Hepatic gene expression involved in glucose and lipid metabolism in transition cows: effects of fat mobilization during early lactation in relation to milk performance and metabolic changes.

Insufficient feed intake during early lactation results in elevated body fat mobilization to meet energy demands for milk production. Hepatic energy metabolism is involved by increasing endogenous glucose production and hepatic glucose output for milk synthesis and by adaptation of postcalving fuel oxidation. Given that cows differ in their degree of fat mobilization around parturition, indicated by variable total liver fat concentration (LFC), the study investigated the influence of peripartum fat mobilization on hepatic gene expression involved in gluconeogenesis, fatty acid oxidation, ketogenesis, and cholesterol synthesis, as well as transcriptional factors referring to energy metabolism. German Holstein cows were grouped according to mean total LFC on d 1, 14, and 28 after parturition as low [<200mg of total fat/g of dry matter (DM); n=10], medium (200-300 mg of total fat/g of DM; n=10), and high (>300 mg of total fat/g of DM; n=7), indicating fat mobilization during early lactation. Cows were fed total mixed rations ad libitum and held under equal conditions. Liver biopsies were taken at d 56 and 15 before and d 1, 14, 28, and 49 after parturition to measure mRNA abundances of pyruvate carboxylase (PC); phosphoenolpyruvate carboxykinase; glucose-6-phosphatase; propionyl-coenzyme A (CoA) carboxylase α; carnitine palmitoyl-transferase 1A (CPT1A); acyl-CoA synthetase, long chain 1 (ASCL1); acyl-CoA dehydrogenase, very long chain; 3-hydroxy-3-methylglutaryl-CoA synthase 1 and 2; sterol regulatory element-binding factor 1; and peroxisome proliferator-activated factor α. Total LFC postpartum differed greatly among cows, and the mRNA abundance of most enzymes and transcription factors changed with time during the experimental period. Abundance of PC mRNA increased at parturition to a greater extent in high- and medium-LFC groups than in the low-LFC group. Significant LFC × time interactions for ACSL1 and CPT1A during the experimental period indicated variable gene expression depending on LFC after parturition. Correlations between hepatic gene expression and performance data and plasma concentrations of metabolites and hormones showed time-specific relations during the transition period. Elevated body fat mobilization during early lactation affected gene expression involved in gluconeogenesis to a greater extent than gene expression involved in lipid metabolism, indicating the dependence of hepatic glucose metabolism on hepatic lipid status and fat mobilization during early lactation.

Variation in fat mobilization during early lactation differently affects feed intake, body condition, and lipid and glucose metabolism in high-yielding dairy cows.

Fat mobilization to meet energy requirements during early lactation is inevitable because of insufficient feed intake, but differs greatly among high-yielding dairy cows. Therefore, we studied milk production, feed intake, and body condition as well as metabolic and endocrine changes in high-yielding dairy cows to identify variable strategies in metabolic and endocrine adaptation to overcome postpartum metabolic load attributable to milk production. Cows used in this study varied in fat mobilization around calving, as classified by mean total liver fat concentrations (LFC) postpartum. German Holstein cows (n=27) were studied from dry off until d 63 postpartum in their third lactation. All cows were fed the same total mixed rations ad libitum during the dry period and lactation. Plasma concentrations of metabolites and hormones were measured in blood samples taken at d 56, 28, 15, and 5 before expected calving and at d 1 and once weekly up to d 63 postpartum. Liver biopsies were taken on d 56 and 15 before calving, and on d 1, 14, 28, and 49 postpartum to measure LFC and glycogen concentrations. Cows were grouped accordingly to mean total LFC on d 1, 14, and 28 in high, medium, and low fat-mobilizing cows. Mean LFC (±SEM) differed among groups and were 351±14, 250±10, and 159±9 mg/g of dry matter for high, medium, and low fat-mobilizing cows, respectively, whereas hepatic glycogen concentrations postpartum were the highest in low fat-mobilizing cows. Cows in the low group showed the highest dry matter intake and the least negative energy balance postpartum, but energy-corrected milk yield was similar among groups. The decrease in body weight postpartum was greatest in high fat-mobilizing cows, but the decrease in backfat thickness was greatest in medium fat-mobilizing cows. Plasma concentrations of nonesterified fatty acids and ß-hydroxybutyrate were highest around calving in high fat-mobilizing cows. Plasma triglycerides were highest in the medium group and plasma cholesterol concentrations were lowest in the high group at calving. During early lactation, the decrease in plasma glucose concentrations was greatest in the high group, and plasma insulin concentrations postpartum were highest in the low group. The revised quantitative insulin sensitivity check index values decreased during the transition period and postpartum, and were highest in the medium group. Plasma cortisol concentrations during the transition period and postpartum period and plasma leptin concentrations were highest in the medium group. In conclusion, cows adapted differently to the metabolic load and used variable strategies for homeorhetic regulation of milk production. Differences in fat mobilization were part of these strategies and contributed to the individual adaptation of energy metabolism to milk production.

