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The aim of this study was to establish a model to induce cystic ovarian follicles (COFs) in cattle using the cyclooxygenase inhibitor, indomethacin. Eighteen Holstein-Frisian cattle were synchronized with prostaglandin F2alpha (PGF2α) and gonadotropin-releasing hormone (GnRH). Ultrasound-guided transvaginal intrafollicular injections were performed in 23 preovulatory follicles with different concentrations of indomethacin 16 h after GnRH administration. An injection of 0.2 ml 35 µM indomethacin solution (resulting in a final concentration of 8 µg/ml in the follicular fluid) was the minimal dosage leading to COF formation. The induced COFs reached a maximum mean diameter of 36.9 ± 4.5 mm eleven days after injection. The estrous cycle was extended to 25–39 days. Luteinization was first observed 4 days after injection, accompanied by a slight increase in plasma progesterone concentration. The bioactivity of indomethacin was demonstrated by the decrease of prostaglandin E2 in the follicular fluid of three animals. The method presented here is minimally invasive and allows for the generation of defined COFs for further investigations.

Genome wide mRNA expression analysis of small and large luteal cells, isolated from the mature staged corpora lutea (CL), was not performed in any species. In the current study, we have isolated bovine small and large luteal cells from mid-cycle (day 10-11) animals and characterized their transcriptomes using "GeneChip™ Bovine Gene 1.0 ST Arrays". A total of 1276 genes were identified to be differentially expressed between small and large luteal cells. Data evaluation revealed that novel functions, extracellular matrix synthesis and immune cell recruitment, were enriched in small luteal cells. On contrary, functions regarding the regulation of folliculogenesis, luteal regression, fatty acid and branched chain amino acid metabolism were differentially enriched in large luteal cells. Overall, the current data offer a first and detailed insight into the functional roles of small and large luteal cells in the mature bovine corpus luteum.

We have previously mimicked the morphological and functional changes occurring in the oviduct epithelium during the estrous cycle in vitro by using an air-liquid interface (ALI) culture system and basolateral application of 17β-estradiol (E2) and progesterone (P4). In the current study we aimed to explore the transcriptomic changes elicited by E2 and P4 together during estrous cycle simulation and to dissect the individual effects of E2 and P4 on oviduct epithelium physiology. Primary porcine oviduct epithelial cells (POECs) (N = 6 animals) were cultured at the ALI. After differentiation for 11 days, we sequentially simulated diestrus (10 days) and estrus (2.5 days) by adding serum levels of E2 and P4 to the basolateral compartment either in combination (mix trial) or separately (P4 trial and E2 trial, respectively). Cell response was evaluated by microarray analysis (mix and P4 trials), quantitative RT-PCR, and histomorphometry (all trials). When we compared simulated diestrus with estrus stage in the mix trial, there were 169 (142 upregulated and 27 downregulated) differentially expressed genes (DEGs; fold change ≥1.5). In the P4 trial, 108 DEGs (83 upregulated and 25 downregulated) were detected. Gene enrichment analysis revealed that immune-related pathways were exclusively affected in the mix trial. In both mix and P4 trials, POECs exhibited in vivo-like morphological changes regarding epithelium height and portion of ciliated cells. However, E2 alone did not trigger morphological changes. We deduce that P4 mainly drives structural variations, and E2 is imperative for regulating immune function of the oviduct epithelium during estrous cycle.

