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The synepitheliochorial placenta of ruminants: Binucleate cell fusions and hormone production

The objective was to characterize transcriptome changes associated with elongation in bovine conceptuses during preimplantation stages. Nonlactating Holstein cows were euthanized 17 d after artificial insemination (AI) and the uterine horn ipsilateral to the CL was flushed with saline solution. Recovered conceptuses were classified as small (1.2 to 6.9 cm; n = 9), medium (10.5 to 16.0 cm; n = 9), or large (18.0 to 26.4 cm; n = 10). Total mRNA was extracted and subjected to transcriptome analyses using the Affymetrix Gene Chip Bovine array. Data were normalized using the GCRMA method and analyzed by robust regression using the Linear Models for Microarray library within Bioconductor in R. Transcripts with P ≤ 0.05 after adjustment for false discovery rate and fold change ≥1.5 were considered differentially expressed. Functional analyses were conducted using the Ingenuity Pathway Analysis platform. Comparisons between large versus small (LvsS), large versus medium (LvsM), and medium versus small (MvsS) conceptuses yielded a total of 634, 240, and 63 differentially expressed transcripts, respectively. Top canonical pathways of known involvement with embryo growth that were upregulated in large conceptuses included actin cytoskeleton (LvsS), integrin signaling (LvsS and LvsM), ephrin receptor (LvsS), mesenchymal transition by growth factor (LvsM), and regulation of calpain protease (LvsS). Transcripts involved with lipid metabolism pathways (LXR/RXR, FXR/RXR, hepatic fibrosis) were associated with the LvsS and LvsM, and some transcripts such as APOC2, APOH, APOM, RARA, RBP4, and PPARGC1A, were involved in these pathways. An overall network summary associated biological downstream effects of invasion of cells, proliferation of embryonic cells, and inhibition of organismal death in the LvsS. In conclusion, differently expressed transcripts in the LvsS comparison were associated with the cell growth, adhesion, and organismal development, although part of these findings could be attributed to differences in circulatory concentrations of progesterone of the cows that bore large and small conceptuses. The large and medium conceptuses developed under similar concentrations of progesterone and presented 240 differently expressed transcripts, associated with cell differentiation, metabolite regulation, and other biological processes.

Reproductive failure is one of the main performance constraints in ruminant livestock. Transmissible agents such as Toxoplasma gondii and Neospora caninum are commonly involved in the occurrence of abortion in ruminants, but little is known about the mechanisms involved. While in vivo models are optimal for the study of abortion pathogenesis, they have a high economic cost and come with ethical concerns. Unfortunately, alternative in vitro models fail to replicate the complex in vivo placental structure. To overcome the limitations of currently available models, we developed an ex vivo model based on the cultivation of fresh and cryopreserved sheep placental explants, enabling the biobanking of tissues. Reproducible and simple markers of tissue integrity (histology, RNA concentrations), viability (resazurin reduction), and functionality (synthesis of steroid hormones) were also investigated, allowing a clear quality assessment of the model. This work shows that, similar to fresh explants, tissues cryopreserved in ethylene glycol using slow freezing rates maintain not only their structure and function but also their receptivity to T. gondii and N. caninum infection. In addition, the findings demonstrate that explant lifespan is mainly limited by the culture method, with protocols requiring improvements to extend it beyond 2 days. These findings suggest that cryopreserved tissues can be exploited to study the initial host‒pathogen interactions taking place in the placenta, thus deepening the knowledge of the specific mechanisms that trigger reproductive failure in sheep. Importantly, this work paves the way for the development of similar models in related species and contributes to the reduction of experimental animal use in the future.