Truncation of the mRNA cap-binding protein eIF4E is specific for the non-invasive implantation in pigs.

The mechanism of implantation is species specific (pig: epitheliochorial, bovine: synepitheliochorial, mouse: hemochorial). Recently, we have shown that proteolytical cleavage of the prototypical 25 kDa mRNA cap-binding protein eIF4E (eukaryotic initiation factor 4E) produces a stable variant with a molecular mass of approximately 23 kDa in porcine endometrium at the time of implantation. Here, we investigate if an eIF4E truncation also takes place in the endometrium of species with other implantation forms. Thus, eIF4E and its repressor protein 4E-BP1 were investigated in porcine, murine and bovine endometrium during the time of implantation. Our results show that eIF4E truncation is specific for the porcine implantation. In bovine and mouse uterine tissue, no cleavage of eIF4E was observed. Whereas no difference of bovine 4E-BP1 was found, in murine samples, increased phosphorylation during implantation was observed. However, porcine samples exhibit an opposite behaviour, the abundance and mainly the phosphorylation of 4E-BP1 decrease. We propose that the translation initiation in the endometrium is differently regulated by the two eIF4E forms with regard to different 4E-BP1 abundance and phosphorylation as well as different eIF4E/4E-BP1 binding dynamic depending on the type of implantation.

Studies on fixed-time ovulation induction in the pig.

A technology that allows for manipulating of oestrus and ovulation, and would then also allow for fixed-time insemination, can be of great benefit for swine farms that operate using sow batch management due, at least in part, to savings in labour and the production of large batches of evenly developed pigs. Thanks to the current knowledge on endocrine regulation of follicle development and ovulation, and the availability of numerous reproductively active substances such a technology is now available. It covers procedures for synchronising oestrus based on the use of altrenogest in gilts and of batch-wise weaning in sows, for stimulating follicle development using eCG and for inducing of ovulation using hCG or LH as well as GnRH analogues. While the procedures for oestrus synchronisation stand alone, other procedures require additional treatments. If fixed-time insemination is the goal, oestrus needs to be synchronised and follicular development and ovulation induced by the use of GnRH analogues and hCG with ovulation occurring within 36-42 hrs. It is a general recommendation to inseminate those animals twice, i.e. 24 and 40 hrs after ovulation induction. However, the aforementioned technology requires healthy animals and a solid management and cannot be used to compensate for poor management.

Performance and metabolic and endocrine changes with emphasis on glucose metabolism in high-yielding dairy cows with high and low fat content in liver after calving.

