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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 innate immune system has numerous mechanisms to fight against pathogens, including the formation of neutrophil extracellular traps (NETs). By spreading out chromatin, antimicrobial peptides and enzymes, neutrophils efficiently trap pathogens like bacteria and facilitate their elimination. During this process, high concentrations of extracellular histones can be reached. Several researchers have demonstrated that the cytotoxic characteristics of these histones can trigger diseases like sepsis. Interestingly, the carbohydrate polysialic acid (polySia) can bind histones and reduce histone-mediated cytotoxicity in a chain length-dependent manner. In the present study, we examined the chain length of polySia in plasma and tested its ability to decrease the cytotoxic characteristics of extracellular histones. Remarkably, we detected polySia not only in the soluble fraction of plasma, but also on enriched extracellular vesicles (EVs). Chain length analysis revealed that polySia chains originating from human plasma can consists of more than 40 sialic acid residues and show a cytoprotective effect against extracellular histones. Intriguingly, polySia is not only present in human plasma but also in fish and other branches of vertebrates. Thus, polySia is a physiological element in plasma and may represent a natural buffer for extracellular histones.

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.

Postprandial alterations of plasma amino acid (PAA) levels partly reflect a temporal contribution of the feed. How cereal grains affect PAA levels is not known. We hypothesized that a meal of cereal grains causes a temporal increase of PAA, affected by grain species, grain genotype and meal size. Six mares were used in three consecutive trials, receiving four oats, barley and maize genotypes, respectively. Individual grain genotypes were provided as 3 meal sizes corresponding to 1.0, 1.5 or 2.0 g starch/kg body weight. Meadow hay (1.5 kg/100 kg body weight) was offered daily. At the test days, 1 kg hay was fed 60 min prior to the grain meal. Blood samples were taken before grain feeding (0 min) and 30, 60, 90, 120, 180, 240 and 300 min thereafter. Subsequently, the remaining hay was offered. The genotype × starch quantity (i.e., meal size) interaction had a major effect on postprandial PAA concentrations (P

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.

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.

Based on a series of exercise tests which included the estimation of sweat losses, this article proposes a novel sweat scoring system for exercising horses. This provides a practical estimate of individual animal exercise-induced sweat losses, based on visible appearance of sweat on the coat after work, which takes into account the effect of various influencing factors. In terms of accuracy and flexibility, the score seems to provide advantages over estimates based on current general recommendations from reference books. Additional studies are needed to validate this scoring system and its use under more diverse situations.

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 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.

Development of assisted reproductive technologies in horses has been slow compared to other domestic animals, like ruminants and pigs. Not only the scarce availability of oocyte, but also the lacks of interest from horse breeders have been the main reason for this delay. In the last few years there has been increasing interest in obtaining foals via assisted reproduction from mares. Efficient methods for oocyte recovery from live mares have generated clinical interest in methods for in vitro fertilization (IVF). The conventional method of IVF (maturation of oocytes, culture of oocytes with capacitated sperm, cultivation of zygotes), as used in the bovine, has proved to be inefficient in the horse (Alm et al. 2001). Therefore, a method for more efficient production of equine embryos has been developed – the intracytoplasmic sperm injection (ICSI). With ICSI, one spermatozoon is injected into a mature oocyte. Equine embryos produced by in vitro maturation, ICSI, in vitro embryo culture and transferred transcervically to recipient mares have resulted in the birth of normal foals. Fertilized equine oocytes, produced by ICSI, are also being used to develop effective equine embryo culture systems.

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.

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.

Anti-Muellerian hormone (AMH) is synthesized only by granulosa cells in female mammals as an inhibiting factor for early follicular recruitment. Especially in preantral and small antral follicles, expression of AMH is very intensive. However, we hypothesize that AMH affects also the development of large follicles by an estrogenesis modification. This current investigation provides for the first time information about concentrations of AMH in equine follicular fluid and demonstrates their relationship to functional characteristics of follicle development. Therefore, fluids of follicles in different developmental stages were obtained by repeated, transvaginal ultrasound guided-follicle aspirations from 8 mares during dioestrus and from preovulatory follicles (after hCG-inducted ovulation) during oestrus. Concentrations of AMH, 1 713-estradiol-, progesterone- and GE-i were measured in the follicular fluids. Data showed significant negative correlations between intra follicular concentrations of AMH and 1 713-estradiol (r = -0.58; p0.0001) as well as between AMH and follicle diameter (r = -0.76; p0.001), but not between AMH and progesterone or IGF-1. Based on the observed dynamic changes of AMH secretion in large antral follicles during follicle development, a regulatory involvement of AMH in equine follicle development is likely. Due to species-specific features, the results are discussed considering their potential relevance to controlled superovulation in mares.

