Exogenous FSH/LH modulates TGF beta signaling genes in granulosa cells of Simmental heifers without affecting IVP results.

Follicular wave synchronization and follicular superstimulation with FSH are commonly used in OPU-IVP programs to increase oocyte developmental competence. Factors like Growth Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15), from the TGF beta superfamily, are produced by the oocyte and modulate follicular function. The aim of this study was to analyze the FSH-induced effects on (1) embryo production in dual-purpose Simmental cattle, and (2) TGF beta-mediated effects on oocyte-granulosa cell communication. Simmental heifers (n = 12, age 484 ± 62 days) underwent two OPU-IVP cycles in a cross-over design. Follicular waves were synchronized using 0.5 mg cloprostenol on Day 0, followed by 10 µg buserelin on Day 2. Subsequently, half of the heifers were randomly assigned to receive FSH/LH (four injections of 75 IU FSHp and 75IU LHp, 12 h apart on Days 4 and 5) before the first OPU, while the remaining heifers received FSH/LH before the second OPU. At the time of OPU, i.e. 7 days after the start of synchronization, granulosa cells were collected for RT-qPCR analysis. FSH treatment did not affect the number of oocytes collected (17.3 vs. 13.3, P > 0.05), but increased the percentage of quality 1 oocytes compared to controls (45.7 % vs. 22.0 %, P < 0.001). Neither cleavage (86.4 % vs. 85.7 %), nor blastocyst (42.1 % vs. 39.3 %) rate, or the number of transferable embryos produced by IVP (4.1 vs 4.8) was influenced by FSH treatment (P > 0.05 in all cases). FSH treatment increased HIF1A and FSHR levels in granulosa cells, while STAR was decreased (P = 0.008 in all cases). FSH treatment did not affect BMP15 or GDF9 mRNA expression (P > 0.05) but appeared to modulate the expression of genes involved in the BMP signaling pathway. Transcriptional levels of BMP15 receptor (BMPR1A, P = 0.016), and its downstream signaling factor SMAD1 (P = 0.008) were affected by FSH treatment. Our results demonstrated no benefit of this FSH stimulation protocol on IVP results in Simmental heifers. Further, our results suggest that the effects of FSH on bovine oocytes during acquisition of developmental competence may be mediated through BMP, but do not involve the regulation of transcriptional availability of GDF9, providing new insights into possible paracrine effects of the oocyte on granulosa cells.

Relationship between Pelvic Dimensions and Maximum Traction Forces Required during Parturition in Holstein Cows Using a Biomechanical Obstetric Simulator.

The primary aim of this study was to investigate the effect of the pelvic dimensions of Holstein cows on the traction forces during parturition. Additionally, the relationship between calf measurements and traction forces was explored. For this purpose, a modified in vitro biomechanical model simulating obstetric tractions was used. For the requirements of the experiment, six bone pelvises of deceased Holstein cows were collected based on their estimated pelvic inlet area (EPA) and prepared. Additionally, six stillborn calves were collected based on their body weight (BW). The parameters of the pelvic inlet and cavity were measured using computed tomography (CT). Using the simulator, every calf was pulled in a random order through all pelvises, realizing a total of 36 obstetrical tractions, and the required forces were documented with appropriate software. In each extraction, three peaks of forces were recorded, with the first peak occurring at the entrance of the elbows into the maternal pelvic cavity, the second peak at the entrance of the thorax, and the third at the entrance of the calf's pelvis. Logistic regression revealed an exponential relationship between pelvic parameters and traction forces for the entrance of the elbows and the pelvis, with the recorded forces being higher in the two smallest pelvises and stabilizing at a lower level thereafter, while for the entrance of the thorax, the correlations were either exponential or linear. The adjusted coefficients of determination (r2) were generally above the threshold of 0.5 for the entrance of the elbows and pelvis and lower (0.3-0.4) regarding the thorax and were statistically significant (p < 0.05) in all cases. Regarding the relationships between the calf dimensions and the required traction forces, the types of correlations were primarily linear and of lower magnitude. The combination of pelvic and calf parameters in a multivariate model resulted in an r2 of 0.72 for the entrance of the elbows using the pelvic diagonal and calf's body weight, an r2 of 0.62 using the pelvic area and calf's thoracic circumference, and an r2 of 0.75 using the pelvic diagonal and calf's fetlock joint width. In conclusion, under the conditions of the present experimentation, the applied traction forces were mainly influenced by the pelvic dimensions in an exponential manner, whereas the calf body measurements showed a weaker effect. Based on these findings, critical cut-off points exist, different for every pelvic parameter, below which a significant increase in the required traction forces is expected.

