Glutamine supplementation enhances development of in vitro-produced porcine embryos and increases leucine consumption from the medium†

Abstract

Improper composition of culture medium contributes to reduced viability of in vitro-produced embryos. Glutamine (Gln) is a crucial amino acid for preimplantation embryos as it supports proliferation and is involved in many different biosynthetic pathways. Previous transcriptional profiling revealed several upregulated genes related to Gln transport and metabolism in in vitro-produced porcine blastocysts compared to in vivo-produced counterparts, indicating a potential deficiency in the culture medium. Therefore, the objective of this study was to determine the effects of Gln supplementation on in vitro-produced porcine embryo development, gene expression, and metabolism. Cleaved embryos were selected and cultured in MU2 medium supplemented with 1 mM Gln (control), 3.75 mM Gln (+Gln), 3.75 mM GlutaMAX (+Max), or 3.75 mM alanine (+Ala) until day 6. Embryos cultured with +Gln or +Max had increased development to the blastocyst stage and total number of nuclei compared to the control (P < 0.05). Moreover, expression of misregulated transcripts involved in glutamine and glutamate transport and metabolism were corrected when embryos were cultured with +Gln or +Max. Metabolomics analysis revealed increased production of glutamine and glutamate into the medium by embryos cultured with +Max and increased consumption of leucine by embryos cultured with +Gln or +Max. As an indicator of cellular health, mitochondrial membrane potential was increased when embryos were cultured with +Max which was coincident with decreased apoptosis in these blastocysts. Lastly, two embryo transfers by using embryos cultured with +Max resulted in viable piglets, confirming that this treatment is consistent with in vivo developmental competence.

Applications of omics and nanotechnology to improve pig embryo production in vitro

“…Also, it was observed that pig embryos depended more on glutamine metabolism than human, murine, and bovine embryos (Krisher, Heuberger, et al, ). As previously described, uptake and production of amino acids have an important role in preimplantation development (Bauer et al, ; Brison et al, ; Chen et al, ; Hemmings, Leese, & Picton, ; Prather, Redel, Whitworth, & Zhao, ; Redel et al, ). Metabolomics profiling data can indicate potential biomarkers of embryo quality to improve implantation and pregnancy rates after culturing embryos in vitro (Krisher, Schoolcraft, et al, ; Wale & Gardner, ).…”

“…However, in contrast to the arginine‐treated embryos, embryos cultured with supplemental glycine did not establish pregnancies after 11 embryo transfers (Redel et al, ). Similar to the arginine story, supplementation of glutamine to the porcine embryo culture medium improved development to the blastocyst stage, reduced transcript levels of a glutamate transporter, SLC1A1 , to a level more similar to in vivo‐derived embryos, promoted leucine consumption from the medium, and resulted in birth of live piglets (Chen et al, ).Furthermore, the subunit of the granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) receptor (CSFR) was more abundant in IVC embryos when compared to IVV embryos (Bauer et al, ). The addition of GM‐CSF during culture in vitro increased blastocyst rates and total cell number of IVF and SCNT‐derived embryos.…”

“…However, in contrast to the arginine‐treated embryos, embryos cultured with supplemental glycine did not establish pregnancies after 11 embryo transfers (Redel et al, ). Similar to the arginine story, supplementation of glutamine to the porcine embryo culture medium improved development to the blastocyst stage, reduced transcript levels of a glutamate transporter, SLC1A1 , to a level more similar to in vivo‐derived embryos, promoted leucine consumption from the medium, and resulted in birth of live piglets (Chen et al, ).…”

“…In general, porcine gametes have been cultivated in a humidified incubator under 5% CO 2 , 20% O 2 , and 75% N 2 at 38.5–39°C (Romar et al, ). After fertilization, the embryos are cultured at 38.5°C in a humidified atmosphere of 5% CO 2 in air overnight, and then the next day (Day 1), they are moved to a humidified atmosphere of low oxygen (5% O 2 , 5% CO 2 , and 90% N 2 ) until Day 6 (Chen et al, ). Recently, García‐Martínez et al () demonstrated that the use of the same O 2 conditions as those found in the pig reproductive tract (7% O 2 during IVF and embryo culture) were beneficial for embryo production in vitro.…”

“…Besides the importance of controlling culture external conditions, supplementation in vitro to correct specific molecular deficiencies has been reported to increase the efficiency of assisted reproduction techniques (Chen et al, ; Heras et al, ; Moussa, Shu, Zhang, & Zeng, ; Redel et al, ; Yuan et al, ). Recent studies have explored the potential of specific growth factors and amino acids to optimize maturation and embryo culture media (Chen et al, ; Krisher & Prather, ; Redel et al, ; Redel, Tessanne, Spate, Murphy, & Prather, ; Yuan et al, ). However, due to the different chemical and biological composition of supplements, strategies to enable more efficient use of these additives by preventing premature degradation, increasing their bioavailability and facilitating their delivery are desired.…”