How to beat the bull: Lycopene as a tool to improve in vitro fertilization efficiency in bulls with high polyspermy.

High levels of reactive oxygen species (ROS) derived from in vitro conditions compromise oocyte quality and subsequent polyspermy prevention by the zona and membrane block. Antioxidant supplementation, like lycopene, during in vitro maturation (IVM) mitigates ROS effects, yet, its efficacy in blocking polyspermy remains uncertain. This study aims to evaluate the effect of lycopene supplementation during IVM on oocyte maturation, fertilization, and developmental parameters. To this end, bovine oocytes were supplemented with 0.2 µM lycopene and fertilized with semen from three bulls. The three bulls showed different fertilization potential in vitro, with bull 1 showing the highest penetration and polyspermy rates and the lowest in vitro fertilization (IVF) efficiency. Interestingly, in bull 1, the treatment with lycopene improved IVF efficiency (p = 0.043) and reduced the polyspermy rate (p = 0.028). However, none of these effects were observed in bulls 2 and 3. Bulls with higher penetration rates exhibited better blastocyst rates although those rates did not seem to be associated with polyspermy or IVF efficiency. Oocyte mitochondrial distribution and activity and cortical granule migration and distribution were not influenced by lycopene. In conclusion, we demonstrated that lycopene addition during oocyte maturation had a positive impact on IVF efficiency by reducing polyspermy rates in a bull-dependent manner. The reduction in polyspermy rates was not caused by changes in cortical granule migration or oocyte mitochondrial distribution. Lycopene must therefore induce other changes in the oocyte that lower the in vitro penetration rates of specific bulls prone to polyspermy.

The Complex Interplay of Insulin Resistance and Metabolic Inflammation in Transition Dairy Cows.

During the transition period, dairy cows exhibit heightened energy requirements to sustain fetal growth and lactogenesis. The mammary gland and the growing fetus increase their demand for glucose, leading to the mobilization of lipids to support the function of tissues that can use fatty acids as energy substrates. These physiological adaptations lead to negative energy balance, metabolic inflammation, and transient insulin resistance (IR), processes that are part of the normal homeorhetic adaptations related to parturition and subsequent lactation. Insulin resistance is characterized by a reduced biological response of insulin-sensitive tissues to normal physiological concentrations of insulin. Metabolic inflammation is characterized by a chronic, low-level inflammatory state that is strongly associated with metabolic disorders. The relationship between IR and metabolic inflammation in transitioning cows is intricate and mutually influential. On one hand, IR may play a role in the initiation of metabolic inflammation by promoting lipolysis in adipose tissue and increasing the release of free fatty acids. Metabolic inflammation, conversely, triggers inflammatory signaling pathways by pro-inflammatory cytokines, thereby leading to impaired insulin signaling. The interaction of these factors results in a harmful cycle in which IR and metabolic inflammation mutually reinforce each other. This article offers a comprehensive review of recent advancements in the research on IR, metabolic inflammation, and their intricate interrelationship. The text delves into multiple facets of physiological regulation, pathogenesis, and their consequent impacts.

The In Vitro Effects of Carprofen on Lipopolysaccharide-Induced Neutrophil Extracellular Trap Formation in Dairy Cows.

