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.

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.

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.

Activation of cryptic splicing in bovine WDR19 is associated with reduced semen quality and male fertility.

Cattle are ideally suited to investigate the genetics of male reproduction, because semen quality and fertility are recorded for all ejaculates of artificial insemination bulls. We analysed 26,090 ejaculates of 794 Brown Swiss bulls to assess ejaculate volume, sperm concentration, sperm motility, sperm head and tail anomalies and insemination success. The heritability of the six semen traits was between 0 and 0.26. Genome-wide association testing on 607,511 SNPs revealed a QTL on bovine chromosome 6 that was associated with sperm motility (P = 2.5 x 10-27), head (P = 2.0 x 10-44) and tail anomalies (P = 7.2 x 10-49) and insemination success (P = 9.9 x 10-13). The QTL harbors a recessive allele that compromises semen quality and male fertility. We replicated the effect of the QTL on fertility (P = 7.1 x 10-32) in an independent cohort of 2481 Brown Swiss bulls. The analysis of whole-genome sequencing data revealed that a synonymous variant (BTA6:58373887C>T, rs474302732) in WDR19 encoding WD repeat-containing protein 19 was in linkage disequilibrium with the fertility-associated haplotype. WD repeat-containing protein 19 is a constituent of the intraflagellar transport complex that is essential for the physiological function of motile cilia and flagella. Bioinformatic and transcription analyses revealed that the BTA6:58373887 T-allele activates a cryptic exonic splice site that eliminates three evolutionarily conserved amino acids from WDR19. Western blot analysis demonstrated that the BTA6:58373887 T-allele decreases protein expression. We make the remarkable observation that, in spite of negative effects on semen quality and bull fertility, the BTA6:58373887 T-allele has a frequency of 24% in the Brown Swiss population. Our findings are the first to uncover a variant that is associated with quantitative variation in semen quality and male fertility in cattle.

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.

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.

A comparative analysis of the intrauterine transcriptome in fertile and subfertile mares using cytobrush sampling.

BACKGROUND: Subfertility is a major problem in modern horse breeding. Especially, mares without clinical signs of reproductive diseases, without known uterine pathogens and no evidence of inflammation but not becoming pregnant after several breeding attempts are challenging for veterinarians. To obtain new insights into the cause of these fertility problems and aiming at improving diagnosis of subfertile mares, a comparative analysis of the intrauterine transcriptome in subfertile and fertile mares was performed. Uterine cytobrush samples were collected during estrus from 57 mares without clinical signs of uterine diseases. RNA was extracted from the cytobrush samples and samples from 11 selected subfertile and 11 fertile mares were used for Illumina RNA-sequencing. RESULTS: The cytobrush sampling was a suitable technique to isolate enough RNA of high quality for transcriptome analysis. Comparing subfertile and fertile mares, 114 differentially expressed genes (FDR = 10%) were identified. Metascape enrichment analysis revealed that genes with lower mRNA levels in subfertile mares were related to 'extracellular matrix (ECM)', 'ECM-receptor interaction', 'focal adhesion', 'immune response' and 'cytosolic calcium ion concentration', while DEGs with higher levels in subfertile mares were enriched for 'monocarboxyl acid transmembrane transport activity' and 'protein targeting'. CONCLUSION: Our study revealed significant differences in the uterine transcriptome between fertile and subfertile mares and provides leads for potential uterine molecular biomarkers of subfertility in the mare.

The micro-RNA content of unsorted cryopreserved bovine sperm and its relation to the fertility of sperm after sex-sorting.