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.

1 Einleitung und Zielstellung 1 2 Literaturübersicht 3 2.1 Physiologie der Fortpflanzung bei Stuten 3 2.2 Follikulogenese 6 2.3 Funktionelle Follikelentwicklung 9 2.4 Eizellreifung 14 2.5 Bedeutung der Mitochondrien in Oozyten während der Maturation 22 2.6 Bedeutung der Kumuluszellen für die Prozesse der Eizellreifung 24 2.7 Parameter zur Bestimmung der Eizellqualität 26 2.8 In-vivo-Oozytengewinnung bei Stuten 33 3 Material und Methode 37 4\. Ergebnisse 54 4.1 Kumulus-Oozyten-Komplex-Gewinnung mittels transvaginaler ultraschallgeleiteter Follikelpunktion 54 4.2. Steroidkonzentrationen in den Follikelflüssigkeiten der unterschiedlichen Follikelpopulationen 56 4.3 Aktivität der Glukose-6-Phosphat-Dehydrogenase der gewonnenen Oozyten 58 4.4 Untersuchungen zur Chromatinkonfiguration der Oozyten 63 4.5 Untersuchungen zur mitochondrialen Aktivität in den gewonnenen Oozyten 70 4.6 Untersuchungen zur mitochondrialen Aggregation der gewonnenen Oozyten 76 5 Diskussion 82 6 Zusammenfassung 106 7 Summary 108 8 Zitierte Literatur 110 9 Publikationen 124 10 Danksagung 125 11 Selbstständigkeitsversicherung 126, Ziel des tierexperimentellen Designs war es Eizellen in vivo zu gewinnen, die verschiedenen, definierten physiologischen Entwicklungsstadien von Follikelpopulationen zugeordnet werden konnten. Durch die parallele Bestimmung mehrerer Parameter der Eizellqualität an jeder einzelnen Eizelle, sollten Zusammenhänge in vivo zwischen chromosomalen und zytoplasmatischen Reifungsprozessen in den Oozyten untersucht werden. Insbesondere sollte dabei die follikuläre Herkunft als Einflussfaktor auf die Eigenschaften von Oozyten näher beschrieben werden. Die Oozytengewinnung erfolgte durch wiederholte transvaginale ultraschallgeleitete Follikelpunktionen, die in 120 Follikelpunktionssitzungen an 14 Mecklenburger Warmblutstuten während einer Zuchtsaison vorgenommen wurden. Die Eizellgewinnung erfolgte zunächst bei rossigen Stuten, denen 24 Stunden zuvor ein hCG-Präparat appliziert worden war. Die Follikelaspirate wurden getrennt nach präovulatorischen Follikeln und subordinanten Follikeln gesammelt. Nach einer Ablation aller sichtbaren Follikel, wurden die Stuten einer zweiten Follikelaspiration unterzogen, noch bevor sich in der neu herangewachsenen Follikelpopulation ein dominanter Follikel entwickelt hatte. Zur genaueren Charakterisierung der verschiedenen Follikelgruppen erfolgten stichprobenartig Analysen der Östradiol-17β- und Progesteronkonzentrationen in den Follikelflüssigkeiten. Die Kumulus-Oozyten- Komplexe (KOK) wurden nach der Gewinnung unter einem Stereomikroskop morphologisch beurteilt und mit Brillant-Cresyl-Blau (BCB) unter Kulturbedingungen zur Bestimmung der Aktivität der Glukose-6-Phosphat- Dehydrogenase (G-6-PDH) inkubiert. Anschließend wurden die denudierten Oozyten mit dem Fluoreszenzfarbstoff MitoTracker Orange CMTM Ros zur Bestimmung der mitochondrialen Aktivität und Aggregation vital inkubiert und nach einer Fixierung in Paraformaldehyd mit Hoechst 33258 zur Bestimmung der Chromatinkonfiguration parallel gefärbt. Die Östradiol-17β-Konzentrationen waren mit 1911,4±184,5 ng/ml in präovulatorischen Follikeln, mit 885,6±120,6 ng/ml in Follikeln der wachsenden und mit 54,4±155,9 ng/ml in Follikeln der subordinanten Follikelgruppe zwischen den Follikelgruppen signifikant unterschiedlich (p, The aim of this study was to recover equine oocytes in vivo, which could be assigned to follicle populations in different physiologic developmental stages. By a parallel determination of several parameters of oocyte quality for every single oocyte, links between chromosomal and cytoplasmic maturation processes in vivo