Newborn ruminants depend on colostrum intake immediately after birth to obtain immunoglobulins for effective transfer of passive immunity (TPI). As colostrum may also be a vehicle of infectious agents, heat treatment of raw colostrum is a practice aimed at eliminating or reducing its pathogen load. Despite the usefulness of heat treatment in preventing the transmission of infectious colostrum-borne diseases, heat treatment of colostrum may have some side effects. A systematic review and meta-analysis were conducted to summarize the effects of colostrum heat treatment on colostral viscosity and IgG concentration, and serum IgG concentration as a proxy for TPI in newborn calves fed raw versus heat-treated colostrum. Moderators were studied to identify sources of heterogeneity. Literature databases were searched for peer-reviewed articles published between 1946 and 2022. A Master of Science thesis was also included. Five, 21, and 19 original publications were quantitatively evaluated in 3 separate meta-analyses, based on predefined selection criteria. Two-level and 3-level random-effects meta-analysis revealed a significant overall effect of heat treatment on colostral viscosity and IgG concentration, and serum IgG concentration in newborns. Heat-treated colostrum had significantly higher viscosity (21.0 cP, 95% CI: 3.8 to 38.2) and lower IgG concentration (−7.4 g/L, 95% CI: −11.1 to −3.7) compared with raw colostrum. Overall, newborn calves fed heat-treated colostrum had higher serum IgG concentrations (2.8 g/L, 95% CI: 1.4 to 4.0) 24–48 h after birth than those fed with raw colostrum. Particularly, this positive effect on the serum IgG concentrations was seen when colostrum was heat-treated at ≤60°C (2.9 g/L, 95% CI: 0.9 to 4.2) and when the standard low-temperature low-time (LTLT) method was used for heat treatment (2.6 g/L, 95% CI: 0.1 to 5.1). Colostrum treated at >60–63.5°C tended to have higher viscosity (275.6 cP, 95% CI: −37.9 to 589.3) and had lower IgG concentration (−21.7 g/L, 95% CI: −27.3 to −16.1). Calves fed colostrum treated at this temperature range had significantly lower serum IgG (−4.2 g/L, 95% CI: −7.9 to −0.4) compared with those fed raw colostrum. Heat treatment of colostrum at 72–76°C was not associated with a significant increase in colostral viscosity (6.3 cP, 95% CI: −324.3 to 336.9) nor a reduction in IgG colostral concentration (−13.1 g/L, 95% CI: −26.5 to 0.2), but calves fed colostrum treated at this temperature range had a significant reduction in serum IgG (−11.3 g/L, 95% CI: −17.1 to −5.4). Feeding newborn calves with colostrum heat-treated at ≤60°C by the standard LTLT method, particularly within 2 h after birth, resulted in increased serum IgG concentration at 24–48 h of age. Importantly, delaying feeding of heat-treated colostrum to newborns beyond 2 h of age resulted in no significant difference in IgG serum levels compared with feeding raw colostrum, highlighting the importance of early administration of heat-treated colostrum to favor TPI. On-farm colostrum heat treating should achieve an equilibrium between pathogen elimination and the preservation of colostral immunoglobulins while minimizing undesired increases in viscosity. The beneficial effects of colostrum heat treatment on TPI can be negligible if colostrum feeding is not performed within 2 h after birth.

Rumen-protected Lys (RPL) fed to Holstein cows prepartum resulted in a greater intake and improved health of their calves during the first 6 wk of life. However, whether increased supply of Lys in late gestation can influence placental tissue and, if so, which pathways are affected remain to be investigated. Therefore, we hypothesize that feeding RPL during late gestation could modulate placental metabolism, allowing for improved passage of nutrients to the fetus and thus influencing the offspring development. Therefore, we aimed to determine the effects of feeding RPL (AjiPro-L Generation 3, Ajinomoto Health and Nutrition North America) prepartum (0.54% DM of TMR) on mRNA gene expression profiles of placental samples of Holstein cows. Seventy multiparous Holstein cows were randomly assigned to 1 of 2 dietary treatments, consisting of TMR top-dressed with RPL (PRE-L) or without (control, CON), fed from 27 ± 5 d prepartum until calving. After natural delivery (6.87 ± 3.32 h), placentas were rinsed with physiological saline (0.9% sodium chloride solution) to clean any dirtiness from the environment and weighed. Then, 3 placentomes were collected, one from each placental region (cranial, central, and caudal), combined and flash-frozen in liquid nitrogen to evaluate the expression of transcripts and proteins related to protein metabolism and inflammation. Placental weights did not differ from cows in PRE-L (15.5 ± 4.03 kg) and cows in CON (14.5 ± 4.03 kg). Feeding RPL prepartum downregulated the expression of NOS3 (nitric oxide synthase 3), involved in vasodilation processes, and SOD1, which encodes the enzyme superoxide dismutase, involved in oxidative stress processes. Additionally, feeding RPL prepartum upregulated the expression of transcripts involved in energy metabolism (SLC2A3, glucose transporter 3; and PCK1, phosphoenolpyruvate carboxykinase 1), placental metabolism and cell proliferation (FGF2, fibroblast growth factor 2; FGF2R, fibroblast growth factor 2 receptor; and PGF, placental growth factor), Met metabolism (MAT2A, methionine adenosyltransferase 2-α), and tended to upregulate IGF2R (insulin-like growth factor 2 receptor). Placental FGF2 and LRP1 (low-density lipoprotein receptor-related protein 1) protein abundance were greater for cows that received RPL prepartum than cows in CON. In conclusion, feeding RPL to prepartum dairy cows altered uteroplacental expression of genes and proteins involved in cell proliferation, and in metabolism and transport of glucose. Such changes are illustrated by increased expression of SLC2A3 and PCK1 and increased protein abundance of FGF2 and LRP1 in uteroplacental tissue of cows consuming RPL.

Here, we report that a chemical cocktail (LCDM: leukemia inhibitory factor [LIF], CHIR99021, dimethinedene maleate [DiM], minocycline hydrochloride), previously developed for extended pluripotent stem cells (EPSCs) in mice and humans, enables de novo derivation and long-term culture of bovine trophoblast stem cells (TSCs). Bovine TSCs retain developmental potency to differentiate into mature trophoblast cells and exhibit transcriptomic and epigenetic (chromatin accessibility and DNA methylome) features characteristic of trophectoderm cells from early bovine embryos. The bovine TSCs established in this study will provide a model to study bovine placentation and early pregnancy failure.