Author's address (for correspondence): E Nestle, 2101 Animal Sciences Center, University of Maryland, College Park, MD, 20742, USA. E-mail: email@example.com
The objective of this study was to assess and compare the quality of cat blastocysts produced in vitro using commercial blastocyst growth media supplemented with different sources of proteins (serum protein substitute from in vitro maturation through embryo development vs 4 mg/ml of bovine serum albumin for maturation and 5% foetal calf serum for fertilization and embryo development). Impact was specifically examined on the proportion of blastocyst formation, total number of blastomeres, proportion of inner cell mass and expression of pluripotency marker proteins NANOG and OCT-4. Blastocyst formation per total cleaved embryos was similar (p > 0.05) regardless of the protein supplementation. There were no differences (p > 0.05) between culture conditions regarding average number of blastomeres and proportion of inner cell mass in each embryo. Presence of OCT-4 protein was detected in nuclei of both trophectoderm and inner cell mass region, with a stronger signal in the latter regardless of the culture medium. NANOG protein also was present in the inner cell mass regardless of the in vitro culture condition. We therefore demonstrated that serum protein substitute was as good as semi-defined protein sources for the production of good-quality blastocysts and embryonic stem cells. In addition, a single defined medium could be successfully used for cat oocyte maturation, in vitro fertilization and embryo development.
The domestic cat (Felis catus) is an excellent biomedical model as well as a good model for rare felid species. Maintenance of genetic diversity in cat populations can be optimized by the use of assisted reproductive techniques including the in vitro production and transfer of embryos at the blastocyst stage. Furthermore, embryonic stem cells may be derived from domestic cat blastocysts, opening a new field of research (Gómez et al. 2010). Both stem cell derivation and embryo transfer necessitate quality blastocysts.
Blastocyst quality can be assessed using several methods. Counting the total number of blastomeres (the higher the better) as well as the ratio between inner cell mass (ICM) and trophectoderm after 7 days of in vitro culture (Gómez et al. 2010) are simple and commonly used methods of determining the quality of a blastocyst. In addition, levels of NANOG and OCT-4 transcription factors in the ICM of a blastocyst can be used to determine blastocyst quality. Studies in the mouse, rat and human have shown that the ICM of a blastocyst can be used to generate embryonic stem cells, the pluripotency of these stem cells being reflected by levels of NANOG and OCT-4 proteins – two critical transcription factors for the maintenance in an undifferentiated stage (Behboodi et al. 2011). Studies have shown that embryos produced in vivo have higher expression of these transcription factors and also produce high-quality embryonic stem cells (Roth et al. 1994; Yu et al. 2008; Behboodi et al. 2011; du Puy et al. 2011). Therefore, in vitro produced embryos that have high levels of NANOG and OCT-4 are closer to the in vivo phenotype and thus are considered of higher quality. However, when culturing embryos in vitro, the composition of the culture medium can significantly affect the quality of produced embryos mainly in terms of morphology, metabolism and gene expression. The quality of the blastocyst subsequently influences the success of the implantation, foetal growth and general health of produced offspring (Dobrinsky et al. 1996).
Studying in vitro production media in various species has revealed that the requirements of pre-implantation embryos are species and stage specific. Multiple protocols have been developed to generate cat embryos in vitro with varying success in blastocyst formation (Hribal et al. 2012). One interesting study sought to make a feline optimized culture medium by identifying precise molar concentrations of ions, carbohydrates, amino acids, vitamins and serum necessary for early embryonic growth (Herrick et al. 2007). A commercially available defined medium would actually be preferable to better investigate and understand the embryo formation and to standardize embryo culture between laboratories.
Previous studies have examined the importance of protein source when culturing embryos of cat and pig in vitro (Wood et al. 1995; Dobrinsky et al.1996; Karja et al. 2002). Complex biological macromolecules are used in many culture systems across species as a source of protein. However, foetal calf serum (FCS) has been found to cause poorer oocyte maturation than bovine serum albumin (BSA) in cat, bovine and rat, yet FCS allowed more oocytes to be fertilized (Wood et al. 1995). Interestingly, more embryos reach the hatching blastocyst stage and have higher cell number with FCS rather than BSA for fertilization and culture (Dobrinsky et al. 1996; Karja et al. 2002; Han and Niwa 2003; Herrick et al. 2007).
The objective of this study was to measure the impact of protein supplementations (defined vs semi-defined) on the success of blastocyst formation. The hypothesis was that a defined medium (serum protein substitute) could provide blastocysts with high cell number more consistently than a culture system that is semi-defined and needs additional protein supplementation (i.e. FCS and/or BSA).
