 3351.6 ± 328 AFU [no additive], Figure 3). However, when all additives were used in combination, there was no increase in mitochondrial polarity compared to the no additive treatment group (3552.5 ± 227 AFU vs. 3351.6 ± 328 AFU, respectively, P < 0.05).

Figure 3.

Effect of culture additives on embryonic mitochondrial polarity. (A) Flowchart depicting experimental design and generation of IVP embryos in experiment 2 part 2. (B) Embryos were cultured with given additives (l-carnitine, coenzyme Q [CoQ], Vitamin K₂ [VitK], and forskolin) and the mitochondrial polarity was assessed using Mitotracker Red staining. Mean mitochondrial polarity of embryos cultured in the presence of no additive (n = 9), l-carnitine (n = 9), CoQ (n = 8), VitK (10), forskolin (9), and a combination of all additives (n = 18) (mean AFU ± SEM, ^a,bP < 0.05).

Experiment 3: Effects of metabolic regulators on embryonic development and metabolism

In part 1 of this experiment, the effects of l-carnitine and CM supplementation on embryonic development, metabolism, and cryopreservation were determined. There was no difference in blastocyst rates between any of the treatment groups (P > 0.05, Figure 4A). Embryos cultured with l-carnitine decreased lipid content compared to the control (214.78 ± 13.85 AFU vs. 266.43 ± 14.44 AFU, respectively, P < 0.05), which coincided with an increase in cryopreservation survival (72.32 ± 9.46%, l-carnitine vs. 42.02 ± 9.46%, control [P < 0.05]). l-carnitine supplementation did not affect mitochondrial polarity (P < 0.05). Embryos cultured with CM had decreased mitochondrial polarity compared to the control (P < 0.05); however, embryos cultured with both CM and l-carnitine had increased mitochondrial polarity (P < 0.05). There was no difference in post-thaw apoptosis in any treatment group.

Figure 4.

Effects of l-carnitine, conditioned medium (CM), and Vitamin K₂ (VitK) on embryonic development, metabolism, and cryotolerance. (A) Flowchart depicting experimental design and generation of IVP embryos in experiment 3 part 1. (B) Blastocyst rates were determined on days 7 and 8 and did not differ between groups n = 8 replicates (P > 0.05). Error bars represent the standard error of the mean. (C) Lipid content, as expressed by mean arbitrary fluorescent units (AFU) of Nile Red, of embryos cultured with no additives (n = 16), l-carnitine (n = 18), CM (n = 18), or l-carnitine plus CM (n = 16). Error bars represent the standard error of the mean, and values without common labels differ (P < 0.05). (D) Mitochondrial polarity, as expressed by mean AFU of Mitotracker Red, of embryos cultured with no culture additives (n = 18), l-carnitine (n = 20), conditioned medium (CM) (n = 20), and l-carnitine plus CM (n = 18). Error bars represent the standard error of the mean, and values without common labels differ (P < 0.05). (E) Post-thaw re-expansion rate in embryos cultured with no additives, l-carnitine, CM, or l-carnitine plus CM (n = 6 replicates for all treatments). Error bars represent the standard error of the mean, and values without common labels differ (P < 0.05). CM, conditioned media. (F) Percentage of apoptotic cells in embryos cultured with no additives (n = 25), l-carnitine (n = 33), CM (n = 22), and l-carnitine plus CM (n = 30) treatment groups. Apoptosis was assessed by TUNEL staining. Error bars represent the standard error of the mean, and values without common labels differ (P < 0.05).

In part 2 of this experiment, l-carnitine was held constant due to its observed beneficial effects on embryonic development. We then tested all combinations of CM, VitK, and forskolin to a control media and tested blastocyst rate, lipid content, mitochondrial polarity, post-thaw re-expansion, and apoptosis (Roberts et al., 2016b). The combination of all three regulators produced the highest blastocyst rate (39 ± 5%), and the highest post-thaw re-expansion rate (98 ± 14%) though it did not lead to a significant improvement in post-thaw apoptosis (P > 0.05, Figure 5). CM added alone was the only treatment that did not improve lipid content (P > 0.05, Figure 5). Blastocysts cultured in control media alone or with CM added had the highest mitochondrial activity (P < 0.05, Figure 5). Based on the improvements seen in blastocyst rate and post-thaw re-expansion, we determined that the addition of all three metabolic regulators had the best effects on embryonic development.