The objective of this study was to develop an in vitro model that mimics inflammatory reactions and neutrophil extracellular traps (NETs) formation by polymorphonuclear leukocytes (PMNs) in dairy cows. This model was used to examine the effect of carprofen (CA) on lipopolysaccharide (LPS)-induced NETs formation and expression of inflammatory factors. Peripheral blood samples were collected from 24 Holstein cows (3-11 days postpartum) and PMNs were isolated. In three replicates, PMNs were exposed to various treatments to establish an appropriate in vitro model, including 80 µg/mL of LPS for 2 h, followed by co-incubation for 1 h with 60 µmol/L CA and 80 µg/mL LPS. The effects of these treatments were evaluated by assessing NETs formation by extracellular DNA release, gene expression of pro-inflammatory cytokines, reactive oxygen species (ROS) production, and the expression of NETs-related proteins, including histone3 (H3), citrullinated histone (Cit-H3), cathepsin G (CG), and peptidyl arginine deiminase 4 (PAD4). The assessment of these parameters would elucidate the specific mechanism by which CA inhibits the formation of NETs through the PAD4 pathway instead of modulating the Nox2 pathway. This highlights CA's effect on chromatin decondensation during NETs formation. Statistical analyses were performed utilizing one-way ANOVA with Bonferroni correction. The results demonstrated that LPS led to an elevated formation of NETs, while CA mitigated most of these effects, concurrent the PAD4 protein level increased with LPS stimulating and decreased after CA administration. Nevertheless, the intracellular levels of ROS did not change under the presence of LPS. LPS supplementation resulted in an upregulation of H3 and Cit-H3 protein expression levels. Conversely, the CA administration inhibited their expression. Additionally, there was no change in the expression of CG with either LPS or LPS + CA co-stimulation. The gene expression of pro-inflammatory cytokines (tumor necrosis factor -α, interleukin (IL)-18, IL-1ß, and IL-6) upregulated with LPS stimulation, while the treatment with CA inhibited this phenomenon. In conclusion, CA demonstrated a pronounced inhibitory effect on both LPS-induced NETs formation as well as the associated inflammatory response.

Progesterone-independent endometrial mRNA expression in dairy cows with clinical or subclinical endometritis.

Up to 50 % of dairy cows fail to resolve uterine involution and develop chronic clinical (CE) or subclinical endometritis (SE) 21 days after calving. Clinical endometritis is associated with purulent discharge, while SE is not associated with overt clinical signs. Along with numerous knowledge gaps related to its pathogenesis, SE does not allow for a straightforward and effective therapy. Therefore, it is crucial to unravel differences in the expression of genes among healthy, CE, and SE cows. This might contribute to the discovery of new drug candidates and, in consequence, a potentially effective treatment. In the present study, cows between 21 and 28 days postpartum (PP) were examined using vaginoscopy for the presence of vaginal discharge and endometrial cytology for the determination of the endometrial polymorphonuclear cell (PMN) percentage. Next, an endometrial biopsy sample was taken to investigate the expression of 13 selected candidate genes by qPCR. Uterine health status was assigned to healthy (absence of abnormal vaginal discharge and ≤5 % PMN, n = 13), SE (absence of abnormal vaginal discharge and >5 % PMN, n = 30), and CE (mucopurulent or purulent vaginal discharge and >5 % PMN, n = 9). At the same time, a blood sample was collected to assess serum progesterone concentration and to categorize cows as low (≤1 ng/mL) or high (>1 ng/mL) in progesterone. High expression of IL1B, IL6, IL17A, CXCL8, PTGES, PTGS1, PTGS2, and INHBA genes and low expression of FST was noted in the endometrium of CE compared to healthy cows. Increased endometrial INHBA expression was observed in both SE and CE compared to healthy cows. Interestingly, greater expression of PTGES and PRXL2B genes and lower expression of PTGS2 were characteristic of SE versus CE or healthy. Among cows with no overt clinical symptoms of uterine disease (healthy and SE), the endometrial expression of IL1 B, CXCL8, and PTGES was greater in cows with high versus low serum progesterone. Several genes were differentially expressed among healthy, SE, and CE cows indicating different pathways for the development of different uterine diseases. In conclusion, we found progesterone-independent SE markers, which suggests that low endometrial PTGS2 expression may be indicative of an inadequate immune response and thus contribute to the pathogenesis of SE.

Detection of Chlamydia and Chlamydia-like organisms in bovine placental tissue.