Materials and Methods
Oocyte maturation and embryo production
Oocyte selection and main steps of the in vitro culture were performed using standard protocols developed in our laboratory (Wood et al. 1995; Hribal et al. 2012). Briefly, ovaries were obtained from local veterinary clinics and stored in PBS (Gibco, Grand Island, NY, USA) at 4°C for up to 24 h. Immature cat oocytes (n = 2126 total; 29 replicates) were collected from adult ovaries and matured in 50 μl droplets of Medium A (Sage, Pasadena, CA, USA) or B (Irvine Scientific, Santa Ana CA, USA) supplemented with 0.01% (v/v) FSH [Hormones: National Hormone and Pituitary Program (NHPP), Torrance, CA, USA], 0.01% LH, 0.16% E2 and 4% BSA (Sigma-Aldrich, St. Louis, MO, USA) added to Medium B only. The media used in this study had essentially the same salt composition and components with the exception of protein source. In Medium A, the serum protein substitute contained 88% normal human serum albumin in the presence of 12% α and β globulins. After 26–28 h of maturation, oocytes were fertilized with 5.0 × 105 motile sperm per ml of frozen–thawed epididymal spermatozoa from different males. Oocytes and sperm cells were co-incubated for 16–18 h. A control for parthenogenesis (no sperm cell insemination) was included in each replicate. Presumptive zygotes then were cultured in vitro, and media was changed at day 4. For in vitro fertilization and embryo culture, Medium B was supplemented with 5% FCS (Irvine Scientific), whereas Medium A contained the same protein substitute. After 7 days of embryo development in vitro, proportions of different embryo stages were recorded according to previous criteria (Wood et al. 1995).
Immunostaining was carried out at 38°C unless otherwise noted. Day 7 blastocysts were fixed in 5% paraformaldehyde in PBS for 30 min. Following fixation, embryos were washed in saturation medium (20% FCS, +0.5%TX100 in PBS; Sigma-Aldrich) for 5 min, then blocked in saturation medium for 30 min before being incubated with a primary antibody (anti-OCT-4; Millipore, Billerica, MA, USA, 1 : 200; or anti-NANOG; AbCam, Cambridge, MA, USA, 1 : 200) at 4°C overnight. The following morning embryos were washed in medium (2% FCS + 0.5% TX100 in PBS) for 45 min before being incubated with the secondary antibody (FITC; Sigma-Aldrich, 1 : 100 in washing medium) for 1 h. A 30-min rinse was followed in washing medium. Chromatin was stained using 10 μg/ml Hoechst 33342 (Sigma-Aldrich) and 5 μg/ml propidium iodide (Sigma-Aldrich) for 10 min. Stained embryos were mounted in Vectashield® (Vector Laboratories, Burlingame, CA, USA) on a glass slide, sealed with nail polish and stored at 4°C.
Experimental design and statistical analysis
Domestic cat ovaries were recovered from local veterinary clinics during routine spay procedures. On a given day, recovered oocytes were evenly distributed between the two culture media. Resulting blastocysts (OCT-4, 4 replicates: Medium A, n = 12, Medium B, n = 18; NANOG, 5 replicates: Medium A, n = 27, Medium B, n = 11) were fixed and immunostained for the pluripotency proteins OCT-4 and NANOG (and determination of number of cells in the inner cell mass). Blastocysts from the remaining trials were preserved in lysis buffer for future gene expression analysis. The remaining non-blastocyst embryos were stained with propidium iodide and Hoechst to evaluate embryo stage. The rate of cleavage was recorded as well as the percentage of blastocysts produced from total oocytes fertilized. Proportions of cleaved cells reaching 8 cells, 8–16 cells, morula and blastocyst stages were transformed (arcsin) and analysed using an unpaired two-tailed t-test (Graphpad Prism 5; GraphPad Software, Inc., La Jolla, CA 92037 USA).
The percentages of cleaved embryos obtained with the two media were not different (p > 0.05; Table 1). Parthenogenetic activation was not observed in either culture medium. The proportions of each embryo stage including blastocyst formation were also similar (p > 0.05) between the two media (Table 1). A small proportion of blastocysts (~5%) were hatched after 7 days of culture regardless of the medium. There was no significant difference in the number of hatched blastocysts produced in either media (Fisher's exact test).