Figure 5.

Blastocyst rate, lipid content, mitochondrial polarity, post-thaw apoptosis, and re-expansion of Holstein IVP embryos produced with different combinations of metabolic regulators. Conditioned medium (CM), Vitamin K₂ (VitK), and Forskolin were added at days 0, 3, and 5, respectively. (A) Flowchart depicting experimental design and generation of IVP embryos in experiment 3 part 2. (B) Blastocyst rate was visually determined on days 7 and 8 (n = 8 replicates). (C) Stage 7 embryos were stained with Nile Red (n = 18, 16, 15, 18, 15, 17, 17, and 19 for control, CM, VitK, Forskolin, VitK + Forskolin, CM + VitK, CM + Forskolin, CM + VitK, + Forskolin, respectively) and fluorescence intensity was quantified in ImageJ (Mean±SEM, ^a,b,c,d,eP < 0.05). (D) Stage 7 embryos were stained with Mitotracker Red CMX-Rosamine (n = 20, 18, 19, 16, 18, 19, 20, and 16 for control, CM, VitK, Forskolin, VitK + Forskolin, CM + VitK, CM + Forskolin, CM + VitK, + Forskolin, respectively) and fluorescence intensity was quantified in ImageJ (mean ±SEM, ^a,bP < 0.05). (E) Slow frozen stage 7 blastocysts were examined for post-thaw re-expansion at 48 h (n = 8 replicates, ^a,b,cP < 0.05). (F) Re-expanded blastocysts (n = 33, 30, 30, 37, 38, 47, 36, and 53 control, CM, VitK, Forskolin, VitK + Forskolin, CM + VitK, CM + Forskolin, CM + VitK, + Forskolin, respectively) were analyzed for apoptosis via a TUNEL assay (^a,bP < 0.05).

Experiment 4: Effects of novel media and improved slow freezing techniques on embryo cryotolerance

In the previous experiments, we determined the optimal combination of media type, embryo stage, and culture media type that led to the best results on embryonic development and metabolism. We determined that adding a combination of l-carnitine, CM, forskolin, and VitK to a continuous medium led to the most beneficial effects on development, metabolism, and cryotolerance, and coined the novel culture medium Synthetic oviductal fluid for Conventional Freezing 1 (SCF1) (Owen et al., 2017). Although blastocysts cultured with conditioned medium had lower post-thaw re-expansion rates and apoptosis, we chose to include it in the final culture media because it led to the highest blastocyst rate (Figure 5A). We confirmed that this culture medium led to increased blastocyst rates and mitochondrial polarity and decreased lipid content compared to the control medium (SOF) with no additives (Figure 6).

Figure 6.

Effects of novel culture medium and slow freezing technique on embryonic development, metabolism, and cryopreservation. (A) Blastocyst rate of embryos cultured in SCF1 or SOF (Mean±SEM). Embryos were cultured in either SCF1 (n = 1781) or SOF (n = 1391) for up to 8 d, and then blastocyst rate was visually assessed. (B and C) Stage 7 embryos (n = 17 per treatment) were stained with Mitotracker Red CMX-Rosamine (red) and fluorescence intensity was quantified in ImageJ (mean ±SEM, ^a,bP < 0.01). (D and E) Stage 7 embryos (n = 16 per treatment) were stained with Nile Red (green) and fluorescence intensity was quantified in ImageJ (Mean±SEM, ^a,bP < 0.01). (F) Main effects of culture media (SCF1 vs. SOF, n = 358 and 198, respectively), sucrose rehydration (0 vs. 0.6 M sucrose, n = 277 and 279, respectively), and equilibration time in EG (10 vs. 20 min, n = 286 vs. 270, respectively) on the re-expansion rate of slow frozen embryos (Mean±SEM, ^a,bP < 0.01, ^c,dP < 0.1). (G) Representative images of apoptotic cells in re-expanded embryos. Nuclei are stained blue with DAPI, and fragmented DNA is stained red with TUNEL. The overlapping pink nuclei represent apoptotic cells, which were counted as a percent of total cells. (H) Main effects of culture media (SCF1 vs. SOF, n = 85 and 63, respectively), sucrose dehydration (0 or 0.6M sucrose, n = 72 and 76, respectively), and equilibration time in EG (10 vs. 20 min, n = 76 and 72, respectively) on post-thaw apoptosis. The number of apoptotic cells was counted in re-expanded embryos (mean±SEM, ^a,bP < 0.01).