Effects of natural mating, artificial insemination and intravaginal deposition of raw semen or seminal plasma on vaginal and uterine blood flow in German Holstein cows.

AIM: The present study was performed to characterize and compare the perfusion of vaginal and uterine arteries after challenging the reproductive tract of dairy cows via natural mating, artificial insemination (AI), or intravaginal deposition (vaginal fundus) of different biological fluids or a placebo. MATERIALS AND METHODS: In a double-blind study, six German Holstein cows were administered PGF2α during dioestrus and 48 h later treated with GnRH. Intravaginal or intrauterine treatments were carried out 12 h after GnRH was administered. Animals served as their controls, using a cross-over design with an interval of 14 days between experiments. The experimental animals were allocated to receive the following treatments: natural mating (N), intrauterine artificial insemination (A), intravaginal deposition (vaginal fundus) of 6 mL raw semen (R) or 6 mL seminal plasma (S), and compared to their controls [control 1: 6 mL placebo (P: physiological saline); control 2: no treatment (C)). Corresponding time intervals were chosen for the untreated control oestrus. Blood flow volume (BFV) in the uterine (u) and vaginal (v) arteries ipsilateral to the ovary bearing the preovulatory follicle was determined using transrectal Doppler sonography. RESULTS: All animals exhibited oestrus and ovulated between 30 and 36 h after GnRH. Transient increases (P < 0.05) in vaginal blood flow occurred between 3 and 12 h following mating as well as 3 to 9 h after deposition of raw semen and seminal plasma, respectively. The most distinct increases (199%) in vBFV occurred 6 h after mating compared to values immediately before mating (= time 0 h). Neither AI nor deposition of a placebo into the vagina affected vBFV (P > 0.05). Only mating and deposition of either raw semen, seminal plasma or AI increased uBFV (P < 0.003). The greatest rise in uBFV occurred after natural mating. Maximum uBFV values were detected 9 h after mating when values were 79% greater (P < 0.05) than at 0 h. CONCLUSIONS: The natural mating, deposition of raw semen or seminal plasma and conventional AI affect vaginal and/or uterine blood flow to different degrees. The factors responsible for these alterations in blood flow and their effects on fertility remain to be clarified in future studies.

Impact of bull age, sperm processing, and microclimatic conditions on the viability and DNA integrity of cryopreserved bovine sperm.

Context Seasonal microclimatic fluctuations can cause changes in sperm quality even in dairy bulls bred under temperate climate. These changes can vary between sires of different age and affect sperm freezability. Aims We aimed to evaluate the modulating effect of bull age and equilibration time before freezing on the seasonal pattern of sperm viability and DNA integrity post-thaw. Methods In the frame of systematic sperm quality control, we assessed the integrity of sperm plasma membrane and acrosome (PMAI) in 15,496 cryopreserved bovine batches, and the percentage of sperm with high DNA fragmentation index (%DFI) after 0h and 3h incubation at 38°C post-thaw (3h) in 3422 batches. Semen was equilibrated for 24h before freezing if collected on Monday or Wednesday and 72h if produced on Friday. We investigated the effect of season, bull age, equilibration, and temperature-humidity index (THI) on the day of semen collection on sperm traits using mixed-effects linear models. Key results PMAI and %DFI (0h and 3h) deteriorated with increasing THI. The effect of THI on %DFI was detected with a 30-day time lag. Seasonal fluctuations of sperm quality were similar between young, mature, and older sires. Prolonged equilibration did not affect PMAI but was linked to elevated %DFI (3h) in summer. Conclusions Extending equilibration from 24 to 72h is compatible with commercial standards of bovine sperm quality post-thaw; however, it could interfere with the seasonal pattern of the latter. Implications Systematic monitoring of bovine sperm quality enables the prompt detection of stress factors related to microclimate and semen processing.

Oviductal extracellular vesicles miRNA cargo varies in response to embryos and their quality.