should be investigated. Especially the impact of the different follicular origins on the characteristics of oocytes should be described in detail. Oocyte recovery was done by repeated transvaginal ultrasound guided follicle aspiration in 14 Mecklenburger Warmblood mares, which underwent 120 follicle aspiration sessions during one breeding season. Follicle aspiration sessions were performed first during the heat of the mares, 24 hours after they had received hCG. The aspirates of the preovulatory follicles and the subordinate follicle populations were collected separately. After an ablation of all visible follicles of the mare, a second follicle aspiration session was done just before a dominant follicle had developed in the newly grown progressive follicle population. To characterise the follicle populations more closely random samples of follicle fluid from the different follicle populations were taken simultaneously and analysed for their oestradiol and progesterone content. Immediately after recovery the cumulus- oocytes-complexes (COCs) were divided in groups depending on their cumulus morphology under a stereomicroscope and incubated under culture conditions with brilliant cresyl blue (BCB), to evaluate the activity of glucose-6-phosphate dehydrogenase (G-6-PDH) enzyme. The denuded oocytes were then incubated with the Mito Tracker Orange CMTM Ros vital stain to measure the mitochondrial activity and aggregation. Following a fixation with paraformaldehyde the oocytes were also subjected to Hoechst 33258 stain to detect the chromatin configuration. The oestradiol concentrations showed a level of 1911.7 ± 185.5 ng/ml in preovulatory follicles, 885.6 ± 120.6 ng/ml in growing and 54.4 ± 155.9 ng/ml in subordinate follicle populations indicating significant differences between the follicular fluid samples of the different follicle populations (p < 0.05). Significantly more oocytes with compact cumuli were found in the progressive follicle population (p < 0.05), whereas the subordinate follicle population tended to contain more oocytes, which were only covered by corona radiata cells (p = 0.06). By the measurement with BCB stain, altogether one third of the recovered oocytes showed a high G-6-PDH activity. No significant impacts of the follicle population of origin, the cumulus morphology, the mitochondrial aggregation patterns or the mitochondrial activity of the oocytes were observed on the distribution patterns of oocytes with different G-6-PDH activity. The evaluation of the chromatin configuration showed that follicles of growing follicle populations contained significantly higher proportions of oocytes with a fibrillar diplotene, while subordinante follicle populations had higher proportions of oocytes with a condensed diplotene (p < 0.05). While oocytes with the chromatin configuration of a fibrillar diplotene showed a significantly higher amount of oocytes with a low G-6-PDH activity, oocytes with pycnotic chromatin had a significantly higher number of oocytes with a high G-6-PDH activity (p < 0.05). Oocytes with a pycnotic chromatin were found in subordinate follicle populations only. The mitochondrial activity (fluorescence intensity/oocyte) was significantly increased in oocytes of growing follicle populations as compared to oocytes from subordinate follicle populations (p < 0.05). Higher levels of mitochondrial activity were found also in oocytes with an expanded cumulus and in oocytes with only corona radiata cells than in the oocytes with a compact cumulus (p < 0.05). The mitochondrial activity was significantly higher in oocytes which showed mitochondria in granulated aggregation pattern than in oocytes with fine mitochondrial aggregation pattern (P