Background: Relevant literature indicate that more than 0.8 g starch/kg body weight from compounded feed composed of different starch sources induces disproportionate glycaemic and insulinaemic responses in horses. Objective: It should be investigated whether crushed oats, barley and maize also cause a disproportionate increase in plasma glucose and insulin when fed as the only concentrate in quantities equal to and above 0.8 g starch/kg body weight. Method: Four mares received hay plus oats, barley and maize, respectively, in quantities equal to 0.8, 1.0 and 2.0 g starch/kg body weight. At the test days, chewing parameters were detected and blood sampled before and 30, 60, 90 and 120 min after the concentrate meal. Plasma glucose and insulin were measured and areas under the curve were calculated. Results: Maize was ingested particularly slowly (dry matter basis; P < 0.05), but glycaemic and insulinaemic responses were particularly low (starch basis; P < 0.05). In general, the glycaemic responses were highest with 1 g starch/kg body weight (P < 0.05). The quantity of starch had no effect on the insulinaemic response (P > 0.05). A defined increase in plasma glucose induced the highest insulinaemic response with oat grains. Conclusion: Oats and barley are ingested faster and induce higher glycaemic and insulinaemic responses than maize. Until 120 min postprandial, elevated quantities of starch from these grains seem to induce no disproportionate or at least linear increase of plasma glucose and insulin. The insulinaemic response to a defined increase of plasma glucose is particularly pronounced with oats.

Experimental setups for physiological research, in which acute operative interventions need to be performed, can require inclusion of general anesthesia (GA), which may interfere or confound with the effects of the experimental factors of interest on measured variables. It was recently shown that the most commonly used sedatives/anesthetics in pigs (e.g., ketamine, xylazine, azaperone) affect physiological responses and thus the primary metabolic readouts have the potential to be confounded. To extend the search for a physiologically-friendly anesthesia regime for such studies, we investigated effects of GA induced by propofol (Prop) or pentobarbital (Pent) or propofol plus isoflurane (Prop + Isof) on plasma concentrations of commonly measured metabolites and hormones. In 2 experimental runs, 6 female pigs fitted with jugular vein catheters were used. Fasted pigs received either no drug (CON) or anesthetized rotationally either with Prop, Pent or Prop + Isof on different days, separated with washout periods of sufficient length (2 to 3 d). Six-h profiles of glucose, lactate, non-esterified fatty acids (NEFA), triglycerides (TG), cholesterol, urea as well as hormones including glucagon, insulin and cortisol were determined. Concentrations of cholesterol, urea and glucagon remained unaffected by any of the treatments ( > 0.05). Pent tended to increase cortisol from 30 to 90 min after drug administration. Glucose and lactate concentrations were increased ( < 0.05) by Prop and Pent within the first hour of GA ( < 0.05). Propofol and Pent reduced NEFA concentrations, which were more pronounced during the last 2 h of the studied period. Triglyceride concentrations were increased by all 3 agents within the first 45 min with Prop containing treatments exerting a stronger effect than Pent. Our data suggest that GA with Prop and particularly with Pent adulterate plasma metabolite and hormone profiles of pigs acutely, and thus has the potential to confound the effects of experimental factors of interest. Although Prop + Isof anesthesia did not differ from the controls, providing a physiologically-friendly GA, both single and the isoflurane-combined treatment of Prop induced hypertriglyceridemia due to the lipid adjuvant of the Prop drugs. It is concluded that readouts obtained under GA may be influenced both by physiological adulterations as response to anesthesia as well as by artifacts due to accompanying ingredients of the drug formulations.

The proliferation of blood lymphocytes from nonpregnant, nonlactating heifers was comparable with that of nonpregnant cows in their first lactation. Both low and high levels of β-hydroxybutyrate and low levels of prolactin, but not isoproterenol, reduced the proliferative response of dairy heifers and cows in their first or second lactation., Canadian Journal of Animal Science, 98 (4), ISSN:0008-3984