This study aimed to investigate the presence of Chlamydia spp. and Parachlamydia acanthamoebae in bovine placental tissue originating from abortion and non-abortion cases in Belgium. Placentas of 164 late term bovine abortions (last trimester of gestation) and 41 non-abortion (collected after calving) cases were analysed by PCR for Chlamydia spp., Chlamydia abortus, C. psittaci and P. acanthamoebae. Additionally, a subset of 101 (75 abortion and 26 non-abortion cases) of these placenta samples were also analysed by histopathology to detect possible Chlamydia-induced lesions. In 5.4% (11/205) of the cases, Chlamydia spp. were detected, and three of those cases were positive for C. psittaci. Parachlamydia acanthamoebae was detected in 36% (75/205) of the cases, being 44% (n = 72) in abortions and 7.3% (n = 3) in non-abortions cases (p < .001). None of the cases was positive for C. abortus. Purulent and/or necrotizing placentitis with or without vasculitis was observed in 18.8% (19/101) of the histopathologically analysed placenta samples. In 5.9% (6/101) of the cases, placentitis was observed along with vasculitis. In the abortion cases, 24% (18/75) of the samples showed purulent and/or necrotizing placentitis, while purulent and/or necrotizing placentitis was visible in 3.9% (1/26) of the non-abortion cases. Placental lesions of inflammation and/or necrosis were present in 44% (15/34) of the cases where P. acanthamoebae was detected, while inflammation and/or necrosis was present in 20.9% (14/67) of the negative cases (p < .05). The detection of Chlamydia spp. and especially P. acanthamoebae, in combination with correlated histological lesions such as purulent and/or necrotizing placentitis and/or vasculitis in placental tissue following abortion, suggests a potential role of this pathogen in cases of bovine abortion in Belgium. Further in-depth studies are necessary to unravel the role of these species as abortifacient agents in cattle and to include them in bovine abortion monitoring programmes.

Development of a Microfluidic Chip Powered by EWOD for In Vitro Manipulation of Bovine Embryos.

Digital microfluidics (DMF) holds great potential for the alleviation of laboratory procedures in assisted reproductive technologies (ARTs). The electrowetting on dielectric (EWOD) technology provides dynamic culture conditions in vitro that may better mimic the natural embryo microenvironment. Thus far, EWOD microdevices have been proposed for in vitro gamete and embryo handling in mice and for analyzing the human embryo secretome. This article presents the development of the first microfluidic chip utilizing EWOD technology designed for the manipulation of bovine embryos in vitro. The prototype sustains the cell cycles of embryos manipulated individually on the chips during in vitro culture (IVC). Challenges related to the chip fabrication as well as to its application during bovine embryo IVC in accordance with the adapted on-chip protocol are thoroughly discussed, and future directions for DMF in ARTs are indicated.

Alternative Culture Systems for Bovine Oocyte In Vitro Maturation: Liquid Marbles and Differentially Shaped 96-Well Plates.

In vivo-matured oocytes exhibit higher developmental competence than those matured in vitro but mimicking the in vivo environment by in vitro conditions has been challenging. Until now, conventional two-dimensional (2D) systems have been used for in vitro maturation of bovine cumulus-oocytes-complexes (COCs). However, using such systems present certain limitations. Therefore, alternative low-cost methodologies may help to optimize oocyte in vitro maturation. Here, we used two different systems to culture COCs and evaluate their potential influence on embryo development and quality. In the first system, we used treated fumed silica particles to create a 3D microenvironment (liquid marbles; LM) to mature COCs. In the second system, we cultured COCs in 96-well plates with different dimensions (flat, ultra-low attachment round-bottom, and v-shaped 96-well plates). In both systems, the nuclear maturation rate remained similar to the control in 2D, showing that most oocytes reached metaphase II. However, the subsequent blastocyst rate remained lower in the liquid marble system compared with the 96-well plates and control 2D systems. Interestingly, a lower total cell number was found in the resulting embryos from both systems (LM and 96-well plates) compared with the control. In conclusion, oocytes matured in liquid marbles or 96-well plates showed no remarkable change in terms of meiotic resumption. None of the surface geometries influenced embryo development while oocyte maturation in liquid marbles led to reduced embryo development. These findings show that different geometry during maturation did not have a large impact on oocyte and embryo development. Lower embryo production after in vitro maturation in liquid marbles was probably detected because in vitro maturation was performed in serum-free medium, which makes oocytes more sensitive to possible toxic effects from the environment.

Detection of Anaplasma phagocytophilum in fetal and placental tissue of bovine abortions and perinatal mortalities.