BACKGROUND: The use of sex-sorted sperm in cattle assisted reproduction is constantly increasing. However, sperm fertility can substantially differ between unsorted (conventional) and sex-sorted semen batches of the same sire. Sperm microRNAs (miRNA) have been suggested as promising biomarkers of bull fertility the last years. In this study, we hypothesized that the miRNA profile of cryopreserved conventional sperm is related to bull fertility after artificial insemination with X-bearing sperm. For this purpose, we analyzed the miRNA profile of 18 conventional sperm samples obtained from nine high- (HF) and nine low-fertility (LF) bulls that were contemporaneously used to produce conventional and sex-sorted semen batches. The annual 56-day non-return rate for each semen type (NRRconv and NRRss, respectively) was recorded for each bull. RESULTS: In total, 85 miRNAs were detected. MiR-34b-3p and miR-100-5p were the two most highly expressed miRNAs with their relative abundance reaching 30% in total. MiR-10a-5p and miR-9-5p were differentially expressed in LF and HF samples (false discovery rate < 10%). The expression levels of miR-9-5p, miR-34c, miR-423-5p, miR-449a, miR-5193-5p, miR-1246, miR-2483-5p, miR-92a, miR-21-5p were significantly correlated to NRRss but not to NRRconv. Based on robust regression analysis, miR-34c, miR-7859 and miR-342 showed the highest contribution to the prediction of NRRss. CONCLUSIONS: A set of miRNAs detected in conventionally produced semen batches were linked to the fertilizing potential of bovine sperm after sex-sorting. These miRNAs should be further evaluated as potential biomarkers of a sire's suitability for the production of sex-sorted sperm.

Isolation and Characterization of Equine Uterine Extracellular Vesicles: A Comparative Methodological Study.

Extracellular vesicles (EVs) have been identified in the uterine fluid in different species and have been pointed as key players in the embryo-maternal dialogue, maternal recognition of pregnancy and establishment of pregnancy. However, little is known about the uterine EVs in the mare. Therefore, the present study aimed at characterizing EVs from uterine lavage of cyclic mares by comparing five EVs isolation methods and the combination of them: (1) ultracentrifugation (UC); (2) concentration of lavage volume by Centricon ultrafiltration (CE); (3) the use of CE with different washing steps (phosphate-buffered saline with or without trehalose); (4) size-exclusion chromatography with iZON-qEV columns, and (5) a combination of the methods with best results based on EVs yield, purity, and protein cargo profiles. Transmission electron microscopy and Western blotting confirmed the isolation of EVs by all methods but with quantitative and qualitative differences. Mass spectrometry provided differences in protein profiles between methods, number of identified proteins, and protein classes. Our results indicate that the combination of CE/trehalose/iZON/UC is an optimal method to isolate equine uterine EVs with good yield and purity that can be applied in future studies to determine the role of equine uterine EVs in embryo-maternal interactions.

Cell type-specific endometrial transcriptome changes during initial recognition of pregnancy in the mare.

Previous endometrial gene expression studies during the time of conceptus migration did not provide final conclusions on the mechanisms of maternal recognition of pregnancy (MRP) in the mare. This called for a cell type-specific endometrial gene expression analysis in response to embryo signals to improve the understanding of gene expression regulation in the context of MRP. Laser capture microdissection was used to collect luminal epithelium (LE), glandular epithelium and stroma from endometrial biopsies from Day 12 of pregnancy and Day 12 of the oestrous cycle. RNA sequencing (RNA-Seq) showed greater expression differences between cell types than between pregnant and cyclic states; differences between the pregnant and cyclic states were mainly found in LE. Comparison with a previous RNA-Seq dataset for whole biopsy samples revealed the specific origin of gene expression differences. Furthermore, genes specifically differentially expressed (DE) in one cell type were found that were not detectable as DE in biopsies. Overall, this study revealed spatial information about endometrial gene expression during the phase of initial MRP. The conceptus induced changes in the expression of genes involved in blood vessel development, specific spatial regulation of the immune system, growth factors, regulation of prostaglandin synthesis, transport prostaglandin receptors, specifically prostaglandin F receptor (PTGFR) in the context of prevention of luteolysis.

"Effect of the addition of different catalase concentrations to a TRIS-egg yolk extender on quality and in vitro fertilization rate of frozen-thawed bull sperm''.