BACKGROUND: Increasing evidence points to an active role of oviductal extracellular vesicles (oEVs) in the early embryo-maternal dialogue. However, it remains unclear whether oEVs contribute to the recognition of the presence of embryos and their quality in the oviduct. Hence, we examined whether the molecular cargo of oEVs secreted by bovine oviduct epithelial cells (BOEC) differs depending on the presence of good (≥ 8 cells, G) or poor (< 8 cells, P) quality embryos. In addition, differences in RNA profiles between G and P embryos were analyzed in attempt to distinguish oEVs and embryonic EVs cargos. METHODS: For this purpose, primary BOEC were co-cultured with in vitro produced embryos (IVP) 53 h post fertilization as follows: BOEC with G embryos (BGE); BOEC with P embryos (BPE); G embryos alone (GE); P embryos alone (PE); BOEC alone (B) and medium control (M). After 24 h of co-culture, conditioned media were collected from all groups and EVs were isolated and characterized. MicroRNA profiling of EVs and embryos was performed by small RNA-sequencing. RESULTS: In EVs, 84 miRNAs were identified, with 8 differentially abundant (DA) miRNAs for BGE vs. B and 4 for BPE vs. B (P-value < 0.01). In embryos, 187 miRNAs were identified, with 12 DA miRNAs for BGE vs. BPE, 3 for G vs. P, 8 for BGE vs. GE, and 11 for BPE vs. PE (P-value < 0.01). CONCLUSIONS: These results indicated that oEVs are involved in the oviductal-embryo recognition and pointed to specific miRNAs with signaling and supporting roles during early embryo development.

Lipopolysaccharide-binding protein in follicular fluid is associated with the follicular inflammatory status and granulosa cell steroidogenesis in dairy cows.

Metabolic stress and subsequent hepatic dysfunction in high-producing dairy cows are associated with inflammatory diseases and declining fertility. Lipopolysaccharide (LPS)-binding protein (LBP) is produced by hepatocytes and controls the immune response, suggesting that it is involved in the pathophysiology of inflammation-related attenuation of reproductive functions during metabolic stress. This study investigated the effect of LBP on the inflammatory status, oocyte quality, and steroidogenesis in the follicular microenvironment of dairy cows. Using bovine ovaries obtained from a slaughterhouse, follicular fluid and granulosa cells were collected from large follicles to evaluate the follicular status of metabolism, inflammation, and steroidogenesis. Cumulus-oocyte complexes were aspirated from small follicles and subjected to in vitro embryo production. The results showed that follicular fluid LBP concentrations were significantly higher in cows with fatty livers and hepatitis than in those with healthy livers. Follicular fluid LBP and LPS concentrations were negatively correlated, whereas LPS concentration showed a positive correlation with the concentrations of non-esterified fatty acids (NEFA) and ß-hydroxybutyric acid in follicular fluid. The blastulation rate of oocytes after in vitro fertilization was impaired in cows in which coexisting large follicles had high NEFA levels. Follicular fluid NEFA concentration was negatively correlated with granulosa cell expression of the estradiol (E2) synthesis-related gene (CYP19A1). Follicular fluid LBP concentration was positively correlated with follicular fluid E2 concentration and granulosa cell CYP19A1 expression. In conclusion, follicular fluid LBP may be associated with favorable conditions in the follicular microenvironment, including low LPS levels and high E2 production by granulosa cells.

Brilliant Cresyl Blue Negative Oocytes Show a Reduced Competence for Embryo Development after In Vitro Fertilisation with Sperm Exposed to Oxidative Stress.

The extent of oxidative damage transferred by the damaged sperm to the progeny is likely to be limited by the oocyte's repair and antioxidative capacity. We aimed to assess the association between Brilliant Cresyl Blue (BCB) staining in oocytes and their competence for embryo development after in vitro fertilisation (IVF) with damaged sperm. For this purpose, bovine sperm were incubated without (non-oxidised sperm, NOX S) or with 100 µM H2O2 (oxidised sperm, OX S) and were used to fertilise in-vitro-matured bovine oocytes (BCB-pos./BCB-neg.). Unstained oocytes served as controls (US). Development was assessed at 30, 46, 60 h and on Days (D) 7 and 8 after IVF. Total cell number and apoptotic index were analysed in D7 blastocysts. BCB-neg. oocytes showed lower cleavage rates and blastocyst rates than unstained oocytes after IVF with NOX S (p < 0.05). They showed the highest reduction in D7 blastocyst rate upon fertilisation with OX S and showed a delayed embryo development at 46 and 60 h after IVF compared to embryos produced with NOX S (p < 0.05). Total cell number in blastocysts produced with BCB-neg. oocytes was lower (p < 0.05) in the embryos produced with OX S than in embryos after IVF with NOX S. In conclusion, BCB-neg. oocytes have a lower competence to support embryo development after in vitro fertilisation with oxidised sperm.