Elevated liver fat content occurs in high-yielding dairy cows during the transition from pregnancy to lactation after fat mobilization and may affect hepatic glucose metabolism, but the degree of liver fat storage is highly variable. Therefore, we studied metabolic and endocrine changes and hepatic glucose metabolism in cows that markedly differ in liver fat content. Multiparous cows from the same herd with high (HFL; n = 10) and low (LFL; n = 10) liver fat contents (mean of d 1, 10, and 21 after calving for each cow, respectively) were studied from 60 d before expected calving to 56 d in milk. Cows were fed ad libitum and all cows received the same diets. Liver samples were taken on d 1, 10, and 21 after calving; mean fat content (+/-SEM) in liver of HFL cows was 174 +/- 9.6 mg/g, whereas mean liver fat content in LFL cows was 77 +/- 3.3 mg/g. Blood samples were taken 20 and 7 d before expected calving and 0, 7, 14, 28, and 56 d after calving to measure plasma concentrations of nonesterified fatty acids, beta-hydroxybutyrate, glucose, insulin, glucagon, insulin-like growth factor-I, and leptin. In liver, glycogen content as well as mRNA levels of phosphoenolpyruvate carboxykinase, pyruvate carboxylase, glucose-6-phosphatase, and glucose transporter were measured by quantitative real-time PCR. Back fat thickness decreased and dry matter intake increased with onset of lactation, and back fat thickness was higher but dry matter intake was lower in HFL than in LFL. Energy-corrected milk yield did not differ between groups, but milk fat content was higher and lactose content was lower in HFL than LFL at the beginning of lactation. Energy balance was more negative in HFL than in LFL. Plasma nonesterified fatty acids and beta-hydroxybutyrate concentrations increased and plasma glucose concentration tended to decrease more in HFL than LFL with onset of lactation. Glucagon to insulin ratios increased more in HFL than LFL with onset of lactation. Hepatic glycogen content was higher in LFL than HFL, whereas mRNA levels of glucose-6-phosphatase and pyruvate carboxylase were higher in HFL than in LFL, and cytosolic phosphoenolpyruvate carboxykinase mRNA level increased similarly after parturition in both groups. In conclusion, an elevated liver fat content was related to greater fat mobilization and reduced feed intake and was associated with effects on hepatic glucose metabolism. As environment and feeding management were the same, individual cow factors were responsible for differences in energy metabolism during the transition period.

Qualitative and quantitative differences of cytoskeleton proteins in embryos produced in vitro, in vivo, and by somatic nuclear transfer.

The cytoskeleton, consisting of complex and dynamic systems of structural filaments, intermediate filaments and microtubules, is not only a structural element but also contributes to many cellular processes such as functional compartments, transportation, mitosis, secretion, formation of cell extensions, and intercellular communication. Suggestions in rat 2-cell embryos that abnormal distributions of cytoskeletal proteins occurred following the initiations of developmental arrest and our former studies showing reduced intercellular contact zones in cloned bovine embryos prompted us to conduct comparative studies on 8-cell stage bovine embryos from nuclear transfer (NT), in vitro, and in vivo production. Immunohistochemistry and Laser-Scanning-Microscopy facilitated detection of cytoskeleton proteins--alpha-tubulin, F-actin, beta-catenin, and the cell adhesion protein cadherin; image and cluster analysis were subsequently used to study the distribution pattern of the proteins, whereas Western blot was carried out for their qualitative and quantitative analysis. The maximum fluorescence intensity of stained alpha-tubulin was observed in the cloned and the in vitro embryos. A significant higher intensity of staining for F-actin was observed in the in vivo and in vitro embryos. In contrast, Western blot revealed no differences of actin, tubulin, and catenin between the three tested groups whereas a lower abundance of cadherin proteins in the cloned embryos was visible. The distribution of actin filaments in cloned embryos was more centric or one-sided and not peripheral whereas the stained spots of catenin were smaller in comparison to in vivo or in vitro produced embryos. These differences recorded in the distribution patterns may be associated with cell physiological processes related to an influenced actin-catenin-cadherin system. In conclusion, reduced intercellular contacts coupled with abnormal distribution of cytoskeletal proteins seem to play an important role in the developmental arrest encountered normally at the 8-cell stage in bovine cloned embryos.

Effect of initial cumulus morphology on meiotic dynamic and status of mitochondria in horse oocytes during IVM.

