A simple and fast alternative method to remove glycerol from chicken semen after cryopreservation.

A concentration of 11% of glycerol is the standard one for sperm cryopreservation in chickens, however, the presence of just 2% glycerol already causes severe fertility reduction, suggesting the necessity of removing glycerol before artificial insemination (AI). The major approach developed for this purpose is serial dilution followed by centrifugation (SDC), which demands special equipment (such as a refrigerate room) to maintain post-thaw semen at 4 °C, besides being time consuming. Therefore, we attempted to develop a simple method to remove glycerol from chicken frozen-thawed semen based on a colloidal gel, Percoll, which is ordinarily used to select motile and viable sperm in mammals as well as in fresh chicken semen. In this study, we used a Percoll based glycerol removal solution (GRS) containing sucrose to avoid frozen-thawed sperm suffering from osmotic stress. Subsequently, several conditions including GRS compositions (GRS A, B, C and D) and centrifugation temperatures (4 and 20 °C) were compared by their influence on sperm in vitro parameters. Afterwards, GRS A and D were selected for fertility evaluation, compared to conventional SDC method. Our results showed that the fertility with GRS A at both 4 and 20 °C were higher than GRS D (p < 0.05) and similar or even superior to the fertility obtained with SDC method. Altogether, our novel GRS protocol is a valuable method for chicken sperm cryobanking policy, supported by its notable results of fertility as well as saving 44% of time, with a simple equipment at flexible operation temperatures of 4 or 20 °C.

Exploring how sucrose-colloid selection improves the fertilizing ability of chicken sperm after cryopreservation with glycerol.

Currently, glycerol is the most effective cryoprotectant when combined with straw packaging for preserving chicken sperm. Glycerol, however, has toxic effects on sperm cells, which can reduce fertility when present in inseminated semen. Historically, the serial dilution (SD) method was developed to eliminate glycerol and mitigate its adverse effects. We have recently developed a new method for removing glycerol called sucrose-Percoll (SP), that can be performed at either 4°C (4°C-SP) or 20°C (20°C-SP). This SP protocol has been found to be simpler and faster to improve fertility compared to the traditional SD method. Nevertheless, the reasons for such effectiveness differences between glycerol removal procedures remained unclear and required more comprehensive understandings for future protocol developments. Here, we examined the effects of SP and SD protocols on the fertility duration. We also investigated the potential causes of varying effects of these methods by analyzing sperm quality parameters and sperm storage in the hen's reproductive tract. The fertility was significantly higher in 4°C-SP than 20°C-SP during the first 6 d after insemination, and also higher than sperm processed using SD. No difference was observed between 20°C-SP and SD between 7 and 13 d. However, a 2.7-time higher fertility was shown with 4°C-SP. In addition, the SP method demonstrated a 2-fold greater ability to remove glycerol than the SD method. Sperm centrifuged at 4°C-SP exhibited higher sperm storage compared to 20°C-SP and were higher than sperm treated with SD. Overall, our findings revealed that the differences in efficiencies between SP and SD methods were not related to in vitro sperm quality but resulted from a higher ability to remove glycerol, a higher storage capacity in the female reproductive tract, and a longer fertility ability. Since no impacts were observed in sperm cellular characteristics, further experiments are necessary to investigate the influences of glycerol removal treatments at the molecular level.

Is glycerol a good cryoprotectant for sperm cells? New exploration of its toxicity using avian model.

