Safety concerns over cell-derived pharmaceutical products being manufactured in supplements of fetal bovine serum (FBS) have ignited pleas to replace FBS. Herein, four newly marketed alternatives to FBS were compared: a xeno-free product called Cell-Ess®, a human platelet lysate marketed as GroPro®, and two mixtures of adult bovine serum varying in their proportions of neonatal growth factors, called Liporo® and FetalGro®. An endothelial cell line (C2BBe1) and a neuronal cell line (SHSY5Y) near confluency in media with 10% FBS were selectively scraped and taken through a 25-day step-wise algorithm to replace FBS, and another human endothelial cell line (HRA-19) was studied to replicate C2BBe1. Cells were stained, counted, and compared for viability, migration, and spheroids. The C2BBe1 and HRA-19 cell lines failed to proliferate in 10% Cell-Ess® but grew in 10% GroPro® or 10% FetalGro® reasonably well compared to reference 10% FBS. With SH-SY5Y, only FetalGro® approached FBS's efficacy. These were all inferior to 11 different branded lots of FBS (positive controls), but five days into switching just amongst the FBS brands, 4 of 11 supported less proliferation than reference FBS in endothelial HRA-19 (p < 0.004). Moreover, neurospheres were enriched in two branded lots of FBS and FetalGro® (each p < 0.004), neurospheres being an unwanted phenotype for any neuronal cell application. Because platelet-derived GroPro® stood out amongst the non-FBS growth supplements to allow proliferation without inducing spheroids, it seems the best (mindful that the cells still grew slower in it compared to FBS). While no perfect replacement was found amongst the alternatives to FBS, the algorithm for switching should be useful in future testing of new alternatives to FBS as the need arises to switch from FBS and expand pharmaceutical products with safety for human use.

Interferon tau (IFNT) is the cytokine responsible for the maternal recognition of pregnancy in ruminants and plays a role modulating embryo–maternal communication in the oviduct inducing a local response from immune cells. We aimed to investigate IFNT production, reactive oxygen species, and oxidative stress under the influence of heat stress (HS) during different stages of bovine in vitro embryo production. HS was established when the temperature was gradually raised from 38.5°C to 40.5°C in laboratory incubator, sustained for 6 hr, and decreased back to 38.5°C. To address the HS effects on IFNT production, reactive oxygen species, and oxidative stress, ovaries from a slaughterhouse were used according to treatments: control group (38.5°C); oocytes matured under HS; oocytes fertilized under HS; zygotes cultured in the first day under HS; and cells submitted to HS at oocyte maturation, fertilization, and the first day of zygote culture. The HS negatively affected cleavage and blastocyst rates, in all HS groups. On Day 7, all HS‐treated embryos showed decrease IFNT gene and protein expressions, whereas reactive oxygen species were increased in comparison to the control. In conclusion, the compromised early embryo development due to higher temperatures during in vitro oocyte maturation, fertilization, and/or zygote stage have diminished IFNT expression and increased reactive oxygen species in bovine.

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Summary The Hippo pathway is involved in the proliferation of intrafollicular cells and in early embryonic development, mainly because effectors of this pathway are key transcription regulators of genes such as CTGF and CYR61, which are involved in cell proliferation. Recent studies by our group found that fibroblast growth factor 18 (FGF18) is present in the fallopian tube during early embryonic development, leading to the hypothesis that FGF18 may have a role during embryonic development. Therefore, the aim of the following study was to determine whether FGF18 modulates the expression of Hippo pathway target genes, CTGF and CYR61, during oocyte maturation and early embryonic development. Three experiments were carried out, with in vitro maturation (IVM) of cumulus–oocyte complexes (COCs) and embryo culture. In experiment one, FGF18 (100 ng/ml) induced an increase (P < 0.05) in CTGF gene expression at 12 h post-exposure. In experiment two, FGF18 (100 ng/ml) induced a reduction (P < 0.05) in CTGF expression at 3 h post-exposure. In the third experiment, day 7 embryos exposed to FGF18 during oocyte IVM expressed greater CTGF mRNA abundance, whereas FGF18 exposure during embryo in vitro embryo culture did not alter CTGF expression in comparison with untreated controls. The preliminary data presented here show that FGF18 modulates CTGF expression in critical periods of oocyte nuclear maturation, cumulus expansion and early embryonic development in cattle.