The aim of this study was to investigate the effects of different concentrations of catalase in a TRIS-egg yolk extender on sperm quality and embryonic development after in vitro fertilization of frozen-thawed bull sperm. For this purpose, from each of 7 bulls 2 ejaculates were collected and diluted with a TRIS-egg yolk extender containing 0, 5, 10, 15 or 20 IU catalase/mL. Sperm quality was evaluated 0, 3, 6, 12 and 24 h after thawing by using computer assisted analysis of motility and by flow cytometric assays. Embryonic development was determined after in vitro fertilization of bovine oocytes. Semen diluted with TRIS-egg yolk extender containing different concentrations of catalase showed more motile sperm, more sperm with intact plasma membranes, acrosomes and DNA, a high mitochondrial membrane potential, a high esterase activity, a low calcium level, a lower amount of synthesis of reactive oxygen species and lower degree of lipid peroxidation of sperm compared to semen frozen without catalase (P < 0.05), but not before 3 h after thawing. There was a dose-response relationship with the most prominent effect of 20 IU catalase/mL. However, the improvement of sperm quality had no effect (P ≥ 0.05) on embryonic development after in vitro fertilization with 20 IU catalase/mL. In conclusion, the addition of catalase to the sperm extender improved sperm quality with no obvious effect on in vitro fertility.

Luteal blood flow measured by Doppler ultrasonography during the first three weeks after artificial insemination in pregnant and non-pregnant Bos indicus dairy cows.

The objective of this study was to determine if there are differences in luteal size (LS), progesterone (P4), and luteal blood flow (LBF) between pregnant and non-pregnant Bos indicus dairy cows during the first three weeks after insemination, and whether these parameters are related to each other. Lactating cows (n = 13) of mixed parity with a body weight of 430 ± 18 kg (mean ± SD), showing regular estrous cycle were used in the study. All cows were artificially inseminated and were classified as pregnant (embryonic heartbeat on day 30; n = 8) or non-pregnant (inter-estrus interval 17 to 21 days, n = 5). In order to compare the LS and LBF after artificial insemination, B-mode and color Doppler ultrasonography of ovaries were performed on days 4, 5, 6, 7 (first week), 8, 10, 12, 14, (second week), and 16, 17, 18, 19, 20, 21 (third week) in pregnant and non-pregnant cows. Results revealed that the mean LBF was consistently higher (P < 0.05) during days 7 through 21 in pregnant cows than in non-pregnant cows. The mean LS was higher (P < 0.05) on days 6 and 7, and from day 17 onwards, and the mean concentration of P4 was higher (P < 0.05) on days 19, 20, and 21 in pregnant cows. In conclusion, LBF is a more sensitive parameter than LS and P4 for detection of differences in luteal function between pregnant and non-pregnant Bos indicus dairy cows during the first three weeks after AI.

Cluster analysis reveals seasonal variation of sperm subpopulations in extended boar semen.

This study aimed to identify motile sperm subpopulations in extended boar semen and to observe the presumptive seasonal variation in their distribution. Data from 4837 boar ejaculates collected over a two-year period were analyzed in terms of kinematic parameters by Computer Assisted Sperm Analysis (CASA). Individual sperm data were used to determine subgroups of motile sperm within the ejaculates using cluster analysis. Four motile sperm subpopulations (SP) were identified, with distinct movement patterns: SP1 sperm with high velocity and high linearity; SP2 sperm with high velocity but low linearity; SP3 sperm with low velocity but high linearity; and SP4 sperm with low velocity and low linearity. SP1 constituted the least overall proportion within the ejaculates (P < 0.05). Season of semen collection significantly influenced the different proportions of sperm subpopulations. Spring was characterized by similar proportions of SP1 and SP4 (NS) and higher proportions of SP3. Summer brought a decrease in both subgroups containing fast sperm (SP1 and SP2) (P < 0.05). During autumn, increases in SP2 and SP4 were recorded. Winter substantially affected the proportions of all sperm subpopulations (P < 0.05) and SP2 became the most represented subgroup, while SP1 (fast and linear) reached its highest proportion compared to other seasons. In conclusion, extended boar semen is structured in distinct motile sperm subpopulations whose proportions vary according to the season of collection. Summer and autumn seem to have a negative impact on the fast and linear subpopulation. Cluster analysis can be useful in revealing differences in semen quality that are not normally detected by classical evaluation based on mean values.