Physiological research with swine often includes sedation or general anesthesia (GA), which may influence the basal physiological responses of experimental animals and may have the potential to confound or interfere with the effects of experimental factors of interest. Using 6 adult female pigs, we investigated whether selected plasma metabolites and hormones are influenced by GA induced with ketamine (K) and 2 neuroleptic sedatives, namely azaperone (A) and xylazine (X). Fasted pigs rotationally received either no drug, a single intravenous administration of A or X, or A or X combined with ketamine (AK or XK, respectively), and plasma concentrations of glucose, lactate, non-esterified fatty acids (NEFA), triglycerides (TG), glucagon, insulin, and cortisol were determined for a 5-h period following administration. Azaperone and X induced deep sedation, whereas AK and XK induced GA. Overall, the average plasma glucose concentrations were increased by A and X, with the latter exerting a stronger effect that was also associated with hypoinsulinemia ( < 0.05). Time-dependent effects indicated a more rapid increase in glucose concentration due to X or XK than AK. Plasma NEFA concentrations were elevated by A and AK and to a lesser extent by X and XK ( < 0.05). Plasma lactate and TG levels were elevated by A and AK and remained unaffected by X or XK. Plasma cortisol concentrations were elevated ( < 0.05) by X and XK and even more so with a single administration of A ( < 0.05), while the combined effect of A with ketamine resulted in the highest cortisol concentrations ( < 0.05). Our data suggest that the effects of azaperone are mediated by cortisol but less so for xylazine, which also indicates that azaperone elicits a stronger stress response in pigs. Xylazine probably induces long-lasting, fasting-state hyperglycemia through the stimulation of hepatic glucose production associated with hypoinsulinemia. A discriminant analysis based on the variation in all of the measured metabolites and hormones, collectively, indicated that ketamine induced no additional effect on the overall physiological response patterns than that of the individual sedatives. In conclusion, the neuroleptic sedatives azaperone, and to a lesser extent, xylazine, acutely affect the metabolism of pigs, so primary metabolic readouts obtained under these drugs may be confounded.

The aim of this study was to investigate whether plasma anti-Muellerian hormone (AMH) levels of Holstein-Friesian heifers could be used to predict ovum pick-up (OPU) and embryo production outcomes. Plasma samples and data were collected from 64 heifers, which underwent repeated OPU with subsequent in vitro embryo production followed by embryo flushing after superovulation. AMH levels were significantly positively correlated with the number of follicles aspirated per OPU session (r = 0.45), recovered oocytes per OPU (r =0.43) and in vitro produced embryos per OPU (r = 0.28). No significant correlations between AMH and in vivo produced embryos were ascertained. Our results suggest that correlations between AMH and outcomes of an OPU-IVF program are too low to use AMH as a precise predictive parameter for the success of a particular OPU procedure in Holstein-Friesian heifers. However, AMH can help to identify groups of very good or very poor oocyte donors.

In female mammals, granulosa cells of the ovarian follicle differentiate into the corpus luteum after ovulation of the pregnable oocyte into the fallopian tube. During these differentiation processes several morphological alterations have to occur and the molecular basis is not fully understood. As an endpoint estradiol production from granulosa cells has to switch off in favor for progesterone production from the proceeding corpus luteum to sustain the developing embryo. Previously, we demonstrated that the multiligand receptor LOX-1 plays a critical role in steroid hormone synthesis of granulosa cells via intracellular calcium release from endoplasmic (ER)-dependent and ER-independent calcium pools. In the present study, we show that inhibition of LOX-1 leads to a rearrangement of ceramide from the basal membrane toward the Golgi apparatus. This activity is accomplished by a calcium-dependent phosphorylation of aromatase, the key step in estradiol production. Phosphorylated aromatase increased estradiol production in a dose-dependent manner. Our data indicate that the ceramide cascade is essential for proper granulosa cell function and ceramide redistribution serves as a first step in order to proceed with the prosperous differentiation into a corpus luteum.