The aim of this investigation was to examine the chromatin configuration of the nucleus, pattern of mitochondrial aggregation and mitochondrial activity in parallel studies in the same horse oocytes. Horse oocytes recovered by ultrasound-guided follicle aspiration in vivo were classified according to two main initial cumulus morphologies as having compact or expanded cumulus. The percentage of oocytes with a diplotene meiotic configuration at the time of recovery from the follicles was highest in compact oocytes. Oocytes with expanded cumulus layers at the time of recovery matured more rapidly in vitro and reached a proportion >50% at the metaphase II stage (M 2) sooner during in vitro maturation (IVM), than did compact oocytes. The mitochondrial aggregation pattern changed from finely distributed (Type 1) through crystalline (Type 2) to an aggregated, granulated appearance (Type 3) during IVM. The pattern of mitochondrial aggregation at the time of recovery was associated with the initial cumulus morphology of the oocyte, in that compact oocytes had a higher proportion of Type 1 aggregation, whereas expanded oocytes had a higher proportion of Type 3. The fluorescence intensity of metabolic active mitochondria, measured by fluorescence intensity (Em 570) per oocyte after MitoTracker CMTM Ros orange labelling, increased in the oocytes during IVM and depended on initial cumulus investment. Oocytes with the granulated type of aggregated mitochondria Type 3 had the highest level of metabolic activity and were in more progressed stages of meiosis (A 1-M 2). Oocytes initially having expanded layers of cumulus reached significantly higher levels of mitochondrial activity after IVM than did oocytes initially having compact cumuli. During resumption of meiosis the mitochondrial activity of oocytes with initially expanded cumulus increased continuously up to M 2, whereas in oocytes from compact cumulus-oocyte complex (COC), the activity declined after A 1/T 1 stages of meiosis.

Selection of developmentally competent oocytes through brilliant cresyl blue stain enhances blastocyst development rate after bovine nuclear transfer.

The aim of the present investigation was to study the effect of oocyte selection on the efficiency of bovine nuclear transfer in terms of increased blastocyst production. For this purpose, prior to in vitro maturation (IVM), oocytes were selected for their developmental competence on the basis of glucose-6-phosphate dehydrogenase (G6PDH) activity indicated by brilliant cresyl blue (BCB) staining. It has been hypothesized that growing oocytes have a higher level of active G6PDH in comparison to the mature oocytes. Compact cumulus oocyte complexes (COCs) were recovered from slaughterhouse-collected bovine ovaries and classified either as control group, which were placed immediately into culture without exposure to BCB stain, or treatment group, which were stained with BCB for 90min before culture. Treated oocytes were then divided into BCB- (colourless cytoplasm, increased G6PDH) and BCB+ (coloured cytoplasm, low G6PDH) based on their ability to metabolize the stain. After IVM, oocytes were subjected to nuclear transfer procedure for the production of cloned embryos which were then cultured for a period of 8 days to determine the blastocyst rate. The BCB+ oocytes yielded a significantly higher blastocyst rate (39%) than the control (21%) or BCB- oocytes (4%). These results show that the staining of bovine cumulus-oocyte complexes with BCB before in vitro maturation could be used to select developmentally competent oocytes for nuclear transfer. In addition, G6PDH activity could prove to be a useful marker for determining the oocyte quality in future.

Pregnancy rates, LH and progesterone concentrations in mares treated with a GnRH agonist.

The aim of the study was to investigate the effect of the GnRH agonist Buserelin given on day 10 after ovulation on pregnancy rate and concentrations of progesterone and LH. Altogether 191 warmblood mares were used for two trials. Fresh or frozen/thawed semen from 27 stallions was used for A.I. In trial A 171 mares received either Buserelin (Receptal, Hoechst, Germany, 40 microg/animal) or 10 ml 0.9% NaCl (placebo). On day 16 after A.I. pregnancy diagnosis was performed by ultrasound scanning of the uterus. For statistical analysis, data were analyzed by a mixed model, with four fixed factors (treatment, type of spermatozoa, A.I. number, reproductive status of the mare) and a random factor (stallion). Least Square Means (LSM) for pregnancy rate were 46.0% in GnRH agonist treated mares and 36.4% in the control group (P=0.22). In trial B 20 lactating and cycling mares were used for endocrine studies. Blood samples were recovered for analyses of progesterone and LH from days 0 to 11. The mean progesterone concentrations increased continuously from days 0 to 8 after ovulation in both groups (GnRH group: from 0.81+/-0.48 to 5.47+/-0.48 ng/ml, control group: from 0.63+/-0.68 to 5.83+/-0.68 ng/ml). Moreover, the progesterone concentrations from days 9 to 11 were not different between the GnRH and the control group. In contrast to this LH concentrations were markedly influenced by the GnRH agonist. On day 10 LH concentrations were significantly higher in GnRH agonist treated than in placebo treated animals. From the data obtained from individual animals it can be concluded that GnRH agonist, given during luteal phase may have different effect on luteal function.