The interaction between early embryo and maternal immune system for the establishment of pregnancy is the focus of several studies; however, it remains unclear. The maternal immune response needs to keep a balance between avoiding any damage to the conceptus and maintaining its function in combating microbes as well. When conceptus-maternal crosstalk cannot achieve this balance, pregnancy losses might occur. Intercommunication between mother and conceptus is fundamental during early pregnancy to dictate the outcome of pregnancy. In ruminants, the embryo reacts with the maternal system mainly via interferon tau (IFNT) release. IFNT can act locally on the embryo and endometrial cells and systemically in several tissues and cells to regulate their response via the expression of interferon-stimulated genes (ISGs). Also, IFNT can induce the expression of inflammatory-related genes in immune cells. Day 7 embryo induces a shift in the maternal immune response towards anti-inflammatory (Th2) immune responses. During maternal recognition of pregnancy, peripheral mononuclear cells (PBMCs) and polymorphonuclear cells (PMNs) express markers that configure an anti-inflammatory response. However, PMNs response is more sensitive to the effects of IFNT. PMNs are more likely to express interferon-stimulated genes (ISGs), transforming growth factor-beta (TGFB), interleukin 10 (IL10), and arginase-1 (ARG1), configuring one of the most rapid immune responses to early pregnancy. This review focus on the local and peripheral immune responses during early pregnancy in ruminants, mainly the PMNs function in the immune system.

This study aimed to assess liver damage and interferon-stimulated gene 15 (ISG15) blood expression as a consequence of embryonic signaling on maternal recognition of pregnancy in beef cattle presenting natural ingestion of Senecio spp. Epidemiological aspects, as the presence of the plant, associated to gamma glutamyl transferase (GGT) activity can be used as Senecio spp. poisoning diagnosis. Maternal recognition of pregnancy period occurs when the embryo secretes interferon tau (IFNT) to signal its presence to the mother and eventually extend corpus luteum (CL) lifespan. In our study, liver damage was determined by concentration serum GGT, cytological and histopathological examinations. Reproductive status was evaluated by concentration of progesterone, CL diameter and ISG15 mRNA expression on Day 19 following fixed-time artificial insemination (FTAI). Cows were categorized into two groups based on concentration of GGT: Group 1 (GGT<30U/L) and 2 (GGT>31U/L). No difference on body condition scores was observed. All the cows presented liver damage based on cytology and histopathological exams. Cows from the Group 1 had higher pregnancy rate, presenting larger CL diameter and greater concentration of progesterone. Interestingly, ISG15 mRNA expression had no difference between Groups 1 and 2, even presenting difference in pregnancy status. These findings suggest embryonic loss beyond Day 19. It suggests late embryonic mortality may be associated to liver insufficiency. In conclusion, liver injury and/or concentration of GGT does not alter ISG15 expression on blood neutrophils, however cows presenting lower concentration of GGT (<30U/L) had increased pregnancy status. Therefore, the concentration of GGT allow us to screen liver status and foresee a successful pregnancy in beef cattle.

Context The establishment of pregnancy in cows requires uterine activity regulation of the main Hippo signalling effector yes-associated protein 1 (YAP). It remains unknown (1) how YAP activity at the corpus luteum (CL) correlates with early pregnancy-related events in ruminants; and (2) if YAP activity in the uterus and CL can be affected by metabolic disorders that may lead to pregnancy failure in ruminants. Aims and methods To determine the effect of early pregnancy on total and phospho-YAP expression and its transcriptional activity in the CL, we compared non-pregnant vs pregnant ewes. To understand the YAP activity dysregulation with disorders that may result in pregnancy loss, we induced negative energy balance in pregnant ewes. Key results and conclusions Our main results indicate that early pregnancy alters the expression and activity patterns of YAP in the ovine CL but not in the endometrium. In addition, while our NEB-induced model fails to alter YAP activity at the endometrium level, we found that fasting during the first but not second week of pregnancy affects YAP activity in the CL of pregnant ewes. Implications The data presented herein provide considerable insight into the activity of a signalling pathway that may be a key player in pregnancy recognition and establishment in ewes.