Estradiol produced by ovarian granulosa cells triggers the luteinizing hormone surge which in turn initiates ovulation in female mammals. Disturbances in estradiol production from granulosa cells are a major reason for reproductive dysfunctions in dairy cows. Endogenous estradiol production might be altered by reactive oxygen species (ROS) such as oxidized low-density lipoprotein (ox-LDL). Inhibition of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), a receptor of ox-LDL, leads to increased estrogenesis in granulosa cells. This activity is mediated by calcium release from endoplasmic reticulum (ER)-dependent and ER-independent calcium pools. Inhibition of the LOX-1 signal transduction pathway is followed by mitochondrial alterations. The membrane potential ΔΨ increases and the ROS production decreases in mitochondria after blocking LOX-1. Our data indicate that blocking the LOX-1 receptor signal pathway might be a promising way to improve steroid hormone concentrations in metabolically highly active female mammals and, therefore, to defend against reproductive dysfunctions in humans and animals. © 2013 International Society for Advancement of Cytometry

Double fixed-time insemination after ovulation induction is commonly used in pigs to obtain in vivo produced embryos at defined stages of development for downstream biotechnological applications. How - ever, variations in the time of ovulation and fertilisation of the ovulated oocytes by spermatozoa, mainly in one of the inseminations, can cause diversities in embryo development. The aim of the present study was to reduce embryo diversity and to achieve a 'uniform outcome' of porcine embryo stages using single laparoscopic fixed- time insemination (LIUI). Altogether, 48 puberal German Landrace gilts were included in the study. Estrus of gilts was synchronized by 15-day long altrenogest (Regumate ® ) feeding and follicle development was stimulated with 850 IU eCG 24 h after the final altrenogest application. Ovulation was induced with 500 IU hCG 80 h after eCG. LIUI was performed 31 h after hCG treatment. Gilts under general anaesthesia were fixed in a dorsal position, a pneumoperitoneum was produced and three trocar cannulas were inserted into the abdomen for optics and instru - ments. Each uterine horn was carefully punctured 10-15 cm caudal from the utero-tubal junction with a 2.5 mm trocar. A 2.2 mm catheter was inserted about 3 cm into the uterine lumen and 20 ml of extended fresh boar semen (32.2 × 106 sperm cells/ml) were injected. Embryos were surgically flushed from the genital tract two (Day 2) and three (Day 3) days after insemination. Altogether, 778 oocytes/embryos were recovered (recovery rate 68 ± 17%); 45 of 48 gilts (93.8%) revealed fertilisation and 76.1% of the recovered embryos ( n = 592) were at the 2- and 4-cell stage. On Day 2 ( n = 22 gilts), a higher percentage of gilts (72.7%, P < 0.05) displayed only 2-cell embryos compared with gilts which had 2- and 4-cell (22.7%), or only 4-cell embryos (4.6%). On Day 3 ( n = 23 gilts), the proportion of gilts with 2-cell, 2- and 4-cell, and only 4-cell embryos shifted to 4.3%, 0% and 95.7%, respectively (P < 0.05). The results of the present study demonstrate high rates of fertilisation and homogenously developed embryos after single fixed-time laparoscopic intrauterine insemination in gilts. Additionally, these results were achieved by inseminating a 60% lower number of sperm cells per insemination dose compared to usual doses used for transcervical insemination. In conclusion, LIUI can be recommended for the in vivo production of embryos in a homogeneous developmental stage, and also as an alternative method for low-dose insemination.

Dynamic follicular changes occur during the equine estrus cycle, but little is known about their impact on the properties of recovered oocytes. The aim of this study was to characterize the cytoplasmic and chromatin status of equine oocytes in relation to the time of recovery during the follicle wave. Transvaginal ultrasound-guided follicle aspiration was performed two times in relation to the follicle wave: estrus-subordinate, from the subordinate follicles of mares in estrus, 24 hours after human chorionic gonadotropin stimulation of a dominant preovulatory follicle, and new-wave, from the follicles of the subsequent induced follicular wave, at the time of dominant follicle divergence (largest follicle 23 mm diameter). A total of 1011 follicles were aspirated. The oocyte recovery rate in the new-wave group was significantly lower than that for the estrus-subordinate group (12% vs. 26%, respectively); this was associated with a significantly higher proportion of oocytes with compact cumuli (44% vs. 27%, respectively). Estradiol concentrations were markedly higher in follicular fluid from new-wave follicles (885.6 ± 123.2 ng/mL vs. 54.3 ± 18.9 ng/mL, for estrus-subordinate; P < 0.001), indicating greater viability. Aspiration group did not affect glucose-6-phosphate dehydrogenase activity in recovered oocytes. Fibrillar (more juvenile) chromatin was more prevalent in new-wave oocytes, whereas estrus-subordinate oocytes showed more condensed chromatin or resumption of meiosis (P < 0.05). Mitochondrial activity was higher in oocytes with expanded cumuli in the new-wave group, but not in the estrus-subordinate group. In conclusion, our results clearly showed that the time of aspiration in relation to the follicle wave is associated with significant differences in follicle status and oocyte characteristics: new-wave oocytes were from a more viable follicle population and had more juvenile chromatin and cytoplasmic characteristics, whereas estrus-subordinate oocytes were from a more atretic follicle population and exhibited signs of atresia-related acquisition of meiotic and cytoplasmic competence. These findings will help in effective scheduling of oocyte recovery for equine-assisted reproduction techniques.