Adding metabolic regulators to culture media improved the post-thaw re-expansion rate, but not post-thaw apoptosis (Figures 4 and 5). Since post-thaw survival is critical to successful pregnancies, we tested whether we could improve the slow-freezing protocol. Three factors (culture media, sucrose dehydration, and EG equilibration time) were tested to determine their effects on cryotolerance. The experiment was designed as a factorial, but no significant interactions occurred so only main effects were analyzed. Embryos cultured in SCF1 had significantly higher post-thaw re-expansion (82.3 ± 2.7%) and lower post-thaw apoptosis (20.4 ± 2.6%) than embryos cultured in SOF (49.6 ± 4.5% and 31.6 ± 2.0%, respectively, Figure 6). Embryos equilibrated for 20 min in EG had a lower percentage of apoptotic cells post-thaw than embryos only equilibrated for 10 min (21.4 ± 2.0% vs. 30.6 ± 2.5%, P < 0.01, Figure 6); however, this only tended to increase post-thaw re-expansion (69.8 ± 3.9% vs. 62.1 ± 4.7%, P < 0.1, Figure 6). Neither post-thaw re-expansion rate or apoptosis was not different between embryos dehydrated in 0 or 0.6M sucrose (P > 0.1, Figure 6).

Experiment 5: Effect of novel culture media and slow freezing technique on pregnancy rates

A recent study has suggested that in vitro cryosurvival does not necessarily correlate with improvements in pregnancy rates in vivo (Gómez et al., 2020). Thus, although SCF1 media with the novel slow freezing technique showed improvements in vitro, it was important to evaluate those effects in a clinical trial. Overall, we found that embryos cultured in SCF1 resulted in higher pregnancy rate, regardless of whether they were fresh or frozen (SOF: 34.5 ± 5.31% vs. SCF1: 46.6 ± 4.1%, P = 0.038). The main effect of cryopreservation tended to be lower pregnancy rate than fresh embryos (Fresh: 44.6 ± 4.6% vs. Frozen: 38.9 ± 4.9%, P = 0.07); however, when examining the effects on embryos cultured in SCF1, there was no difference between fresh and frozen embryos (P > 0.05, Table 3). There was no difference in pregnancy rates at different locations (P > 0.1). Overall, embryos cultured in SCF1 had similar pregnancy rates whether they were transferred frozen or fresh (43.6% ± 4.9% and 49.5% ± 5.0%, respectively, Table 3).

Table 3.

The pregnancy rate for embryos cultured in either SOF or SCF1, then either transferred frozen or fresh

Media	Cryopreservation	n	Mean	SEM
SOF	Fresh	173	40.4ab	±5.7
SOF	Frozen	104	28.6b	±7.2
SCF1	Fresh	252	49.5a	±5.0
SCF1	Frozen	228	43.6ab	±4.9

Embryos were cultured in synthetic oviductal fluid (SOF) or synthetic oviductal fluid for conventional freezing 1 (SCF1). Embryos were then either transferred fresh, or slow frozen following a 20-min equilibration in ethylene glycol (EG), then transferred. The pregnancy rate was determined by ultrasound on day 60 by a skilled technician (^a,bP < 0.05).