Glycerol is a cryoprotectant used widely for the cryopreservation of animal sperm, but it is linked to a decrease in fertility. The mechanism underlying the negative effects of glycerol remains unclear. Therefore, in this study, we aimed to gain a better understanding by using the chicken model. First, we investigated the impact of increasing the concentration of glycerol during insemination on hen fertility. Our findings revealed that 2% glycerol resulted in partial infertility, while 6% glycerol led to complete infertility. Subsequently, we examined the ability of sperm to colonize sperm storage tubules (SST) during in vivo insemination and in vitro incubation. The sperm used in the experiment were stained with Hoechst and contained 0, 2, or 6% glycerol. Furthermore, we conducted perivitelline membrane lysis tests and investigated sperm motility, mitochondrial function, ATP concentration, membrane integrity, and apoptosis after 60 min of incubation with different glycerol concentrations (0%, 1%, 2%, 6%, and 11%) at two temperatures to simulate pre-freezing (4 °C) and post-insemination (41 °C) conditions. Whereas 2% glycerol significantly reduced 50% of sperm containing SST, 6% glycerol completely inhibited SST colonization in vivo. On the other hand, in vitro incubation of sperm with SST revealed no effect of 2% glycerol, and 6% glycerol showed only a 17% reduction in sperm-filled SST. Moreover, glycerol reduced sperm-egg penetration rates and also affected sperm motility, bioenergetic metabolism, and cell death at 4 °C. These effects were observed when the concentration of glycerol exceeded 6%. Furthermore, at 41 °C, glycerol caused even greater damage, particularly in terms of reducing sperm motility. These data altogether reveal important effects of glycerol on sperm biology, sperm migration, SST colonization, and oocyte penetration. This suggests that glycerol plays a role in reducing fertility and presents opportunities for improving sperm cryopreservation.

FOXL2 is regulated during the bovine estrous cycle and its expression in the endometrium is independent of conceptus-derived interferon tau.

FOXL2, a winged-helix/forkhead domain transcription factor, is a key gene involved in the differentiation and biological functions of the ovary. In a recent transcriptomic analysis, we found that FOXL2 expression in bovine caruncular endometrium was different from that in intercaruncular endometrium. In order to gain new insights into FOXL2 in this tissue, we determined the expression of this transcription factor during the estrous cycle and the establishment of pregnancy in cattle. The endometrial expression of FOXL2 did not vary during maternal recognition of pregnancy (Days 16-20). Using an in vivo bovine model and primary cell cultures, we showed that FOXL2 was not an interferon-tau target gene. Both FOXL2 transcript and protein were expressed from Day 5 to Day 20 of the estrous cycle, and their levels showed a significant increase during the luteolytic phase. A 2-day progesterone supplementation in heifers led to a clear down-regulation of FOXL2 protein levels, suggesting the negative impact of progesterone on FOXL2 expression. Immunohistochemistry data revealed the localization of FOXL2 in endometrial stromal and glandular cells. FOXL2 subcellular distribution was shown to be nuclear in endometrial samples collected during the luteolytic period, while it was not detected in nuclei during the luteal phase and at implantation. Collectively, our findings provide the first evidence that FOXL2 is involved in the regulation of endometrial tissue physiology.

Microbiota and stress: a loop that impacts memory.

Chronic stress and the gut microbiota appear to comprise a feed-forward loop, which contributes to the development of depressive disorders. Evidence suggests that memory can also be impaired by either chronic stress or microbiota imbalance. However, it remains to be established whether these could be a part of an integrated loop model and be responsible for memory impairments. To shed light on this, we used a two-pronged approach in Japanese quail: first stress-induced alterations in gut microbiota were characterized, then we tested whether this altered microbiota could affect brain and memory function when transferred to a germ-free host. The cecal microbiota of chronically stressed quails was found to be significantly different from that of unstressed individuals with lower α and ß diversities and increased Bacteroidetes abundance largely represented by the Alistipes genus, a well-known stress target in rodents and humans. The transfer of this altered microbiota into germ-free quails decreased their spatial and cue-based memory abilities as previously demonstrated in the stressed donors. The recipients also displayed increased anxiety-like behavior, reduced basal plasma corticosterone levels and differential gene expression in the brain. Furthermore, cecal microbiota transfer from a chronically stressed individual was sufficient to mimic the adverse impact of chronic stress on memory in recipient hosts and this action may be related to the Alistipes genus. Our results provide evidence of a feed-forward loop system linking the microbiota-gut-brain axis to stress and memory function and suggest that maintaining a healthy microbiota could help alleviate memory impairments linked to chronic stress.