BACKGROUND: Anaplasma phagocytophilum is a tick-borne zoonotic bacterium that is the aetiologic pathogen of tick-borne fever (TBF) in ruminants. In clinical bovine cases of TBF, abortion and stillbirth may be observed. However, in this regard, the pathophysiology of TBF has not yet been completely elucidated, and no clear guidelines to diagnose A. phagocytophilum-related abortions and perinatal mortalities (APM) are available. METHODS: This exploratory study aimed to investigate the presence of A. phagocytophilum in bovine cases of APM and determine whether placental or fetal spleen tissue has the greatest sensitivity for A. phagocytophilum identification. The placenta and fetal spleen of 150 late-term bovine APM cases were analysed using real-time PCR to detect A. phagocytophilum. RESULTS: A total of 2.7% of sampled placentas were positive for A. phagocytophilum, while none of the fetal spleen samples was. LIMITATIONS: No histopathology to detect associated lesions was performed. Consequently, no evidence of causality between the detection of A. phagocytophilum and APM events could be achieved. CONCLUSION: The detection of A. phagocytophilum suggests a potential role of this pathogen in bovine APM, and placental tissue seems to be the most suitable tissue for its identification.

Transcriptomics Reveal Molecular Differences in Equine Oocytes Vitrified before and after In Vitro Maturation.

In the last decade, in vitro embryo production in horses has become an established clinical practice, but blastocyst rates from vitrified equine oocytes remain low. Cryopreservation impairs the oocyte developmental potential, which may be reflected in the messenger RNA (mRNA) profile. Therefore, this study aimed to compare the transcriptome profiles of metaphase II equine oocytes vitrified before and after in vitro maturation. To do so, three groups were analyzed with RNA sequencing: (1) fresh in vitro matured oocytes as a control (FR), (2) oocytes vitrified after in vitro maturation (VMAT), and (3) oocytes vitrified immature, warmed, and in vitro matured (VIM). In comparison with fresh oocytes, VIM resulted in 46 differentially expressed (DE) genes (14 upregulated and 32 downregulated), while VMAT showed 36 DE genes (18 in each category). A comparison of VIM vs. VMAT resulted in 44 DE genes (20 upregulated and 24 downregulated). Pathway analyses highlighted cytoskeleton, spindle formation, and calcium and cation ion transport and homeostasis as the main affected pathways in vitrified oocytes. The vitrification of in vitro matured oocytes presented subtle advantages in terms of the mRNA profile over the vitrification of immature oocytes. Therefore, this study provides a new perspective for understanding the impact of vitrification on equine oocytes and can be the basis for further improvements in the efficiency of equine oocyte vitrification.

Studying the pre-implantation uterine microbiota in cattle using transabdominal laparoscopic low-volume lavage: Aiming for zero-contamination.

Recent studies have suggested that bacteria associated with the female reproductive tract - the uterine microbiota - may be important for reproductive health and pregnancy success. Therefore, uterine microbiome research gained much interest in the last few years. However, it is challenging to study late postpartum uterine samples, since they hold a low microbial biomass. Next-generation sequencing techniques are very sensitive for microbial identification, but they cannot make a distinction between actual microbiota and contaminant bacteria or their DNA. Our aim was to test a new method to sample the bovine uterine lumen in vivo, while minimizing the risk of cross-contamination. In order to evaluate this method, we performed a descriptive assessment of the microbial composition of the obtained samples. Transabdominal, laparoscopic sampling of the uterine lumen was conducted in five Holstein-Friesian cows. Uterine fluid from the uterine horns was collected by low-volume lavage. DNA from the samples was extracted using two different DNA extraction methods, and negative controls (sampling blank controls and DNA extraction blank controls) were included. Bacteria were identified using 16S rRNA gene amplicon sequencing. In this proof-of-concept study, no evidence for authentically present uterine microbiota could be found. During laparoscopic sampling, some practical challenges were encountered, and the reliability of low-volume-lavage for the collection of a low microbial biomass could be questioned. By comparing two DNA extraction methods, a significant contamination background could be noticed originating from the DNA extraction kits.