Table 1. Percentages of cleaved embryos and proportions of each developmental stage obtained after in vitro culture in the two different media. Values are expressed as mean ± SE
Number of oocytes
Proportion of embryo stages (per total cleaved)
74.1 ± 2.0
24.6 ± 2.3
24.9 ± 2.7
20.9 ± 2.6
29.6 ± 3.8
82.5 ± 2.6
29.5 ± 3.4
30.8 ± 3.1
17.4 ± 3.1
22.4 ± 3.2
There were no differences (p > 0.05) between media regarding average number of blastomeres in blastocysts (Medium A, 158.1 ± 11.1; Medium B, 174.6 ± 10.2) and proportion of inner cell mass in each embryo (Medium A, 23.7% ± 2.0; Medium B, 24.7% ± 1.5). There was no difference in the variance of any cleavage state (F-test).
Immunostaining revealed the presence of OCT-4 protein in nuclei of both trophectoderm and inner cell mass region, with a stronger signal in the latter (Fig. 1b) regardless of the culture medium. NANOG protein also was detected in the inner cell mass (Fig. 1e) of embryos from both media.
The quality of in vitro blastocysts can be defined by total number and uniformity of cells and the presence of pluripotency proteins NANOG and OCT-4. In this study, we found no difference between media and protein sources for the number of blastomeres in a given blastocyst. There was no observed difference in the proportion of cells in the inner cell mass between media and protein sources either. Concordantly, expression of the pluripotency proteins NANOG and OCT-4 was similar between the two culture conditions. We also found that the proportion of cleaved embryos for either medium was not significantly different. The cleavage proportions for each embryo stage (<8-cells, 8–16 cells, morulae and blastocyst) were similar between the two media. Both the rates of cleavage and proportion of embryos to reach the blastocyst stage were comparable to similar studies in the cat (Gómez et al. 2010). Both media produced good-quality blastocysts of high cell number that expressed NANOG and OCT-4 proteins. Our results confirmed that in a blastocyst, OCT-4 protein was present in the nuclei of both the trophectoderm and inner cell mass; however, it was more prominently in the inner cell mass. NANOG protein was found to be present in the inner cell mass only. Thus, cat blastocysts expressed pluripotency proteins in a pattern similar to the bovine model (Berg et al. 2011), porcine model (Kirchhof et al. 2000) as well as to previous results reported in the domestic cat (Gómez et al. 2010).
Protein source in culture media has been shown to have a critical effect on the maturation of oocytes and success of blastocyst formation. Previous studies have shown that BSA is best used for oocyte maturation, followed by FCS for fertilization and culture to the blastocyst stage, resulting in more hatched blastocysts with higher cell number comparatively (Wood et al. 1995; Dobrinsky et al. 1996; Karja et al. 2002; Han and Niwa 2003; Herrick et al. 2007). The media used in this study had the same components and salt composition, with the exception of protein source. In Medium A, the serum protein substitute contains 88% normal human serum albumin in the presence of 12% α and β globulins. The high content of polyhydroxy domains in the α and β globulins produces a weak gel-like environment that can enhance embryonic development both in human and mouse embryos (Weathersbee et al. 1995). The serum albumin protein sequences for human and cat are more similar than for cat and bovine, (H:C 82%; B:C 78%); however, there was no beneficial effect of using human serum over bovine. To the author's knowledge, this is the first time human serum (present in the serum protein substitute) has been used successfully for cat embryo culture. Interestingly, the media containing serum protein substitute performed equally well as the media that used BSA for maturation and FCS for fertilization and culture. Using the same protein source from in vitro maturation through embryo culture did not influence the quality of the resulting blastocysts.
This study demonstrates that high-quality blastocysts can be cultured in vitro for the domestic cat utilizing two commercially available media. Producing high-quality blastocysts consistently and abundantly in vitro will provide superior starting material for performing embryo transfer, derivation of embryonic stem cells and for additional research. Further advancement in the understanding of the nutritional requirements of pre-implantation embryos will enable improved media to be developed and generation of blastocysts that may more closely resemble in vivo.
We would like to thank Drs. Michael Cranfield and Brent Whitaker (Maryland Line Animal Rescue), Susan McDonough, DVM, and staff from Animal Birth Control, LLC, as well as the staff and volunteers of Baltimore Animal Rescue and Care Shelter for tissue donation. Funding for this project was provided by the University of Maryland-Smithsonian Institute Joint SEED Grant.
Conflicts of interest
The authors declare that there are no conflicts of interest that could be perceived as prejudicing the impartiality of the research reported.
EN, JG, CK, and PC provided substantial contributions to conception and design, analysis and interpretation of data, critical revision and final approval of the version to be published. EN was also responsible for acquisition of data and drafting the article.