Discussion

Despite the importance of IVP embryos in cattle production, IVP embryos are still inferior to their IVD counterparts in development and cryotolerance (Ferré et al., 2020). The differences between IVP and IVD embryos likely stem from improper culture conditions which inadequately replicate the in vivo environment and lead to changes in embryonic metabolism. Although recent advances in embryo culture (Stoecklein et al., 2021) and slow freezing (Gómez et al., 2020) have led to improvements in pregnancy rates, frozen IVP embryos are still suboptimal for use in commercial settings and pregnancy rates still have high variation. Therefore, the goal of this study was to determine the optimal culture conditions and slow-freezing techniques to improve IVP embryo development and cryotolerance such that transferring frozen IVP embryos would result in pregnancy rates comparable to fresh IVP embryos. We tested the effects of different combinations of metabolic regulators, embryonic stage, and continuous and sequential culture medium on embryonic development, metabolism, and cryotolerance. Overall, we found that the use of a combination of metabolic regulators added to SOF medium, which we termed SCF1, was beneficial for embryo development, metabolism, and cryopreservation, and led to increased pregnancy rates in both fresh and frozen embryos after transfer.

Supplementation of l-carnitine and forskolin decreased lipid content in IVP embryos of both Holstein and Jersey cattle, though the Jersey embryos overall had higher lipid content. Jersey embryos are more lipid dense than Holstein, regardless of housing and feed conditions, perhaps due to differences in lipid metabolism (Steel et al., 2003; Baldoceda et al., 2015a; Baldoceda et al., 2015b). The increased lipid content of Jersey embryos could explain why cryopreserved IVP Jersey embryos produce significantly fewer pregnancies than cryopreserved Holstein embryos (Steel et al., 2003). Interestingly, a previous study supplementing culture medium with l-carnitine found reductions in lipid content in both Holstein and Jersey embryos, but the effect was reduced in Jersey embryos (Baldoceda et al., 2015a). In this study, supplementing embryo culture with both l-carnitine and forskolin led to a similar reduction in lipid content in both Jersey and Holstein embryos, likely due to an increase in activation of lipases via multiple pathways (Xw et al., 2010; Dunning and Robker, 2012; Chankitisakul et al., 2013; Sanches et al., 2013; Takahashi et al., 2013; Paschoal et al., 2017). Although the subsequent studies were only performed in Holstein embryos, this suggests that the use of multiple metabolic regulators may also be beneficial in Jersey embryos, and this should be further investigated in future studies.

Previous studies have indicated that the use of continuous or sequential embryo culture medium does not affect embryonic development (Reed et al., 2009; Sfontouris et al., 2016; Dieamant et al., 2017); however, the effects on lipid content have not yet been evaluated. Embryos require specific nutrients at various stages of development due to activation of the embryonic genome (Graf et al., 2014). Activation of the embryonic genome in IVP embryos leads to the expression of glycolytic enzymes (García-Herreros et al., 2018), and the embryo no longer relies on pyruvate-based oxidative phosphorylation (Thompson, 2000). Altering the availability of preferred energy sources at developmental stages may change the embryo metabolism (Gardner et al., 2000; Sutton-McDowall et al., 2012). Here, we evaluated total embryo lipid content and found that the use of continuous or sequential medium did not affect embryonic lipid content, regardless of embryo stage or the use of culture additives. Preimplantation embryos show high levels of plasticity and can survive high stress environments (Betts and King, 2001; Ramos-Ibeas et al., 2020); therefore, it is plausible that the embryos can overcome the unideal energy substrates provided in a continuous culture medium.

