The Puma concolor population has been decreasing during the last 30 years. Semen cryopreservation of this species has been accomplished successfully and offers the possibility of preserving endangered species. We previously showed that fertilizing capability of wild felid spermatozoa can be evaluated using intracytoplasmic sperm injection (ICSI) with in vitro-matured domestic cat oocytes (Moro et al. 2014 Reprod. Domest. Anim. 49, 693-700). Due to the lack of homologous oocytes, we evaluated the capability of the Puma concolor sperm to induce domestic cat oocyte fertilization and subsequent pre-implantation embryo development. In the present study, cryopreserved sperm obtained by electroejaculation from five different males were used for IVF of in vitro-matured (IVM) domestic cat oocytes. Straws were thawed by exposing them to air for 10 s and then immersing in a 37°C water bath for 30 s. The contents of the straws were poured into a sterile 1.5-mL microtube pre-warmed to 37°C. The sperm suspension was diluted (1:3 v/v) by the slow (drop-by-drop) addition of a modified Tyrode’s solution. For IVF, IVM oocytes (n = 370) were co-incubated with 0.5 × 105 motile spermatozoa mL−1 in an atmosphere of 21% O2 in air at 38.5°C for 18 to 20 h. Presumptive zygotes were cultured in vitro in 50-μL drops of modified Tyrode’s medium on 6.5% CO2 in air at 38.5°C. Cleavage was determined at 48 h post-fertilization, and 5% FBS was added at Day 5 of in vitro culture. Blastocyst stage was evaluated at Day 8. Results (mean ± SEM) showed a high cleavage rate (179/370, 49.0 ± 4.0%), and a high development to morula stage (137/370, 34.4 ± 7.2%), and to blastocyst stage (94/370, 23.4 ± 4.7%) for all males. These results indicated that Puma concolor spermatozoa can induce domestic cat oocyte activation and development to blastocyst stage in similar rates to domestic cat homologous IVF: IVM oocytes (n = 291), cleavage rate (199/291, 67.1 ± 6.1%), development to morula stage (144/291, 47.8 ± 4.9%), and to blastocyst stage (86/291, 30.1 ± 1.6%). In conclusion, we demonstrated that domestic cat oocyte can be used to evaluated cryopreserve sperm samples from another felid species.
Different feeder cells (FC) influence the isolation, proliferation, and self-renewal of cat embryonic stem cells (cat ESC; Gómez et al. 2010 Theriogenology 74, 498–515) possibly by secretion of growth factors that affect intracellular signalling pathways involved in self-renewal. Supplementation of the culture medium with fibroblast growth factor (FGF) stimulates the secretion of Activin A in mouse and human FC, which enhances undifferentiation in human ESC (Eiselleova et al. 2008 Int. J. Dev. Biol. 52, 353-363). Moreover, the Activin/Nodal pathway plays an important role in maintaining pluripotency of hESC through mechanism(s) in which FGF acts as a competence factor (Vallier et al. 2005 J. Cell Sci. 118, 4495–4509). Little is known about secretion of growth factors by cat FC and whether cat ESC use the activin/nodal pathway for their self-renewal. Our previous work has indicated that culturing cat ESC with bFGF enhances the stem cell replication and self-renewal (Gómez et al. 2010 Theriogenology 74, 498–515). Here we evaluated the effect of bFGF supplementation in the culture medium on the abilities of cat embryonic fibroblast (CEF) and mouse embryonic fibroblast (MEF) FC to: (1) secrete Activin A and (2) support undifferentiated growth of cat ESC. For experiment 1, mitomycin-C-treated CEF (n = 2) and MEF (n = 2) were, respectively, cultured with ESC medium supplemented with (1) LIF (1000 IU), (2) bFGF (10 ng mL–1), (3) LIF + bFGF, or (4) no factors. The medium for each condition was collected at 24 h after culture and Activin A protein concentration was detected with a feline Activin A-ELISA kit. Results showed that supplementation of ESC medium with bFGF with or without LIF significantly increased the secretion of Activin A in MEF (5256 and 7048 ng mL–1, respectively; P < 0.001), but not in CEF (150 and 131 ng mL–1, respectively). Moreover, differences in Activin A secretion were observed between both MEF cell lines (10 269 v. 2034 ng mL–1; P < 0.001). For experiment 2, cat ESC were cultured in CEF or MEF in the