Circulating and endometrial cell oxidative stress in dairy cows diagnosed with metritis.

Dairy cows diagnosed with metritis may experience a greater degree of oxidative stress (OS) and a deficit in the antioxidative capacity compared to healthy cows. We aimed to assess circulating OS markers and endometrial cell mitochondrial function, intracellular reactive oxygen species (ROS) production, and mean endometrial nuclear cell area in postpartum cows diagnosed with metritis or as healthy. From an initial pool of 121 Holstein cows, we retrospectively selected 34 cows and balanced for metritis (n = 17) or healthy (n = 17). Metritis was defined as an enlarged uterus with red-brown watery or thick off-white purulent discharge occurring within 21 days postpartum. Cows with no signs of clinical disease (including dystocia or retained placenta) were referred to as healthy. Blood samples for serum reactive oxygen metabolites (d-ROM), antioxidants (OXY), and oxidative status index (OSI) tests, evaluated via photometric determination of plasma thiols, were performed at 7, 14, 21, 28, and 35 days postpartum. Furthermore, from the initial pool, a random subset of 5 cows diagnosed with metritis and 6 diagnosed as healthy we collected (at the same time points as for the blood samples) endometrial cytology samples using the cytobrush technique. From the uterine samples, we evaluated the endometrial cell mitochondrial function, intracellular ROS levels, and the endometrial cell nuclear area using MitoTracker Orange, dichlorodihydrofluorescein diacetate, and Hoechst 33258, respectively. Mixed linear regression models, accounting for repeated measurements, were fitted to assess the effect of metritis versus healthy on circulating and endometrial cell OS parameters and endometrial cell size. The effect of days postpartum and its interaction with uterine health status were forced into each model. Serum concentrations of d-ROMs and OSI were greater in metritis at 7, 14, and 35 days postpartum than in healthy cows. Interestingly, the mean endometrial cell nuclear area was lower in metritis than healthy cows at 14 and 21 days postpartum. We found no differences between metritis and healthy for endometrial cell mitochondrial function and intracellular ROS production. In conclusion, cows diagnosed with metritis experienced greater systemic OS levels than healthy cows, but their OS was not higher in the uterine milieu.

Oocyte developmental capacity is influenced by intrinsic ovarian factors in a bovine model for individual embryo production.

The ovary and its hormones may have major effects on the in vitro developmental capacity of the oocytes it contains. We related intrinsic ovarian factors namely the presence of corpus luteum (CL) and/or dominant follicle (>8 mm) and the follicular count to cumulus expansion (CE), embryo development, and blastocyst quality in a bovine model. Cumulus-oocyte-complexes (COCs) were aspirated from follicles between 4 and 8 mm in diameter. In vitro embryo production was performed in a fully individual production system. The follicular fluid from which COCs were collected was pooled (per ovary) to evaluate the estrogen, progesterone, and insulin-like growth factor-1 (IGF-1) concentrations. Cumulus oocyte complexes collected from ovaries without a CL presented a greater CE than COCs derived from ovaries bearing CL. The absence of ovarian structures increased the blastocyst rate when compared to oocytes derived from ovaries with a CL, a dominant follicle, or both. Blastocysts derived from ovaries without a dominant follicle presented higher total cell numbers and a lower proportion of apoptosis than blastocysts derived from ovaries containing a dominant follicle. Cumulus oocyte complexes collected from ovaries with high follicular count resulted in higher cleavage than from ovaries with low follicular count, but the blastocyst rate was similar between groups. Ovaries bearing a CL had greater progesterone and IGF-1 follicular fluid concentrations in neighboring follicles than ovaries without a CL. Selection for bovine ovaries without CL or dominant follicle can have positive effects on CE, embryo development, and blastocyst quality in an individual embryo production system set-up.

Maladaptation to the transition period and consequences on fertility of dairy cows.