Altered vocalization rate during the estrous cycle in dairy cattle.

Correct detection of estrus is a problem in dairy herds. In practice, several procedures exist for detection of estrus besides conventional visual observation by humans. These procedures deliver very different results regarding detection of estrus. It is known that the calls of female mammals can contain information about reproductive status. It is also suspected that the vocalizations of cattle contain information about age, sex, dominance status, and stage in the estrous cycle. In the present study, a methodology for the continuous automatic recording of vocalization of heifers during the periestrous period is presented. It was shown in 10 tethered heifers that the estrous climax results in an increase in vocalization rate. Vocalization rate of heifers increased approximately 84% from d -2 to 0 (related to observed estrus) and approximately 59% from d -1 to d 0. After d 0, vocalization rate decreased about 79%. Increased vocalization was correlated with the visual observation of estrus by humans. We also found 2 different structures in the vocalization of heifers. The harmonic structure showed regular frequency bands, whereas the nonharmonic structure was noisy. The hypothesis that the disharmonic structure increases near the estrous climax was confirmed. Hence, it seems possible to get information about stage of the estrous cycle of dairy cattle by means of monitoring vocalization. The presented method of automatically detecting the rate of cattle vocalization (patent pending) could be used solely or in combination with other automated systems for detecting estrus and could considerably increase current estrus detection rates once its applicability can be demonstrated in nontethered cattle.

Luteinizing hormone release after administration of the gonadotropin-releasing hormone agonist Fertilan (goserelin) for synchronization of ovulation in pigs.

The generic GnRH agonist, Fertilan (goserelin), was tested for the ability to induce an LH surge and ovulation in estrus-synchronized gilts. Three experiments were performed to 1) examine the effect of various doses of Fertilan on secretion of LH in barrows, to select doses to investigate in gilts (Exp. 1); 2) determine doses of Fertilan that would induce a preovulatory-like rise of LH in gilts (Exp. 2); and 3) determine the time of ovulation after Fertilan treatment (Exp. 3). In Exp. 1, 10 barrows were injected on d 1, 4, 7, 10, and 13 with 10, 20, or 40 microg of Fertilan; 50 microg of Gonavet (depherelin; GnRH control) or saline (negative control); and sequential blood samples were collected for 480 min. There was a dose-dependent stimulation (P < 0.05) of LH release. Maximal plasma concentrations of LH (LH(MAX)) were 2.1 +/- 0.2, 4.1 +/- 0.3, 2.6 +/- 0.4, and 3.4 +/- 0.3 ng/mL after 10, 20, and 40 microg of Fertilan and 50 microg of Gonavet, respectively, and duration of release was 78 +/- 9, 177 +/- 12, 138 +/- 7, and 180 +/- 11 min, respectively. Fertilan doses of 10 and 20 microg were deemed to be the most suitable for testing in gilts. In Exp. 2, 12 gilts received (after estrus synchronization with Regumate and eCG) injections of 10 or 20 microg of Fertilan or 50 microg of Gonavet 80 h after eCG to stimulate a preovulatory-like LH surge and ovulation. An LH surge was induced in 3 of the 4 gilts in both of the Fertilan groups and in all of the Gonavet-treated gilts. Characteristics of induced release of LH did not differ among groups: LH(MAX), 5.0 +/- 0.9 vs. 4.6 +/- 1.8 vs. 6.6 +/- 1.1 ng/mL; duration, 11.7 +/- 2.0 vs. 12.3 +/- 2.2 vs. 14.3 +/- 0.5 h; interval from GnRH injection to LH(MAX), 4.0 +/- 2.0 vs. 6.7 +/- 1.3 vs. 5.8 +/- 1.6 h. In Exp. 3, estrus-synchronized gilts were injected with 20 microg of Fertilan (n = 8) or 50 microg of Gonavet (n = 4), and the time of ovulation was determined by repeated endoscopic examination. Time of ovulation ranged from 34 to 42 h postGnRH; however, ovulation occurred earlier in the Gonavet compared with the other groups (P < 0.05). Results of these experiments indicate that 1) barrows are an appropriate model for determining GnRH doses that can be effective in inducing a preovulatory-like LH surge in females; 2) the generic GnRH agonist Fertilan, at doses of 10 to 20 microg, can stimulate an LH surge in gilts, with subsequent ovulation; and 3) Fertilan at doses of 10 and 20 microg should be examined further for use in fixed-time insemination protocols.