Effects of postpartum diseases on antral follicle count and serum concentration of Anti-Müllerian hormone in dairy cows.

The antral follicle count (AFC) and Anti-Müllerian hormone (AMH) concentration are validated markers for ovarian reserve in cattle, but their use as fertility markers is controverse. Here we assessed the effects of postpartum diseases on AFC and AMH concentration, as well as the influence of parity and breed on these parameters. Cows (n = 513, mostly Holstein Friesian and Brown Swiss, parity 3.0 ± 1.8) underwent a single ultrasonography examination 28-56 days after parturition and categorized as having low (n ≤ 15 follicles), intermediate (n = 16-24 follicles), or high (n ≥ 25 follicles) AFC based on objective video analysis of recorded sequences. Blood samples for AMH determination were collected at the time of examination and animals divided into low (< 0.05 ng/ml) and high AMH (≥ 0.05 ng/ml) group, respectively. No effects of postpartum diseases or breed on either AFC or AMH groups could be observed. There was a strong interaction between parity and AFC, primiparous cows having less follicles (13.6 ± 6.2 vs. 17.1 ± 7.0, P < 0.001) than pluriparous cows. The AFC did not affect reproductive parameters or productivity of the cows. In comparison, pluriparous cows with high AMH concentration had shorter calving to first service (86.0 ± 37.6 vs. 97.1 ± 46.7 days, P < 0.05) and calving to conception (123.8 ± 51.9 vs. 135.8 ± 54.4 days, P < 0.05) intervals, but lower milk yield (8440.3 ± 2292.9 vs. 8927.9 ± 2192.5 kg, P < 0.05) compared to cows with low AMH. In conclusion, no effect of postpartum diseases on AFC or AMH concentration of dairy cows could be observed. However, an interaction between parity and AFC, as well as associations of AMH with fertility and productivity in pluriparous cows, were demonstrated.

Relationship between Clinical Uterine Findings, Therapy, and Fertility in the Mare.

Persistent breeding-induced endometritis (PBIE) is a major cause of subfertility in horses and the susceptibility is increased by several factors. The aim of this study was to determine the effects of clinical uterine findings and PBIE therapies, respectively, on pregnancy rate in mares. The analysis included records from 220 mares (390 cycles) inseminated at an artificial insemination (AI) center in Switzerland. Gynecological examinations were performed repeatedly before and after AI to determine cervical tone, uterine edema, and intrauterine fluid accumulation. Pregnancy rate was lower (p < 0.001) in barren mares compared to mares of other reproductive status. A more flaccid cervix (p = 0.009) was observed at the time of ovulation in pregnant cycles, but there was no difference (p > 0.05) regarding uterine edema. Intrauterine fluid accumulation reduced pregnancy rate (p = 0.002). Oxytocin administration had beneficial effects on pregnancy rate (p = 0.015), especially for barren mares, while uterine lavage did not have any effect (p > 0.05). The results show that cervical tone and intrauterine fluid accumulation, but not its degree, are useful parameters for assessment of fertility in mares. Oxytocin treatment improved pregnancy rates in mares with PBIE while uterine lavage had a limited effect.

Impact of negative energy balance and postpartum diseases during the transition period on oocyte quality and embryonic development in dairy cows.