After parturition, dairy cows undergo a plethora of metabolic, inflammatory, and immunologic changes to adapt to the onset of lactation. These changes are mainly due to the homeorhetic shift to support milk production when nutrient demand exceeds dietary intake, resulting in a state of negative energy balance. Negative energy balance in postpartum dairy cows is characterized by upregulated adipose tissue modelling, insulin resistance, and systemic inflammation. However, half of the postpartum cows fail to adapt to these changes and develop one or more types of clinical and subclinical disease within 5 weeks after calving, and this is escorted by impaired reproductive performance in the same lactation. Maladaptation to the transition period exerts molecular and structural changes in the follicular and reproductive tract fluids, the microenvironment in which oocyte maturation, fertilization, and embryo development occur. Although the negative effects of transition diseases on fertility are well-known, the involved pathways are only partially understood. This review reconstructs the mechanism of maladaptation to lactation in the transition period, explores their key (patho)physiological effects on reproductive organs, and briefly describes potential carryover effects on fertility in the same lactation.

Lycopene supplementation to serum-free embryo culture medium and its effect on development and quality of bovine blastocysts produced in vitro.

Selenium is commonly used as an antioxidant in a serum-free culture medium setting. However, lycopene has emerged as a potent antioxidant being twice as efficient as ß-carotene and 10 times as efficient as α-tocopherol with beneficial effects when supplemented in a serum-free maturation medium. Here, we aimed to evaluate the effect of lycopene supplementation in a serum-free culture medium on blastocyst development and quality. After in vitro maturation and fertilization, presumed zygotes were cultured in groups of 25 in 50 µl droplets of synthetic oviductal fluid. Culture medium supplementation was done using four experimental groups: insulin, transferrin, selenium (ITS, control); ITS + DMSO (diluent control); ITS + DMSO-lycopene 0.1 µM (ITSL); and IT + DMSO-lycopene 0.1 µM (ITL). DMSO was used as a diluent for lycopene. Blastocyst development among experimental groups was fitted in mixed-effects models, and blastocyst quality parameters (assessed via differential apoptotic staining) were evaluated in mixed linear regression models. The cleavage (85.3 ± 2.4, 82.6 ± 2.7, 86 ± 2.3 and 86.4 ± 2.3% for control, diluent control, ITSL and ITL, respectively) and day 8 blastocyst rates (37.4 ± 3.3, 36.9 ± 3.4, 39.7 ± 3.3 and 46.2 ± 3.4% for control, diluent control, ITSL and ITL, respectively) were not different (p > .1) among experimental groups. Embryos produced in the ITL group resulted in blastocysts with higher total cell numbers (TCN; 141 ± 19.2), inner cell mass (ICM; 65.3 ± 11.6) and trophectoderm cells (TE; 75.2 ± 8.8) compared with the control (129 ± 19.2, 56.3 ± 11.6 and 72.7 ± 8.8, for TCN, ICM and TE; p < .01, respectively). Lycopene-supplemented groups (ITSL and ITL) resulted in blastocysts with similar TCN, ICM and TE (p > .2). The number of apoptotic cells was not different among experimental groups (p > .1). Lycopene supplementation to the culture medium only produced a numerical increase in the blastocyst rate but replacing selenium with lycopene in a serum-free culture medium resulted in blastocysts with more cells.

In Vitro Production of Neutrophils Extracellular Traps Is Affected by the Lactational Stage of Dairy Cows.

We aimed to research the neutrophil extracellular traps (NETs) and reactive oxygen species (ROS) formation capacity of polymorphonuclear cells (PMN) during different lactational stages of Holstein cows. We also aimed to validate a model which could mimic infection and inflammation in vitro by adding increasing concentrations of lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) to PMN suspensions isolated from nulliparous heifers and evaluate their capacity to produce NETs and ROS. In 3 replicates, we collected blood from nulliparous heifers (n = 3), cows at the end of gestation (n = 3), early postpartum (n = 3) and in mid-lactation (n = 3) in which PMN were isolated. The production of ROS in PMN were assessed using the 2',7'-Dichlorofluorescein diacetate method, while the SYTOX Orange and Quant-iT™ PicoGreen dsDNA ultra-sensitive nucleic fluorescent acid staining methods were applied in order to quantitatively analyze the formation of NETs. Statistical analyses were performed via linear regression models using the replicate as a random. ROS values of PMN harvested from peripartum cows were 1.3 times increased compared with those in nulliparous heifers (p < 0.01). Compared with nulliparous heifers, the production of NETs by PMN isolated from mid-lactation and postpartum cows was 2.1 and 2.5 times higher (p < 0.01), respectively. In 3 replicates, in vitro stimulation of PMN isolated from nulliparous heifers (n = 3) with LPS linearly increased the production of ROS and NETs (R2 = 0.96 and 0.86, respectively). Similarly, when PMN isolated from nulliparous heifers were stimulated with PMA, a linear increase in the production of ROS (R2 = 0.99) and NETs (R2 = 0.78) was observed. The basal NETs and ROS production is lower in nulliparous heifers. Thus, they are an excellent model to mimic inflammation and study fundamental aspects of the production of NETs and ROS in vitro.