Embryos utilize stores of endogenous lipid throughout development (Abe et al., 2002; Ibayashi et al., 2021), which explains why late-stage embryos show decreased lipid content compared to early-stage embryos in experiment 2 part 1. Despite this stage-specific reduction in lipid content, IVP embryos still possess higher lipid content than IVD embryos of the same stage, as demonstrated in experiment 1 of this study. The addition of forskolin to embryo culture medium activates lipase enzymes to generate free fatty acids within the cytosol. When coupled with the addition of L-carnitine, which facilitates the rate-limiting step of fatty acid transport into the mitochondria, these two additives can increase β-oxidation and potentially decrease the accumulation of intracellular lipid (Men et al., 2006; Xw et al., 2010; Dunning and Robker, 2012). Improving mitochondrial function could also reduce ROS levels, so for part 2 of this experiment we aimed to improve electron transport efficiency. CoQ and VitK have both been shown to increase the efficiency of electron flow between protein complexes of the electron transport chain (ETC) (Stojkovic et al., 1999; Baldoceda-Baldeon et al., 2014). This should reduce the premature escape of electrons from the ETC, which is a major source of ROS generation, particularly in dysfunctional mitochondria. The endpoint used to measure electron transport efficiency for this study was inner mitochondrial membrane potential. Although both CoQ and VitK were found to increase mitochondrial membrane potential when used individually, there was no effect when the regulators were used in combination with conditioned medium, l-carnitine, and forskolin. Despite a lack of increase in mitochondrial membrane potential with all additives used in combination, we did see a reduction in lipid content and, most importantly, an increase in blastocyst development rate. Due to the simultaneous manipulation of various metabolic pathways carried out in this study, it is possible that interactions exist between the additives used which may be contributing to the inconsistent results in mitochondrial membrane potential observed. Since membrane potential was the only endpoint used to determine mitochondrial function, we cannot say conclusively that this was an adequate measure of embryo quality as used in this study.

Several factors could explain the inconsistent results of mitochondrial membrane potential measurements observed between experiments 2, 3, and 4. In experiments 2 and 3, the use of individual regulators increased polarity, whereas a combination of regulators did not. These results directly contradict the results in experiment 4 when SCF1, containing a combination of multiple metabolic regulators, increased mitochondrial polarity compared to the standard SOF medium. Previous studies have suggested that greater inner mitochondrial membrane polarity indicates increased mitochondrial function and thus improved embryo viability (Baldoceda-Baldeon et al., 2014). However, several groups have also claimed that optimal metabolic activity for developing embryos may exist as a range rather than as an absolute measure (Leese, 2012; de Lima, 2020). This theory is in part supported by the quiet embryo hypothesis, which posits that embryo viability is associated with lower levels of oxygen consumption, as viable cells require less energy for genome and proteome repair processes (Leese, 2002). Given the lack of consensus on whether metabolic activity is an adequate marker of embryo viability, we do not conclude that mitochondrial membrane polarity was an accurate indicator of embryo quality in this study, as it did not consistently correlate with increased blastocyst or post-thaw re-expansion rates.

A more appropriate measure of improved mitochondrial function could be quantification of ROS, which has previously been correlated with embryo quality (Yoon et al., 2014) and may provide a more direct readout of mitochondrial dysfunction. Oxygen consumption rate (OCR) and extracellular acidification rate may also give a better representation of the overall metabolic competency of the embryo. OCR measures mitochondrial function by quantifying cellular respiration, and higher OCR directly correlates with improved fertility outcomes in human oocytes and embryos (Yamanaka et al., 2011; Tejera et al., 2012; Hashimoto et al., 2017) and equine oocytes (Catandi et al., 2021). The stage of development at which mitochondrial activity is measured may also be relevant in determining how this parameter correlates to developmental competence. It is known that a drastic shift in energy demand occurs as the embryo transitions from the morula to blastocyst stage, which is marked by a sharp increase in ATP production and oxygen consumption as the embryo undergoes compaction and blastocoel formation (Leese, 2012). Future studies should not only consider additional measures of mitochondrial function such as ROS production and OCR, but also assess mitochondrial membrane potential at different stages of development.