Transition period is a critical time for dairy cows because a large proportion of clinical and subclinical diseases are observed in the first month after parturition. Occurrence of negative energy balance is associated with depressed immunity and these conditions can affect oocyte quality and further embryonic development. The aim of this study was to assess the effects of negative energy balance-associated disorders on in vitro embryo production (IVP) in dairy cattle. We hypothesized that subclinical metabolic and/or inflammatory disorders have a negative effect on oocyte developmental competence and morphokinetic parameters of the resulting embryos. The study was conducted on 30 lactating Holstein-Friesian cows which were assigned into four groups: healthy (HEAL, n = 6), metabolic disease (META, n = 8), inflammatory disease (INFL, n = 8), or combined metabolic and inflammatory disease (COMB, n = 8). Ovum pick-up (OPU) was performed twice weekly on all cows over a period of four weeks (n = 8 OPU sessions/cow) starting on the fifth week postpartum, and the collected oocytes were subjected to routine IVP. Donor's health status did not affect the number of oocytes/OPU or the recovery rate (p > 0.05). The number of quality 1 oocytes collected from INFL and COMB cows was lower compared to HEAL cows (p < 0.05). Also, the percentage of quality 1 embryos was reduced in META and COMB compared to HEAL cows (p < 0.05). Cleavage, blastocyst and hatching rates were similar among groups (p > 0.05). Presence of disease did not affect the time required by zygotes to reach specific developmental stages, as recorded by means of time-lapse monitoring. Nevertheless, there was a higher probability of direct cleavage after IVF in oocytes of COMB cows compared to those of HEAL cows (p < 0.05). In conclusion, oocytes and embryos derived from dairy cows diagnosed with subclinical metabolic and/or inflammatory diseases during the transition period showed reduced quality but similar developmental potential and morphokinetics when compared to healthy cows. These results shed light on the consequences of subclinical disease on embryonic development in dairy cows which might be important for embryo transfer programs.

Dynamic regulation of the transcriptome and proteome of the equine embryo during maternal recognition of pregnancy.

During initial maternal recognition of pregnancy (MRP), the equine embryo displays a series of unique events characterized by rapid blastocyst expansion, secretion of a diverse array of molecules, and transuterine migration to interact with the uterine surface. Up to date, the intricate transcriptome and proteome changes of the embryo underlying these events have not been critically studied in horses. Thus, the objective of this study was to perform an integrative transcriptomic (including mRNA, miRNAs, and other small non-coding RNAs) and proteomic analysis of embryos collected from days 10 to 13 of gestation. The results revealed dynamic transcriptome profiles with a total of 1311 differentially expressed genes, including 18 microRNAs (miRNAs). Two main profiles for mRNAs and miRNAs were identified, one with higher expression in embryos ≤5 mm and the second with higher expression in embryos ≥7 mm. At the protein level, similar results were obtained, with 259 differentially abundant proteins between small and large embryos. Overall, the findings demonstrated fine-tuned transcriptomic and proteomic regulations in the developing embryo associated with embryo growth. The identification of specific regulation of mRNAs, proteins, and miRNAs on days 12 and 13 of gestation suggested these molecules as pivotal for embryo development and as involved in MRP, and in establishment of pregnancy in general. In addition, the results revealed new insights into prostaglandin synthesis by the equine embryo, miRNAs and genes potentially involved in modulation of the maternal immune response, regulation of endometrial receptivity and of late implantation in the mare.

Uterine extracellular vesicles as multi-signal messengers during maternal recognition of pregnancy in the mare.

In contrast to other domestic mammals, the embryo-derived signal(s) leading to maternal recognition of pregnancy (MRP) are still unknow in the mare. We hypothesize that these embryonic signals could be packed into uterine extracellular vesicles (uEVs), acting as multi-signal messengers between the conceptus and the maternal tract, and contributing to MRP. To unveil these signals, the RNA and protein cargos of uEVs isolated from uterine lavages collected from pregnant mares (P; day 10, 11, 12 and 13 after ovulation) and cyclic control mares (C; day 10 and 13 after ovulation) were analyzed. Our results showed a fine-tuned regulation of the uEV cargo (RNAs and proteins), by the day of pregnancy, the estrous cycle, and even the size of the embryo. A particular RNA pattern was identified with specific increase on P12 related to immune system and hormonal response. Besides, a set of proteins as well as RNAs was highly enriched in EVs on P12 and P13. Differential abundance of miRNAs was also identified in P13-derived uEVs. Their target genes were linked to down- or upregulated genes in the embryo and the endometrium, exposing their potential origin. Our study identified for first time specific molecules packed in uEVs, which were previously associated to MRP in the mare, and thus bringing added value to the current knowledge. Further integrative and functional analyses will help to confirm the role of these molecules in uEVs during MRP in the mare.