Effect of immune modulators on in vitro activation and proliferation of peripheral blood mononuclear cells from multiparous Holstein cows peripartum.

This in vitro study examined the ability of important immune modulators [ß-hydroxybutyrate (BHB), cortisol, prolactin, isoproterenol and insulin] to influence the responsiveness of peripheral blood mononuclear cells (PBMC) from multiparous dairy cows 29 ± 2 days before and 14 ± 3 days after calving. The activation and proliferation of PBMC in response to the mitogen phytohemagglutinin was estimated by the oxygen consumption rate after 24 hr and the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide) method after 72 hr respectively. In early lactation, the presence of 2 compared to 0.5 mmol/L BHB reduced PBMC activation (p < 0.05) and proliferation (p < 0.10), and the presence of 0.7 compared to 0.2 ng/ml insulin enhanced (p < 0.10) PBMC proliferation. In dry cows, the presence of low concentrations of BHB and insulin and both concentrations of prolactin (20 vs. 300 ng/ml) and isoproterenol (70 vs. 130 ng/L) enhanced activation (p < 0.10), but not proliferation (p ≥ 0.10) compared to cultures with no modulator addition. The presence or absence of high or low concentrations of hydrocortisone (20 vs. 45 nmol/L) did not (p ≥ 0.10) influence the activation and proliferation of PBMC from dry and early lactating cows. It is tempting to speculate that in antepartum PBMC the modulators represented an energy source or positive extrinsic signals to use nutrients for the activation process. On the other hand, PBMC from postpartum cows are known to be exposed to a metabolic challenging endocrine background. Under such conditions, high BHB concentrations and high insulin concentrations seem to act as negative and positive signals for PBMC, respectively, to utilize nutrients for activation and proliferation.

Clinical and subclinical endometritis induced alterations in bovine endometrial transcriptome and miRNome profile.

BACKGROUND: Clinical and subclinical endometritis are known to affect the fertility of dairy cows by inducing uterine inflammation. We hypothesized that clinical or subclinical endometritis could affect the fertility of cows by disturbing the molecular milieu of the uterine environment. Here we aimed to investigate the endometrial molecular signatures and pathways affected by clinical and subclinical endometritis. For this, Holstein Frisian cows at 42-60 days postpartum were classified as healthy (HE), subclinical endometritis (SE) or clinical endometritis (CE) based on veterinary clinical examination of the animals and histological evaluation the corresponding endometrial biopsies. Endometrial transcriptome and miRNome profile changes and associated molecular pathways induced by subclinical or clinical endometritis were then investigated using GeneChip® Bovine Genome Array and Exiqon microRNA PCR Human Panel arrays, respectively. The results were further validated in vitro using endometrial stromal and epithelial cells challenged with subclinical and clinical doses of lipopolysaccharide (LPS). RESULT: Transcriptome profile analysis revealed altered expression level of 203 genes in CE compared to HE animals. Of these, 92 genes including PTHLH, INHBA, DAPL1 and SERPINA1 were significantly upregulated, whereas the expression level of 111 genes including MAOB, CXCR4, HSD11B and, BOLA, were significantly downregulated in CE compared to the HE animal group. However, in SE group, the expression patterns of only 28 genes were found to be significantly altered, of which 26 genes including PTHLH, INHBA, DAPL1, MAOB, CXCR4 and TGIF1 were common to the CE group. Gene annotation analysis indicated the immune system processes; G-protein coupled receptor signaling pathway and chemotaxis to be among the affected functions in endometritis animal groups. In addition, miRNA expression analysis indicated the dysregulation of 35 miRNAs including miR-608, miR-526b* and miR-1265 in CE animals and 102 miRNAs including let-7 family (let-7a, let-7c, let-7d, let-7d*, let-7e, let-7f, let-7i) in SE animals. Interestingly, 14 miRNAs including let-7e, miR-92b, miR-337-3p, let-7f and miR-145 were affected in both SE and CE animal groups. Further in vitro analysis of selected differentially expressed genes and miRNAs in endometrial stroma and epithelial cells challenged with SE and CE doses of LPS showed similar results to that of the array data generated using samples collected from SE and CE animals. CONCLUSION: The results of this study unraveled endometrial transcriptome and miRNome profile alterations in cows affected by subclinical or clinical endometritis which may have a significant effect on the uterine homeostasis and uterine receptivity.