Ultrastructural analysis reveals striking differences of intercellular contact lengths in bovine embryos produced in vivo, in vitro and by somatic cell nuclear transfer.

Cellular coherence and communication, thus cell-to-cell contact is an indispensable premise to sustain the formation of complex, multi-cellular organisms. We have analyzed intercellular contact lengths in NT-cloned bovine embryos compared to the in vivo or in vitro produced counterparts. Therefore, ultrastructural analysis was carried out by transmission electron microscopy (TEM) at the 8-cell and blastocyst stage of development. To obtain embryos generated in vivo, oviducts of superovulated cows were flushed 3 days after insemination, subsequent to slaughter. Standard in vitro maturation (IVM) and -fertilization (IVF) were utilized to obtain in vitro embryos. Cloned embryos by somatic nuclear transfer were produced by the handmade cloning (HMC) procedure. The points of apposition/focal contact points (CPs) between the blastomeres were of the shortest order in cloned embryos (236 +/- 135 nm) and of highest order in the in vivo produced embryos (2,085 +/- 1,540 nm), although no significant differences regarding the blastomere sizes in the various groups of 8-cell embryos could be established. In summary, the CP lengths in case of in vitro and in vivo 8-cell embryos were, on an average, five or nine times longer, respectively, than in the case of the cloned embryos. These differences of CP lengths vanished in embryos reaching the blastocyst stage of embryonic development in all the three groups of embryos. The observed differences of intercellular contact length at distinct stages of embryonic development could be responsible for differences in intercellular communication between the blastomeres at the beginning of cellular differentiation. These may be one reason for the lower developmental competence of cloned (NT) embryos.

Measurement of redox potential and steroid concentrations in the follicular fluid of growing and regressing follicles of mares.

The aim of this study was to prove if oxidation-reduction levels in the follicular fluid were new functional indices of follicular health and whether there was a high level of accordance with endocrinological parameters and with the growth stage as detected by ultrasound monitoring of individual follicles during the oestrous cycle in mares. Follicles were classified as growing and regressing follicles using ultrasonography. Altogether 48 follicles with a diameter from 20 to 56 mm were aspirated by transvaginal ultrasound guided follicular aspiration. Follicular concentration of oestradiol and progesterone in relation to the diameter of growing follicles showed correlations of r = 0.64 and r = 0.57, respectively. The redox potential derived index D2 varied from -448 to +431 in the collected fluids of the follicles. The accordance of the judgement of all follicles using both complexes of methods - endocrinological and ultrasonographic parameters vs. analysis of oxidation and reduction levels - reached 72.5%. This finding has shown that parameters of redox reactions do not correlate closely with the stage of follicular growth or regression as determined by in vivo scanning of ovaries or by assessment of follicular steroid concentrations. However, the measurement of redox potentials offers an opportunity to examine the whole process of metabolism in follicular cells and to forecast impairments of cellular performances. Changes of redox parameters in growing follicles enable an earlier prediction of their further development. The data demonstrate that growing and regressing follicles do not represent nonatretic, early atretic and atretic follicles, respectively.

Endocrine, morphological, and cytological effects of a depot GnRH agonist in bovine.