Effect of Acetylsalicylic Acid on Uterine Blood Flow, Gestation Length, Foal Birth Weight and Placental Weight in Pregnant Thoroughbred Mares - A Clinical Pilot Study.

The aim of this double-blinded placebo-controlled study was to investigate the effect of acetylsalicylic acid (ASA) on uterine blood flow, gestation length, placental and foal weights in pregnant mares. Sixteen Thoroughbred mares of different age (13.3 ± 4.1) and parity (7.4 ± 3.1) were randomly assigned to three treatment groups. Mares in group C (n = 4) served as controls and received 5,000 mg lactose orally once daily from D 120 (D 0 = day of ovulation) until parturition. Mares in group ASA1 (n = 7) received 5,000 mg ASA orally once daily from D 120 until parturition. Mares in group ASA2 (n = 5) received the same dose ASA as group ASA1 from D 120 to D 285, but twice daily from D 285 until parturition. Mares were examined by ultrasonography on D 14, 28, and 60, and in 21-days intervals from D 120 until parturition. The cross-sectional area, time average maximum velocity (TAMV), and pulsatility index were measured in both uterine arteries and the blood flow volume was calculated for each uterine artery and then summarized. All 16 mares carried a normal pregnancy and delivered live foals. In group ASA2 TAMV in the ipsilateral artery was significantly higher (P = .03) and these mares showed a tendency of increased total blood flow volume (P = .07) during late pregnancy (D 305-346). Results indicate that oral administration of 5,000 mg of ASA twice daily in pregnant mares causes a rise in uterine blood flow during late pregnancy.

Structural variant-based pangenome construction has low sensitivity to variability of haplotype-resolved bovine assemblies.

Advantages of pangenomes over linear reference assemblies for genome research have recently been established. However, potential effects of sequence platform and assembly approach, or of combining assemblies created by different approaches, on pangenome construction have not been investigated. Here we generate haplotype-resolved assemblies from the offspring of three bovine trios representing increasing levels of heterozygosity that each demonstrate a substantial improvement in contiguity, completeness, and accuracy over the current Bos taurus reference genome. Diploid coverage as low as 20x for HiFi or 60x for ONT is sufficient to produce two haplotype-resolved assemblies meeting standards set by the Vertebrate Genomes Project. Structural variant-based pangenomes created from the haplotype-resolved assemblies demonstrate significant consensus regardless of sequence platform, assembler algorithm, or coverage. Inspecting pangenome topologies identifies 90 thousand structural variants including 931 overlapping with coding sequences; this approach reveals variants affecting QRICH2, PRDM9, HSPA1A, TAS2R46, and GC that have potential to affect phenotype.

NMR spectroscopy of a single mammalian early stage embryo.

The resolving power, chemical sensitivity and non-invasive nature of NMR have made it an established technique for in vivo studies of large organisms both for research and clinical applications. NMR would clearly be beneficial for analysis of entities at the microscopic scale of about 1 nL (the nanoliter scale), typical of early development of mammalian embryos, microtissues and organoids: the scale where the building blocks of complex organisms could be observed. However, the handling of such small samples (about 100 µm) and sensitivity issues have prevented a widespread adoption of NMR. In this article we show how these limitations can be overcome to obtain NMR spectra of a mammalian embryo in its early stage. To achieve this we employ ultra-compact micro-chip technologies in combination with 3D-printed micro-structures. Such device is packaged for use as plug & play sensor and it shows sufficient sensitivity to resolve NMR signals from individual bovine pre-implantation embryos. The embryos in this study are obtained through In Vitro Fertilization (IVF) techniques, transported cryopreserved to the NMR laboratory, and measured shortly after thawing. In less than 1 h these spherical samples of just 130-190 µm produce distinct spectral peaks, largely originating from lipids contained inside them. We further observe how the spectra vary from one sample to another despite their optical and morphological similarities, suggesting that the method can further develop into a non-invasive embryo assay for selection prior to embryo transfer.

Mitoquinone does not improve sperm cryo-resistance in bulls.