Possible role of interferon tau on the bovine corpus luteum and neutrophils during the early pregnancy.

When pregnancy is established, interferon tau (IFNT), a well-known pregnancy recognition signal in ruminants, is secreted by embryonic trophoblast cells and acts within the uterus to prepare for pregnancy. IFNT acts as an endocrine factor on the corpus luteum (CL) to induce refractory ability against the luteolytic action of PGF2 α. Hypothesising that IFNT may influence not only the uterine environment but also the CL in cows via local or peripheral circulation, we investigated qualitative changes in the CL of pregnant cows during the maternal recognition period (day 16) and the CL of non-pregnant cows. The CL of pregnant animals had a higher number of neutrophils, and the expression of interleukin 8 (IL8) mRNA and its protein was higher as well as compared with the CL of non-pregnant animals. Although IFNT did not affect progesterone (P4) secretion and neutrophil migration directly, it stimulated IL8 mRNA expression on luteal cells (LCs), influencing the neutrophils, resulting in the increased migration of IFNT-activated neutrophils. Moreover, both IFNT-activated neutrophils and IL8 increased P4 secretion from LCs in vitro. Our novel finding was the increase in neutrophils and IL8 within the CL of pregnant cows, suggesting the involvement of IFNT function within the CL toward establishment of pregnancy in cows. The present results suggest that IFNT upregulates neutrophil numbers and function via IL8 on LCs in the CL of early pregnant cows and that both neutrophils and IL8, stimulated by IFNT, are associated with an increase in P4 concentrations during the maternal recognition period in cows.

Tolerance of spermatozoa to hypotonic stress: role of membrane fluidity and correlation with cryosurvival.

The aim of this study was to evaluate inter-individual variability in osmotic properties of stallion spermatozoa and its correlation with cryosurvival. In addition, temperature dependency of hypo-osmotic tolerance and membrane fluidity were studied. Stallion sperm membranes exhibited good resistance towards hypotonic stress in the 15-30 °C temperature range, whereas membrane stability was found to be decreased at 4 and 37 °C. Bull spermatozoa showed greater hypo-osmotic tolerance compared with stallion spermatozoa, especially at temperatures above 30 °C, which coincided with decreased membrane fluidity of bovine spermatozoa in this temperature range. The critical osmolality at 22 °C, at which half of the sperm population survived exposure to hypotonic saline solution, was found to vary between 55 and 170 mOsm kg(-1) among different stallions. Clear correlations were found for pre- versus post-freeze sperm motility and membrane integrity. Pre-freeze percentages of membrane-intact spermatozoa after exposure to hypotonic stress showed a weak correlation with sperm motility after cryopreservation. This correlation, however, was not found when data were corrected for initial numbers of membrane-intact spermatozoa in the sample. We thus conclude that studies on pre-freeze tolerance towards hypotonic stress cannot be used to predict sperm cryosurvival rates for individual stallions.

The effect of metritis and subclinical hypocalcemia on uterine involution in dairy cows evaluated by sonomicrometry.