The present study was conducted to assess effects of the gonadotropin-releasing hormone agonist (GnRHa) triptorelin in dairy heifers. The peptide was released from a commercial 4-week depot formulation (Decapeptyl Depot) administered at animals' estrus (day 0). First experiment (EXP I, n=5), which was aimed to explore the availability of peptide, detected a maximum of triptorelin concentration between day 2 and 5 after depot injection, and the peptide remained detectable by RIA in peripheral blood for about 3 weeks. In further experiments, the peptide release was terminated on day 9 (EXP II, n=16) or day 21 (EXP III, n=47). Treatment effects were studied on follicular development, the characteristics of cumulus-oocyte complexes (COCs) (EXP II; EXP IIIa) and secretions of LH and progesterone (EXP IIIb). Results showed that the occurrence of the pre-ovulatory LH surge was more uniform in treated heifers than that in controls. The duration of ovulation periods was similar amongst the heifers of EXP II, but more compact amongst those of EXP III each compared with the respective controls. Post-ovulatory, the number of LH pulses was significantly reduced by treatment, whereas both basal LH and progesterone concentrations were elevated on a few days. Follicular growth was reduced only by the prolonged influence of the GnRHa. There were increased proportions of both degenerated COCs and immature oocytes from small follicles (<3mm in diameter), and meiotic configuration and quality of oocytes isolated from follicles 3-5mm were changed after the prolonged, 21-day treatment. These results indicate that a continuous influence of a GnRHa over more than 1 week may increasingly impair the development of bovine follicles and oocytes. This may have some significance for the development of novel GnRH-based techniques in regulating the reproductive function in cattle.

[Utilization of glucose and long-chain fatty acids in lactating dairy cows fed a fat-enriched diet]. / Verwertung von Glucose und langkettigen Fettsäuren durch die laktierende Milchkuh bei Fütterung einer fettangereicherten Diät.

The fate of carbon from long-chain fatty acids and glucose in dairy cows which were fed with protected fat was studied using stable isotope technique. The experiment was carried out on two groups of dairy cows (n=16 in each group) during the first 15 weeks of the lactation period. The cows were fed isoenergetic and isoproteinogenous diets based on corn silage. About 1.8 kg of tapioca starch in the diet of the starch group was substituted by about 0.7 kg of rumen protected fat (Ca salts of palm oil and soybean oil) in the diet of the fat group. The carbon atoms of dietary fat were naturally depleted in 13C as compared to carbon atoms of starch. Daily milk performance and lactose output were significantly (P < 0.05) higher among the cows fed with fat diet. In comparison to the starch group, the enrichment of milk fat with 13C was significantly lower, while that of breath CO2 was significantly higher in the fat group (P < 0.05). This means the fatty acids were incorporated into milk fat in preference to metabolic oxidation. Further studies showed that blood glucose is oxidized to a lower extent and is used for the synthesis of lactose to a higher proportion if the cows were fed with the fat diet. The glucose entry rate into the body glucose pool was not different between the diets. In conclusion, the dietary fatty acids perform a glucose sparing effect and improve the glucose supply for the mammary gland.

Influence of ovarian steroid hormones or platelet-activating factor on mRNA of platelet-activating factor receptor in endometrial explant perfusion cultures from ovariectomized bovine.

Platelet-activating factor (PAF) and its receptors are involved in inflammatory-like processes of the uterus associated with increased vascular permeability. PAF is supposed to be influenced by ovarian steroid hormones. The present study was undertaken to examine whether progesterone (P(4)), estradiol (E(2)) or PAF influence the PAF receptor gene expression in perfused endometrial explants derived from ovariectomized bovine. Furthermore, we identified the cell types in which the PAF receptor gene and protein are expressed. In endometrial explants, applications of 10 nM P(4) or 10nM P(4) plus 10 nM E(2) for 24 h induced elevated transcript levels of PAF receptor in comparison to the controls or after treatment with 1 nM E(2). When explants were administered 10 nM E(2), a slight decrease in the transcript level was recorded. After treatment of explants with PAF, no significant changes in PAF receptor mRNA expression was observed compared to the control group. We demonstrate that PAF receptor immunoreactivity and mRNA are detected mainly in the luminal epithelium, epithelial cells of the superficial glands and to a lesser degree in stroma. Levels of PAF receptor mRNA in bovine endometrial explants were correlated with PAF receptor protein localization assessed by immunohistochemistry. The regulation of PAF receptor by progesterone in bovine endometrial explants suggests that PAF is involved in the physiological process of reproduction.