Oxidative stress is associated with impaired post-thaw sperm quality. As mitochondria are the main source of reactive oxygen species (ROS) in sperm, the goal of this study was to evaluate effects of the mitochondria-targeting antioxidant Mitoquinone (MitoQ) during cryopreservation of bull sperm. Semen was collected from 11 Simmental bulls (two ejaculates per bull) and diluted in Triladyl® supplemented with various concentrations of MitoQ (0, 0.2, 2, and 20 nM) to a final concentration of 65 × 106  sperm/ml. After thawing (0 and 3 hr), we assessed the following sperm traits: sperm motility by computer-assisted sperm analysis (CASA), DNA fragmentation index by SCSA® and plasma and acrosome membrane integrity, intracellular calcium concentration, esterase activity, mitochondrial membrane potential and synthesis of ROS using two multicolour flow cytometric assays. After 3 hr of incubation, 20 nM MitoQ increased (p < .05) sperm ROS synthesis compared to Control, whereas none of the other quality parameters were altered (p > .05). Therefore, we concluded that addition of MitoQ to semen extender before cryopreservation of bull sperm was unable to improve post-thaw sperm quality. Furthermore, 20 nM of MitoQ increased frozen-thawed sperm ROS synthesis, without apparent negative effects on the evaluated sperm traits.

Inhibition of lipopolysaccharide-induced suppression of luteal function in isolated perfused bovine ovaries.

Recently, we observed that lipopolysaccharide (LPS) suppresses corpus luteum (CL) function in isolated perfused ovaries. It remained unclear if this suppression was due to increased luteal PGF2α secretion or LPS-induced apoptosis. Therefore, possible impacts of PGF2α and LPS were inhibited by a non-steroidal anti-inflammatory drug (flunixin) and an endotoxin-binding agent (polymyxin B), respectively. Bovine ovaries with a mid-cycle CL were collected immediately after slaughter and perfused for 240 min. After 50 min of equilibration, either flunixin or polymyxin B (5 µg/ml of each) were added to the perfusion medium of six ovaries, respectively. All ovaries (n = 12) were treated with E. coli LPS (0.5 µg/ml) 60 min after the onset of perfusion, and received 500 I.U. of hCG after 210 min of perfusion. Progesterone and PGF2α were measured in the effluent perfusate every 10 and 30 min, respectively. Biopsies of the CL were collected every 60 min to determine the mRNA expression of the cytokine TNFA and factors of apoptosis (CASP3, -8). Flunixin-treatment inhibited the increase of PGF2α after LPS-challenge that was observed in the polymyxin B-treated (PX-LPS) ovaries. After hCG-stimulation, progesterone secretion increased (P < 0.05) in group PX-LPS but not in the flunixin-treated (F-LPS) ovaries. Compared to initial values before LPS-challenge, luteal mRNA expression of TNFA and CASP3 was increased (P < 0.05) in group F-LPS at 120 and 180 min, respectively, and those of CASP8 was decreased (P < 0.05) in PX-LPS at 60 and 120 min after LPS-treatment. In conclusion, although flunixin managed to inhibit PGF2α, it did not suffice to successfully prevent LPS-induced apoptosis. However, endotoxin-binding polymyxin B resulted in luteal responsiveness to hCG after LPS-challenge.

Identification of genes associated with susceptibility to persistent breeding-induced endometritis by RNA-sequencing of uterine cytobrush samples.

This study aimed to investigate the susceptibility to persistent breeding-induced endometritis (PBIE). Cytobrush samples were collected from 81 broodmares 1-3 days before artificial insemination (AI). Susceptibility to PBIE was evaluated by the presence of ≥ 2 cm of intrauterine fluid 24 h after AI, besides the fertility was determined by a sonographic pregnancy diagnosis 2 weeks after ovulation. RNA expressions were compared between susceptible non-pregnant (SNP) mares (n=9) and resistant pregnant (RP) mares (n=9) as well as between susceptible pregnant (SP) mares (n=9) and susceptible non-pregnant (SNP) mares. 66 differentially expressed genes (DEGs) were identified between SNP and RP mares and 60 DEGs between SP and SNP mares. In SNP compared to RP mares, transcript levels of genes regulating steroid hormone metabolism and neutrophil chemotaxis were lower, while higher for genes participating in uterine inflammation.Transcripts of genes related to extracellular matrix degradation, tissue adhesions, and fibrosis were lower in SP mares than in SNP mares, while higher for genes related to uterine cell proliferation, differentiation, and angiogenesis in SP mares than SNP mares. In conclusion, increased transcript levels of apolipoprotein E (APOE) and roundabout 2 (ROBO2), cluster domain 44 (CD44), integrin beta 3 (ITGB3), and epidermal growth factor (EGF) are possible biomarkers for susceptibility to PBIE. While higher expression of fibroblast growth factor 9 (FGF9), kinase domain receptor (KDR), and C-X-C motif chemokine ligand (CXCL) 16, collagen type V alpha 2 (COL5A2) and fibronectin (FN1) are suggested indicators of fertility in susceptible mares if they receive proper breeding management.