Secretion of adequate levels of progesterone from a proper corpus luteum (CL), which develops out of the cells of a healthy preovulatory follicle, is a key-factor for establishment of a pregnancy. The aim of this study was to investigate the relationship between morphological and secretory characteristics of preovulatory follicles and their corresponding corpus luteum with regard to the post-partum period in high-yielding dairy cows. There- fore, ultrasound-guided aspirations of preovulatory follicles were performed repeatedly, using 20 first lactating cows between 26 and 121 days after parturition. Heat was induced with a PGF analogon followed by administra- tion of a GnRH analogon. The dominant follicle was aspirated 21 h after administration of the GnRH analogon. The diameters of the follicles were estimated at aspiration and the morphology of the resulting luteal tissue was examined on day 14 after follicle aspiration using ultrasonographic examinations. Concentrations of progesterone (P 4 ) and 17-beta-oestradiol (E 2 ) were determined in the follicular fluids (FF) and P 4 concentration was estimated at the time of CL examination in plasma. A CL development occurred in 82% after dominant follicle aspiration. The interval of time between parturition and follicle aspiration did not affect the investigated variables. The diameter of the aspirated preovulatory follicle was positively correlated to the cross-section area of the developed luteal tissue (R = 0.60; P < 0.01) as well as to the plasma P 4 concentration on day 14 after follicle aspiration (R = 0.47; P < 0.05). Also, E 2 concentrations in FF were positively correlated to cross-section area of the luteal tissue (R = 0.54; P < 0.05). Comparing the FF of the follicles that gave rise to a CL after aspiration to follicles that did not, both types had comparable P 4 , but the former type harboured higher E 2 concentrations. In conclusion, preovulatory follicle diameter as well as steroid concentrations in the follicular fluid could be used prospectively to identify cows which will have well-developed CLs and high plasma P 4 levels later. On the other hand, CL development after follicle aspiration can be used as a retrospective quality parameter of dominant follicles. These results will help to identify suitable animals for breeding or recipients for embryo transfer.

The provision of NaCl, according to current recommendations, to horses in moderate work has been shown to induce immediate postprandial acidosis. The present study aimed to clarify whether this NaCl induced acidosis i) persists beyond the immediate postprandial period, and ii) is still present after a 2 week adaptation period. Six adult warmblood mares in moderate work received daily 1.00 kg hay per 100 kg body weight (bwt) only together with 0.64 kg unprocessed cereal grains/100 kg bwt.d as fed basis. Using a 3x3 Latin Square, either 0 (NaCl-0), 50 (NaCl-50) or 100 (NaCl-100) g NaCl/d were fed together with the concentrates in two equal doses for 3 weeks. During the final week, a mineral digestibility trial was undertaken. The middle sodium and chloride intake (NaCl-50) at least met the most common recommendations for moderate work. Morning (7:00 AM) urine and venous blood samples were collected on days 0, 1–4, 8, and 15, and analysed for pH, acid-base status, creatinine and electrolyte concentrations. Fractional electrolyte clearances (FC) were determined. Mean apparent sodium digestibility ranged between 60–62% whereas chloride digestibility was consistently above 94%. Supplementing 100 g but not 50 g of NaCl resulted in significant reduction of blood pH and base excess as well as urinary pH and urine acid excretion. Both 50 g and 100 g NaCl supplementation caused a significant reduction in base and net acid-base excretion, urine density and potassium concentration, but increased urine sodium concentration and the FC of sodium and chloride (P < 0.05). This suggests that a high proportion of the recommended salt doses is excreted renally. The above effects of NaCl supplementation persisted over the 2 week measurement period. Results suggest that feeding 100 g NaCl to moderately exercising horses results in mild metabolic acidosis, whereas feeding 50 g according to current recommendations resulted in compensated acidosis.