Co-culturing embryos with conditioned medium or monolayers of Vero cells or oviductal epithelial cells has been shown to improve embryonic development in multiple studies. However, while Vero cells are a continuous cell line that are commercially available, oviductal epithelial cells must be isolated from a primary source and are therefore less convenient for use (Abe and Hoshi, 1997). There is the possibility that the improvements in embryo development seen were due to the chemically defined FBS the Vero cells were grown in. However, this seems unlikely since the cells were grown in 5% chemically defined FBS, and the conditioned medium was added at %% v:v ratio with the final embryo culture medium, leading to a final concentration of 0.0025% chemically defined FBS in the embryo culture medium. It is more likely, however, that embryotrophic factors released into the conditioned medium enhanced embryonic development as has been previously described (Lee et al., 2001, Maeda et al., 1996).

In the present study, two techniques were investigated to determine the effects on cryopreservation: 1) dehydration with sucrose, a nonpermeable cryoprotectant that may further dehydrate the cells (Tominaga et al., 2007) and 2) longer equilibration in EG to further dehydrate the embryos before slow freezing (Li et al., 2012). There was no benefit of sucrose dehydration, potentially due to the low concentration used (0.6 M). There was a tendency for increased cryotolerance in the longer equilibration, and a significant decrease in post-thaw apoptosis which suggests that the longer equilibration may have improved dehydration and led to less ice crystal formation. Interestingly, while the changes in post-thaw apoptosis were significant, it only led to a tendency to increase post-thaw survival. Thawed embryos have altered post-thaw metabolism (Kaidi et al., 2001). Other tissues show increased production of ROS due to oxygen influx upon thawing (El-Wahsh et al., 2003) heat shock proteins upon thawing (Odani et al., 2003), though these responses have not been investigated in embryos. These alternate responses to thawing may lead to decreased survival without activating apoptotic responses, thus leading to the discrepancy between the post-thaw re-expansion and apoptosis rates. It is worth noting that the cell death observed in our study was higher than previous studies (30% compared to 15% in Gómez et al., 2020). However, it has been shown that post-thaw apoptosis does not correlate with post-thaw survival; thus, we do not believe this impacted our results (Gómez et al., 2020).

The improvements that were seen in vitro through IVP embryo culture with SCF1 and the improved slow freezing technique correlated with an increased pregnancy rate. The pregnancy rates achieved in our study with both fresh and frozen embryos cultured in SCF1 are comparable to recent pregnancy rates obtained with fresh embryos transferred to heifer recipients (46%) (Demetrio et al., 2020) and to rates reported in commercial enterprises (45-50%) (Creating embryos from oocytes by fertilizing them with semen in a Petri dish | Trans Ova). These results suggest that culturing embryos in SCF1 media and a longer duration equilibration phase during slow freezing enhances embryo cryotolerance such that frozen embryos can be comparable to fresh IVP embryos. This is the second study to report pregnancy rates in frozen IVP embryos comparable to fresh IVP embryos (Gómez et al., 2020), and suggests that there are multiple ways to improve in vitro embryo culture. Overall, this study provides further evidence that alterations to embryo culture can ultimately affect embryo cryotolerance and subsequent success in frozen embryo transfer. The use of SCF1, a culture media specifically designed to optimize embryo metabolism throughout the entire culture period, enhanced the survival of cryopreserved embryos and led to embryo transfer rates consistent with fresh embryos cultured in SCF1. Future studies involving other breeds including the use of lactating recipients are warranted to corroborate the results obtained in the present study (Sirard, 2022).

Glossary

Abbreviations

IVP

in vitro produced

SCF1

synthetic oviductal fluid for conventional freezing 1

IVD

in vivo derived

SOF

synthetic oviductual fluid

ROS

reactive oxygen species

CM

conditioned medium from Vero cells

CoQ

coenzyme Q

VitK

vitamin K₂

EG

ethylene glycol

FSH

follicle stimulating hormone

FCS

fetal calf serum

GnRH

gonadotropin releasing hormone

CIDR

controlled intravaginal drug release

AFU

arbitrary fluorescence unit

OCR

oxygen consumption rate

Conflict of Interest Statement

J.L.A. and M.B.F. work in separate commercial assisted reproduction clinical practices that could benefit from veterinary case referrals.