Spatiotemporal endometrial transcriptome analysis revealed the luminal epithelium as key player during initial maternal recognition of pregnancy in the mare.

During the period of maternal recognition of pregnancy (MRP) in the mare, the embryo needs to signal its presence to the endometrium to prevent regression of the corpus luteum and prepare for establishment of pregnancy. This is achieved by mechanical stimuli and release of various signaling molecules by the equine embryo while migrating through the uterus. We hypothesized that embryo's signals induce changes in the endometrial gene expression in a highly cell type-specific manner. A spatiotemporal transcriptomics approach was applied combining laser capture microdissection and low-input-RNA sequencing of luminal and glandular epithelium (LE, GE), and stroma of biopsy samples collected from days 10-13 of pregnancy and the estrous cycle. Two comparisons were performed, samples derived from pregnancies with conceptuses ≥ 8 mm in diameter (comparison 1) and conceptuses ≤ 8 mm (comparison 2) versus samples from cyclic controls. The majority of gene expression changes was identified in LE and much lower numbers of differentially expressed genes (DEGs) in GE and stroma. While 1253 DEGs were found for LE in comparison 1, only 248 were found in comparison 2. Data mining mainly focused on DEGs in LE and revealed regulation of genes related to prostaglandin transport, metabolism, and signaling, as well as transcription factor families that could be involved in MRP. In comparison to other mammalian species, differences in regulation of genes involved in epithelial barrier formation and conceptus attachment and implantation reflected the unique features of equine reproduction at the time of MRP at the molecular level.

Transrectal three-dimensional fetal volumetry in early pregnant mares: Relationships between maternal factors and equine fetal volume measurements.

The aim of this study was to investigate the effects of maternal, hormonal, and fetal factors on early fetal volume (FV) measurements in mares obtained by three-dimensional (3D) ultrasound. Furthermore, postpartum parameters were explored in regard to their association with early FV. For this purpose, 149 German warmblood mares that were artificially inseminated and confirmed to be pregnant between days 14-16 of gestation, were examined transrectally at day 45 ± 1 of gestation with the portable 3D ultrasound device Voluson® i (GE, Zipf, Austria). FV was calculated by using the extension software Virtual Organ Computer-aided AnaLysis (VOCAL™). Two different mixed linear models were used to analyze associations between the investigated maternal and fetal factors and the FV. Explanatory variables investigated in the first model were: maternal age, parity, maternal weight, and body condition score, type of pregnancy (recipient or biological mother), barren status (lactating or non-lactating), fetal sex, progesterone (P4) and equine chorionic gonadotropin (eCG) concentrations; and in the second model outcome variables such as gestational length, birth weight, placental weight, fetal sex, and abortion were included in the analysis. The final models revealed a significant relation between FV and eCG (b = 0.011, P = 0.030), as well as with P4 (b = -0.053, P = 0.016), but interestingly P4 was negatively related to FV. Fetal sex showed the most prominent effect on FV (b = -0.256, P = 0.039), with female fetuses being smaller than male fetuses. In the second model none of the investigated parameters were related to early FV except for fetal sex (b = -0.328, P = 0.047), again with female fetuses being smaller. In summary, it was found that FV is related with eCG, P4 and fetal sex, but was no suitable predicting factor for the investigated outcome parameters. Furthermore, the findings suggest that sex specific growth differences exist already in early gestation. The detailed biological mechanism by which P4 and eCG affect fetal size has to be investigated in prospective studies.