ESC medium supplemented with bFGF (10 ng mL–1), LIF (1000 UI), and an inhibitor of glycogen synthase kinase-3 β (GSK3-b), SB 216763 (2.1 µM mL–1). Results showed differences in morphology of cat ESC cultured in CEF or MEF, where colonies cultured in CEF had clearly defined borders and a tightly domed shape, with a high nucleus to cytoplasm ratio and prominent nucleoli. In comparison, ESC cultured in MEF had poorly defined borders and a flattened shape. In addition, the mean cell size of colonies at passage 8 (P8) cultured on CEF was larger (612 ± 0.9 µm) than that of those cultured on MEF (360 ± 0.5 µm; P < 0.001). Colonies cultured on MEF differentiated into fibroblast-like cells and other noncharacterised cell types after P8. These results clearly indicated that CEF do not secrete Activin A. The negative effect of Activin A on the morphology of cat ESC cultured on MEF may suggest a synergism between GSK3b inhibitor and Activin A that may induce differentiation, possibly into mesoendodermal cells (Teo et al. 2014 Stem Cell Rep. 3, 5–14). Studies that evaluate the effects of supplementing ESC medium with a lower concentration of Activin A may help to elucidate the importance of the Activin/Nodal pathway in cat ESC.
Canine oocyte invitro maturation (IVM) is one of the challenges of animal reproduction because of low maturation and high degeneration rates. In the bitch, after ovulation, oocytes remain in an immature stage and acquire their competence in the intra- and extrafollicular (oviductal) environments. Oxidative stress and reactive oxygen species affect canine oocytes, which can be related to the high amount of lipids they contain. Therefore, the use of antioxidants such as insulin-transferrin-selenium (ITS) and lower oxygen tension during IVM could be beneficial for oocyte maturation and survival. The purpose of this study was to determine an optimum IVM culture medium and to evaluate the effect of ITS and lower oxygen tension in canine IVM. In experiment 1, TCM-199 and synthetic oviductal fluid (SOF) media were evaluated for their ability to promote nuclear maturation at 72 and 48h of culture. Also, two protein sources were used: 8% bovine serum albumin (BSA) and 2.5% fetal bovine serum (FBS), and media were supplemented with hormones. The results revealed that SOF with FBS and BSA had similar results to TCM-199 supplemented with FBS after 72 and 48h of IVM (MII rates of 7% and 4% for the 72-h group, and 4% and 10% for the 48-h group). Synthetic oviductal fluid supplemented with BSA but without FBS produced significantly higher degeneration rates compared with SOF with FBS and BSA (44% and 23%, respectively). Forty-eight hours of IVM decreased degeneration rates, with higher MII rates compared with 72h of IVM. In experiment 2, SOF medium supplemented with FBS and BSA was chosen. Oocytes were cultured in SOF with FBS and BSA supplemented at two concentrations of ITS (1 and 10μLmL−1 ITS). Supplementation with 1μLmL−1 ITS demonstrated a beneficial effect by improving maturation rates up to 20%, compared to control and 10μLmL−1 supplemented group (4% and 6% MII, respectively) after 72h of IVM. For experiment 3, oocytes were cultured in SOF medium with or without ITS (0 and 1μLmL−1 ITS) under two oxygen tensions (5% and 20% O2) for 48h. Results from this experiment demonstrated that the combination of low oxygen tension and ITS (5% O2 and 1μLmL−1 ITS) improved maturation rates up to 26.2%, although there were no statistically significant differences compared with high oxygen and ITS (20% O2 and 1μLmL−1 ITS) and low oxygen without ITS (5% O2 and 0μLmL−1 ITS) groups. These treatments were able to increase MII rates compared with the control group (20% O2 and 0μLmL−1 ITS). Parthenogenetic activation was performed on the low oxygen with or without ITS supplemented groups. The untreated group generated higher degeneration rates after 7 days of culture, and cleavage rates were low for both groups. Nevertheless, an oocyte at the 8-cell stage was obtained in the ITS-supplemented group. Taken together, these results indicate that ITS supplementation and low oxygen tension during IVM improve canine oocyte maturation.
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