Removal of hypotaurine from porcine embryo culture medium does not impair development of in vitro‐fertilized or somatic cell nuclear transfer‐derived embryos at low oxygen tension

Abstract Hypotaurine (HT) is a routine component of porcine embryo culture medium, functioning as an antioxidant, but its requirement may be diminished as most embryo culture systems now use 5% O2 instead of atmospheric (20%) O2. Our objective was to determine the effects of removing HT from the culture medium on porcine preimplantation embryo development. Embryos cultured in 20% O2 without HT had decreased blastocyst development compared to culture with HT or in 5% O2 with or without HT. Notably, differences in blastocyst development or total cell number were not detected between embryos cultured in 5% O2 with or without HT. After culture in 5% O2 without HT and embryo transfer, healthy fetuses were retrieved from two pregnancies on Day 42, confirming in vivo developmental competence. Transcript abundance of proapoptotic markers was decreased in embryos cultured without HT regardless of oxygen tension; however, assays for apoptosis did not demonstrate differences between groups. Additionally, no differences were observed in the development or apoptosis of somatic cell nuclear transfer‐derived embryos cultured in 5% O2 with or without HT. With decreased utility in 5% O2, removing HT from porcine embryo culture medium would also have economic advantages because it is undoubtedly the most expensive component.

development to the blastocyst stage in porcine embryos (Petters & Reed, 1991), and bovine blastocyst development increased after culture with 0.5-to 2-mM HT on monolayers of Vero cells (Guyader-Joly et al., 1998).
The majority of studies demonstrating the beneficial effects of HT during embryo culture were conducted in 5% CO 2 in air (20% O 2 ).
However, preimplantation embryo development in vivo occurs at 1.5-8.5% O 2 depending on the species, and oxygen tension decreases around the period of blastocyst development and implantation (Fischer & Bavister, 1993;Ufer & Wang, 2011). Culture in 5% O 2 compared to 20% O 2 has been shown to improve blastocyst development and total cell numbers in embryos of many different species (Batt, Gardner, & Cameron, 1991;Booth, Holm, & Callesen, 2005;Karagenc, Sertkaya, Ciray, Ulug, & Bahçeci, 2004;Lim, Reggio, Godke, & Hansel, 1999;Redel et al., 2011) as well as live birth rates after in vitro fertilization (IVF) in humans (Meintjes et al., 2009). Furthermore, a meta-analysis of 21 studies confirmed a modest increase in the number of clinical pregnancies after transferring human embryos that were cultured in 5% O 2 ; however, more trials are required to confirm the results (Nastri et al., 2016). Redel et al. (2011) observed that the transcriptional profiles of porcine embryos cultured in 5% O 2 were characteristic of the Warburg effect (WE) with an increased message for enzymes of glycolysis. The WE describes the shuttling of glycolytic intermediates away from the tricarboxylic acid cycle and towards the pentose phosphate pathway and lactic acid formation in rapidly proliferating cells to support macromolecule synthesis (Warburg, 1956). This metabolic explanation of reduced respiration is in agreement with the fact that porcine embryos cultured in 5% O 2 have decreased ROS accumulation and DNA damage compared to embryos cultured in 20% O 2 (Kitagawa, Suzuki, Yoneda, & Watanabe, 2004). Therefore, when culturing porcine embryos in 5% O 2 , we hypothesized that addition of HT to the embryo culture medium was unnecessary. First, we investigated the effects of culturing embryos with or without HT in 5% or 20% O 2 on embryo cleavage and blastocyst development. Embryo transfers were performed by using blastocysts that developed after culturing without HT in 5% O 2 to confirm that this treatment could establish pregnancies. Subsequently, the abundance of transcripts related to HT synthesis, oxidative stress, and apoptosis, as well as incidences of apoptosis, were determined in the blastocysts. Finally, developmental parameters were reassessed in somatic cell nuclear transfer (SCNT)-derived embryos cultured with or without HT in 5% O 2 as these embryos are more prone to stress and damage during micromanipulation procedures.

| Establishment of pregnancy after transferring embryos cultured without HT
To ensure that porcine embryos cultured in low O 2 −HT could establish pregnancies, two embryo transfers were performed with 60 IVF-derived D5 blastocysts and morulae each. Fetuses were collected on D42 of gestation. The pregnancies yielded 11 and 9 fetuses, respectively, and the sexes and average crown-rump lengths were measured (Table 2). Importantly, all fetuses appeared to be healthy with no signs of absorption ( Figure 1a). Sexes determined by external genitalia were confirmed by using a polymerase chain reaction (PCR)based sex determination assay ( Figure 1b). These data confirm that culturing embryos without HT can result in the successful establishment of pregnancy.  (Guérin et al., 2001). Specifically, HT is a well-known scavenger of hydroxyl radicals ( • OH) that reduces lipid peroxidation in spermatozoa (Alvarez & Storey, 1983;Aruoma, Halliwell, Hoey, & Butler, 1988). Petters and Reed (1991) reported that addition of HT to porcine embryo culture medium improved the development of one-and two-cell stage embryos to the blastocyst stage.
Thereafter, HT has been routinely added to porcine embryo culture medium as a protective measure against ROS. However, with the advent of embryo culture being performed in 5% O 2 , the necessity for antioxidants, including HT, in the medium decreases because of reduced exposure to ROS. Furthermore, HT is the most expensive component of porcine embryo culture medium, accounting for 68% of the cost of making MU2 on a per-mL basis (Spate, Brown, Redel, Whitworth, & Prather, 2015). Therefore, its removal from formulations would have an economic advantage for embryo culture laboratories as well.  King, 1995). Despite the fact that the current study did not include sufficient pregnancies to determine alterations in sex ratio, the observed ratio of male to female fetuses from both pregnancies was approximately 1:1.
The abundance of transcripts involved in HT synthesis, oxidative stress, and apoptosis was measured to determine if removing HT from the culture medium affected these processes. Interestingly, the abun- has been shown to decrease the formation of the apoptosome, thereby inhibiting the apoptosis cascade (Takatani et al., 2004). Moreover, HT addition to cryopreservation medium for human sperm reduced the percentage of apoptotic sperm after thawing (Brugnon et al., 2013).
Apoptosis assays were utilized in the current study to assess the effects of removing HT instead of ROS assays because of inconsistencies in the latter. For example, the most common ROS probe, dichloro-dihydrofluorescein diacetate (DCFH-DA), forms a free radical during its oxidation that can generate more superoxide ions, and cytochrome c released during apoptosis can oxidize the probe to falsely increase the fluorescence signal (Kalyanaraman et al., 2012