The objective of this study was to examine the effects of metritis and subclinical hypocalcemia on reduction of uterine size in dairy cows using ultrasonography and sonomicrometry. Four piezoelectric crystals were implanted via laparotomy into the myometrium of the pregnant uterine horn of 12 pluriparous Holstein Friesian cows 3 weeks before the calculated calving date. Sonometric measurements were conducted daily from 2 days before parturition (= Day 0) until Day 14 after calving and then every other day until Day 28. Distances between adjacent crystals were expressed in relation to reference values obtained before calving. The diameter of the formerly pregnant uterine horn was measured using transrectal B-Mode sonography starting on Day 10. Cows were retrospectively divided into the following groups: cows without metritis (M-; n = 7), cows with metritis (M+; n = 5), cows with normocalcemia (SH-; Ca > 2.0 mmol/l on Days 1 to 3; n = 5) and cows with subclinical hypocalcemia (SH+; Ca < 2.0 mmol/l in at least one sample between Days 1 and 3; n = 7). Metritis did not affect (P > 0.05) sonometric measurements, but the diameter of the formerly pregnant horn was larger (P ≤ 0.05) between Days 15 and 21 in M+ cows than in M‒ cows. Reduction in uterine length in hypocalcemic cows was delayed (P ≤ 0.05) between Days 8 and 21 compared with normocalcemic cows, but the uterine horn diameter was not related to calcium status. In conclusion, both diseases affected reduction of uterine size until Day 28. Cows with metritis had a larger uterine diameter, possibly attributable to accumulation of lochia, and cows with subclinical hypocalcemia had delayed reduction of uterine length, presumably related to reduction of myometrial contractility.

Different chronological patterns of appearance of blood derived milk components during mastitis indicate different mechanisms of transfer from blood into milk.

This study aimed to describe chronological patterns of changes of various candidate blood components in milk during the acute phase of a mammary immune response in detail. Eight dairy cows were challenged with Escherichia coli lipopolysaccharide in one udder quarter. Milk from challenged and control quarters and blood samples were taken before, and 1 and 2 h after challenge and then every 15 min until 5 h after challenge. The SCC, serum albumin, immunoglobulin (Ig)G1, IgG2, lactate dehydrogenase (LDH), and L-lactate in milk and blood, and α-lactalbumin in blood were analysed. All selected parameters in milk increased in challenged quarters but did not increase in control quarters. Milk IgG1, IgG2, serum albumin, and LDH were already significantly increased at 2 h after challenge whereas a significant increase of SCC was detectable at 2.75 h and L-lactate was increased at 2.25 h after challenge. In blood L-lactate was increased at 3.75 h after challenge, however, other factors in blood did not change significantly within the 5 h of experiment. In conclusion, the increase of blood components in milk during inflammation follows two different patterns: There is a rapid increase for IgG1, IgG2, or LDH, before the increase of SCC, and their concentrations reach a plateau within 3 h. On the other hand, SCC and L-lactate show a slower but consistent increase not reaching a plateau within 5 h after LPS challenge. L-lactate increases to higher concentrations in milk than in blood. This clearly shows that the increase of blood components follows different patterns and is therefore a controlled and compound-specific process and not exclusively an unspecific type of leakage.

Dimethylsulfoxide and conjugated linoleic acids affect bovine embryo development in vitro.

Conjugated linoleic acids (CLA) are employed to overcome the bovine periparturitional negative energy balance. Especially of interest are trans10,cis12 -linoleic acid (t10c12-CLA) and cis9,trans11-linoleic acid (c9t11-CLA). Their impact on embryonic development, though, is not clear. Here, effects of both above-mentioned CLA on bovine in vitro-produced embryos were assessed. Zygotes (n=2098) were allocated to one of seven groups: cultured with 50 or 100µM of either c9t11-CLA or t10c12-CLA, with 14 or 28mM DMSO or without supplement (control). Messenger RNA analysis of target gene transcripts (IGF1R, IGFBP2, IGFBP4, CPT2, ACAA1, ACAA2, FASN, SCD) via RT-qPCR was performed in single blastocysts. Cleavage rates did not differ, whereas development rates were decreased in both t10c12-supplemented groups in comparison to the unsupplemented group (31.7% ±2.2 control vs 20.2% ±2.0 50µM t10c12 vs 21.0% ±2.8 100µM t10c12). Compared with the unsupplemented group, SCD was expressed at a lower level in embryos cultured with 50µM c9t11-CLA. The relative amount of several transcripts was increased in embryos cultured with 14mM DMSO in comparison to those that developed in the presence of 50µM t10c12-CLA (IGFBP2, ACAA1, CPT2, FASN, SCD) or 50µM c9t11-CLA (IGF1R, IGFBP2, ACAA1, CPT2, FASN, SCD). The molecular analyses show that CLA influence embryonic fat metabolism.