Hatching is modulated by microRNA-378a-3p derived from extracellular vesicles secreted by blastocysts.

SignificanceHatching from the zona pellucida is a prerequisite for embryo implantation and is less likely to occur in vitro for reasons unknown. Extracellular vesicles (EVs) are secreted by the embryo into the culture medium. Yet the role that embryonic EVs and their cargo microRNAs (miRNAs) play in blastocyst hatching has not been elucidated, partially due to the difficulties of isolating them from low amounts of culture medium. Here, we optimized EV-miRNA isolation from medium conditioned by individually cultured bovine embryos and subsequently showed that miR-378a-3p, which was up-regulated in EVs secreted by blastocysts, plays a crucial role in promoting blastocyst hatching. This demonstrates the regulatory effect of miR-378-3p on hatching, which is an established embryo quality parameter linked with implantation.

Serum Anti-Müllerian Hormone: A Potential Semen Quality Biomarker in Stud Dogs?

Anti-Müllerian hormone (AMH) has been suggested to be involved in spermatogenesis. The aim of this study was to investigate the relationship between blood serum AMH concentration and semen quality in dogs. Moreover, this study sought to find the optimal cut-off point value of serum AMH with the greatest sensitivity and specificity to predict semen quality. Forty-five clinically healthy dogs were included in the study and their age as well as the following semen parameters were determined and correlated to serum AMH concentration: total sperm output, normal morphology, plasma membrane integrity, total motility, progressive motility, and velocity parameters. Statistical analysis for correlations were performed using Spearman's correlation coefficients. Moderate negative associations were found between serum AMH and semen total motility (r = -0.38, p = 0.01), progressive motility (r = -0.36, p = 0.01), and normal morphology (r = -0.36, p= 0.02). Based on these associations, an AMH concentration of 5.54 µg/L was found to be the optimal cut-off point value to obtain the greatest summation of sensitivity (86%) and specificity (63%) to predict semen quality. The serum AMH assay may therefore be a potential hormonal marker to predict which dogs would require further semen analysis. Future research is however needed to confirm these preliminary results.

Lycopene Supplementation to Serum-Free Maturation Medium Improves In Vitro Bovine Embryo Development and Quality and Modulates Embryonic Transcriptomic Profile.

Bovine embryos are typically cultured at reduced oxygen tension to lower the impact of oxidative stress on embryo development. However, oocyte in vitro maturation (IVM) is performed at atmospheric oxygen tension since low oxygen during maturation has a negative impact on oocyte developmental competence. Lycopene, a carotenoid, acts as a powerful antioxidant and may protect the oocyte against oxidative stress during maturation at atmospheric oxygen conditions. Here, we assessed the effect of adding 0.2 µM lycopene (antioxidant), 5 µM menadione (pro-oxidant), and their combination on the generation of reactive oxygen species (ROS) in matured oocytes and the subsequent development, quality, and transcriptome of the blastocysts in a bovine in vitro model. ROS fluorescent intensity in matured oocytes was significantly lower in the lycopene group, and the resulting embryos showed a significantly higher blastocyst rate on day 8 and a lower apoptotic cell ratio than all other groups. Transcriptomic analysis disclosed a total of 296 differentially expressed genes (Benjamini-Hochberg-adjusted p < 0.05 and ≥ 1-log2-fold change) between the lycopene and control groups, where pathways associated with cellular function, metabolism, DNA repair, and anti-apoptosis were upregulated in the lycopene group. Lycopene supplementation to serum-free maturation medium neutralized excess ROS during maturation, enhanced blastocyst development and quality, and modulated the transcriptomic landscape.