| Chemical components
All chemicals were purchased from Sigma Chemical Company (St. Louis, MO) unless stated otherwise.

| Ethics statement
The use of live animals and collection of ovaries from prepubertal gilts were in accordance with the approved protocol and standard operating procedures by the Animal Care and Use Committee of the University of Missouri.

| Oocyte collection and maturation
Ovaries were collected from prepubertal gilts at a local abattoir Sperms were washed by centrifugation in 45% Percoll solution and then in a modified Tris-buffered medium. The spermatozoa pellet was resuspended in IVF medium to 0.5 × 10 6 cells/ml. Then, 50 μl of the sperm suspension was added to the droplets to obtain a final concentration of 0.25 × 10 6 cells/ml. Gametes were incubated together in a humidified incubator at 38.5°C for 4 hr . Hoechst 33342 (10 μg/ml) for 15 min, and the total number of nuclei was recorded after visualization by using an ultraviolet filter attached to a Nikon Eclipse E600 microscope (Nikon, Tokyo, Japan).

| Embryo transfer and fetal collection
IVF-derived D5 blastocysts and morulae cultured in low O 2 −HT were placed in 3 ml of manipulation medium (9.50 g TCM-199, 0.05 g NaH-CO3, 0.75 g HEPES, 1.76 g NaCl, 3.00 g BSA, 1 ml gentamicin, 1,000 ml  for 10 s, and 72°C for 30 s. A dissociation curve was generated after amplification to ensure that a single product was amplified. The abundance of each mRNA transcript was calculated relative to the housekeeping gene, YWHAG (tyrosine 3-monooxygenase/ tryptophan 5-monooxygenase activation protein, gamma polypeptide) and a reference sample, which consists of pooled cDNA from various porcine tissues (Whitworth et al., 2005). The comparative threshold cycle method (C q ) method was used to determine transcript abundance for each treatment. Trypsin-ethylenediaminetetraacetic acid (Gibco, Denmark). Fibroblasts were centrifuged for 5 min at 500g and resuspended in manipulation medium with 7.0 µg/ml of cytochalasin B for SCNT.
Procedures for SCNT have been previously described (Lai & Prather, 2003). Briefly, MII oocytes were placed in 7 μl drops of manipulation medium with 7.0 μg/ml of cytochalasin B on a micromanipulator with an inverted microscope and enucleated by aspirating the polar body, MII plate, and its surrounding cytoplasm by using a hand-tooled beveled glass pipette. A single donor cell was injected into the perivitelline space next to the oocyte membrane.
Then, the oocyte and donor cell were placed in fusion medium ( for 30 min. Afterward, the embryos were split into two groups and placed in MU2 +HT or −HT with 0.5 μM of scriptaid, a histone deacetylase inhibitor, for 14-16 hr at 38.5°C and 5% CO 2 in air (Whitworth, Zhao, Spate, Li, & Prather, 2011). Embryos were placed into the respective media without scriptaid and cultured to D6 in 5% O 2 , 5% CO 2 , and 90% N 2 . The percentage cleaved on D2, percentage developed to blastocyst stage on D6, and the total number of nuclei were recorded for each treatment.

| Statistical analysis
All experiments were repeated at least four times so that the replicate variation could be assessed. For IVF experiments, percentage data used to quantify embryo cleavage and development to the blastocyst stage was analyzed by a generalized linear model (PROC GENMOD). The total number of nuclei, transcript abundance ( −ΔΔ 2 Ct ), caspase activity, and DNA fragmentation were analyzed by linear mixed models (PROC MIXED). Analyses for developmental parameters and DNA fragmentation of SCNT embryos were conducted by using Student's t test. The Shapiro-Wilk test was used for assessing the normality assumption for each experiment and none of the data deviated from the normality assumption. Treatment was modeled as a fixed factor and the biological replicate was modeled as a random factor. Significance was discovered by testing hypotheses by using least square estimates. The type I error and family-wise error rate were controlled at a level of 0.05. All of these analyses were conducted by using SAS version 9.4 (SAS Institute, Cary, NC).