Protein Cargo of Extracellular Vesicles From Bovine Follicular Fluid and Analysis of Their Origin From Different Ovarian Cells.

Follicular fluid (FF) fills the interior portion of the ovarian antral follicle and provides a suitable microenvironment for the growth of the enclosed oocyte through molecular factors that originate from plasma and the secretions of follicular cells. FF contains extracellular nanovesicles (ffEVs), including 30-100-nm membrane-coated exosomes, which carry different types of RNA, proteins, and lipids and directly influence oocyte competence to develop embryo. In the present study, we aimed to characterize the protein cargo of EVs from the FF of 3-6-mm follicles and uncover the origins of ffEVs by assessing expression levels of corresponding mRNAs in bovine follicular cells and oocyte and cell proteomes. Isolated exosome-like ffEVs were 53.6 + 23.3 nm in size and could be internalized by cumulus-oocyte complex. Proteomes of ffEVs and granulosa cells (GC) were assessed using nanoflow liquid chromatography coupled with high-resolution tandem mass spectrometry after the gel fractionation of total proteins. In total, 460 protein isoforms corresponding to 322 unique proteins were identified in ffEVs; among them, 190 were also identified via GC. Gene Ontology terms related to the ribosome, protein and RNA folding, molecular transport, endocytosis, signal transduction, complement and coagulation cascades, apoptosis, and developmental biology pathways, including PI3K-Akt signaling, were significantly enriched features of ffEV proteins. FfEVs contain numerous ribosome and RNA-binding proteins, which may serve to compact different RNAs to regulate gene expression and RNA degradation, and might transfer ribosomal constituents to the oocyte. Majority of genes encoding ffEV proteins expressed at different levels in follicular cells and oocyte, corroborating with numerous proteins, which were reported in bovine oocyte and cumulus cells in other studies thus indicating possible origin of ffEV proteins. The limited abundance of several mRNAs within follicular cells indicated that corresponding ffEV proteins likely originated from circulating exosomes released by other tissues. Analysis of bovine ffEV transcriptome revealed that mRNAs present in ffEV accounted for only 18.3% of detected ffEV proteins. In conclusion, our study revealed numerous proteins within ffEVs, which originated from follicular and other cells. These proteins are likely involved in the maintenance of follicular homeostasis and may affect oocyte competence.

Maternal recognition of pregnancy and implantation are not associated with an interferon response of the endometrium to the presence of the conceptus in dromedary camel.

Maternal recognition of pregnancy (MRP) and implantation involve appropriate interactions between the elongating conceptus and the receptive endometrium that will condition development of the feto-placental unit to term. Molecular mechanisms that take place at the conceptus-endometrium interface during early pregnancy have been extensively investigated in domestic ungulates but they are still poorly understood in camelids including the dromedary camel (Camelus dromedarius), a domestic species with important economic and social roles in arid and semi-arid areas. In order to better understand how MRP and implantation take place in the left horn of this species, we investigated expression levels of genes encoding steroid hormones (PGR, ESR1), transcription factors (STAT1, FOXL2), interferon stimulated genes (MX1, MX2, OAS1, RSAD2) including SOCS genes (SOCS1, SOCS2, SOCS3 and CISH), previously identified as conceptus regulated genes in the endometrium of other domestic animals. Using endometrial tissue collected from left and right uterine horns of dromedary camel females that were non pregnant or early pregnant, gene expression of these genes was detected and our results provided first insights on their regulation, showing that (i) conceptus implantation is not associated with an IFN response in the pregnant uterine horn (ii) when regulation of classical interferon-stimulated genes (ISG) occurs, it takes place during the formation of the feto-placental unit, and (iii) gene expression can differ between the left and right uterine horns during implantation and early placentation phase. Additional experiments will be required in dromedary camels to understand the unusual regulation of ISG during implantation as well as to determine the molecular processes that drive the systematic implantation of the elongating conceptus in the left uterine horn.