The platelet-activating factor (PAF) is a proinflammatory lipid present in the fluid of ovarian Graafian follicle. Ovarian blockage of the PAF receptor (PAFr) reduces ovulations in the rat whereas underlying mechanism is poorly understood. Mural granulosa cells (MGC) were mechanically isolated from the theca interna of bovine periovulatory follicle. The mRNA abundance for PAFr, progesterone receptor and cyclooxygenase-2 were measured by real-time PCR. Cytosolic calcium (Ca2+) concentration was assayed by microscopy using Fura-2 AM as indicator, 8-isoprostaglandin F2α (8-isoPGF2α) by an ELISA kit. Fluorescent products arising from intracellular oxidation of hydroethidine (HE) and dihydrorhodamine (DHR) were quantified by flow cytometry. The cells expressed PAFr mRNA and PAFr protein and responded to cPAF (nonhydrolyzable form of PAF) with a pulsating increase in Ca2+, demonstrating functional PAFr. Elevation of Ca2+ was reversed by WEB-2086, an inverse PAFr agonist. cPAF elevated the level of 8-isoPGF2α in the medium of MGC cultured with luteinizing hormone (LH). cPAF alone had no significant influence on the oxidation of HE and DHR, or 8-isoPGF2α level. In MGC from vital periovulatory follicle, PAF and LH signaling plays an important role in regulating the production of excessive oxidants. Blockage of PAFr seems to interfere with these regulatory processes essential for ovulation. © 2009 International Society for Advancement of Cytometry

F2-isoprostanes such as 8-iso-prostaglandin F2alpha(8-iso-PGF2α) are formed by free radical-catalyzed mechanisms from membrane phospholipids and from low density lipoproteins through peroxidation of arachidonic acid. Esterified 8-iso-PGF2α is cleaved by phospholipases, circulates in blood and is excreted as putatively harmful oxidatively modified lipid via the kidney into urine. In this study we demonstrate that 8-iso-PGF2α concentrations in plasma samples from heifers are higher (p0.005) compared to those from first-lactating dairy cows at 71 days postpartum. Furthermore, plasma 8-iso-PGF2α concentrations vary with ovarian activity and differ in response to luteolytic initiation as well as activation of the hypothalamic-pituitary-gonadal axis between heifers and first-lactating cows. Sustainable concentrations of 8-iso-PGF2α (50-150 pg/ml) are detectable in the phospholipid fraction of milk, suggesting milk as an additional excretion route for 8-isoprostanes. Plasma levels largely paralleled levels in milk (p0.001). Plasma phospholipid 8-iso-PGF2α concentrations in cyclic cows decreased (p0.05) from day 38 to day 71 postpartum, whereas milk phospholipid 8-iso-PGF2α rather increased (p0.05). Cyclic cows tend to have higher 8-isoprostane levels compared to acyclic animals. In contrast to lipohydroperoxides, concentration of 8-iso-PGF2α were not correlated with milk yield (p0.05). Our data indicate 8-iso-PGF2α may be a novel biomarker of oxidative stress in dairy cow, which is detectable in blood as well as in milk.