The effect of season of birth on the morphometrics of newborn Belgian Blue calves.

Breed type and environmental factors such as breeding season may have a significant impact on neonatal morphometrics. We followed a total of 236 elective cesarean sections in Belgian Blue (BB) cows, from which neonatal calves were morphometrically assessed (in cm) within the first 72 h of delivery using a strictly standardized protocol. The effect of the season of birth on each calf measurement was analyzed using mixed linear regression models, including the farm of origin as a random effect. Calves born in spring had a longer diagonal length (69.7 ± 1.24; P = 0.05) than those born in autumn (66.9 ± 1.16). The tibial length of calves born in spring (35.8 ± 0.48) was longer (P < 0.02) than of those born in autumn (33.1 ± 0.57) or summer (34.1 ± 0.49). Calves born in autumn have a shorter head diameter (12.9 ± 0.23; P < 0.02) than those born in summer (12.6 ± 0.29) or winter (13.5 ± 0.22). For all other parameters, no differences were found (P > 0.08). Based on the results of this study, it can be concluded that the birth season influences the morphometrics of neonatal BB calves, with a tendency for spring to be associated with the largest body size. The latter is important to avoid dystocia when BB cattle are crossed with other breeds.

Quantitative and functional dynamics of circulating and endometrial polymorphonuclear leukocytes in healthy peripartum dairy cows.

The aim of the present study was to assess the counts, viability, and functionality of circulating and endometrial polymorphonuclear leukocytes (PMN) isolated from fourteen clinically and metabolically healthy multiparous dairy cows in the peripartum period. For this, blood samples were collected at -5, +9, +21 and + 37 days (d) relative to calving. Cytology samples were collected from the vagina, cervix, and uterus at +9, +21 and + 37 d, using the cytobrush technique. Additional vaginal samples were collected at -5 d. Cytology smears were prepared and the PMN-to-all nucleated cell proportions (PMN%) were calculated. The endometrial cytobrush samples were also used for flow cytometric assessment of endometrial PMN (ePMN) viability and functionality. Functionality tests for circulating PMN (cPMN) included phagocytosis (PC), oxidative burst, and intracellular proteolytic degradation. For ePMN, we evaluated PC only. The effect of day relative to calving on PMN viability and functionality were fitted in linear regression models, accounting for repeated measures. The endometrial PMN% were higher at +9 d (23.5 ± 0.4%; least-squares means ± standard error) and +21 d (8.5 ± 0.3%) than at +37 d (1.4 ± 0.3%). No changes in PMN% were found on either vaginal or cervical cytology along the peripartum period. The cPMN counts were higher pre- (6.2 ± 0.4 x 106/mL) than postpartum (4.9 ± 0.4 x 106/mL). Upon viability analysis, only the percentage of viable cPMN tended to be lower at -5 d (90.1 ± 1.5%) than at +37 d (94.1 ± 1.4%), and no other changes in the percentage of apoptotic and necrotic cPMN, nor in their functionality were found during the peripartum period. Analysis of ePMN viability showed that the percentage of viable ePMN did not change over time. In marked contrast, the percentage of apoptotic ePMN was higher at +9 d (37.8 ± 5.1%) than at +21 d (20.9 ± 5.1%) and +37 d (11.9 ± 5.3%), while the percentage of necrotic ePMN was lower at +9 d (27.0 ± 6.3%) than at +37 d (54.9 ± 6.6%). The percentage of ePMN PC was higher at +9 d (27.5 ± 3.4%) than at +37 d (13.3 ± 4.9%). In conclusion, during the peripartum period ePMN in the healthy postpartum uterus are highly dynamic in terms of counts, viability, and functionality compared to their circulating counterparts.