Short communication: hydroperoxides in circulating lipids from dairy cows: implications for bioactivity of endogenous-oxidized lipids.

This study was conducted to investigate the potential for increased oxidative stress of high- vs. average-producing dairy cows. Two experiments were performed using 11 and 13 Holstein cows (53 +/- 2 d postpartum). Lipohydroperoxides (LHP) were determined in serum lipids (experiment 1) and low-density lipoprotein (experiment 2) via oxidation of ferrous to ferric ions through LHP using thiocyanate as chromogen. In experiment 1, differing milk yield and milk energy output corresponded to different concentrations of LHP. In experiment 2, analysis of regression resulted in a significant relationship between milk yield and LHP. Phospholipids isolated from lipids with 6.5 microM of LHP evoked in monocytic cells a transient increase in superoxide formation, indicating inflammatory potential. The results show that high milk productivity can associate with oxidative stress indicated by oxidative modifications of circulating lipids and their changed bioactivity.

Bovine blastocyst development rate in vitro is influenced by selection of oocytes by brillant cresyl blue staining before IVM as indicator for glucose-6-phosphate dehydrogenase activity.

The aim of this present study was to increase the efficiency of blastocyst production from cows after in vitro maturation/fertilization (IVM/IVF) by oocyte selection before maturation. Oocytes were selected on the basis of brillant cresyl blue (BCB) staining, used to indicate glucose-6-phosphate dehydrogenase (G6PDH) activity. To re-valuate the hypothesis that growing oocytes are expected to have a high level of active G6PDH, while mature oocytes have low G6PDH activity, cumulus oocyte complexes (COCs) were recovered from slaughterhouse ovaries by slicing the surface of the ovary. Only oocytes with a compact cumulus investment were used. Oocytes were placed into three groups: (1) control--placed immediately into culture; (2) holding control--COCs kept in PBS containing 0.4% BSA for 90 min before placement into culture; and (3) treatment--incubation with BCB for 90 min before culture. Treated oocytes were then divided into BCB- (colorless cytoplasm, increased G6PDH) and BCB+ (colored cytoplasm, low G6PDH) on their ability to metabolize the stain. Activity of G6PDH was determined via measurement of NADP reduction induced by G6P as substrate oxidized by G6PDH in the cytosol of control, BCB- and BCB+ groups; G6PDH activity was significant higher in BCB- COCs than in control and BCB+ COCs. After IVM, oocytes were fertilized in vitro. Embryos were cultured to day 8. The rate of maturation to metaphase II was significantly higher for control and BCB+ oocytes than for BCB- oocytes. The BCB+ oocytes yielded a significantly higher proportion of blastocysts (34.1%) than did control or holding control oocytes (18.3 and 19.2%); and both controls and BCB+ oocytes had significantly higher blastocyst development than did BCB- oocytes (3.9%). These results show that the staining of bovine cumulus oocyte complexes with BCB before in vitro maturation may be used to select developmentally competent oocytes for IVF. In addition, G6PDH activity may be useful as a marker for oocyte quality in future studies on factors affecting developmental competence.

Mitochondrial aggregation patterns and activity in porcine oocytes and apoptosis in surrounding cumulus cells depends on the stage of pre-ovulatory maturation.

In this study, we evaluated the distribution and oxidative activity of mitochondria in ex vivo pre-ovulatory porcine oocytes using the fluorescence probe MitoTracker CMTM Ros Orange. Cumulus-oocyte complexes (COCs) were classified according to cumulus morphology and time from hCG administration. The meiotic configuration of the oocytes and the degree of apoptosis in the surrounding cumulus cells were also evaluated. Estrus was synchronized in 45 crossbred Landrace gilts by feeding altrenogest for 15 days and administering 1000 IU PMSG on Day 16. The LH peak was simulated by treatment with 500 IU hCG, given 80 h after PMSG. Endoscopic oocyte recovery was carried out 2 h before or 10, 22, or 34 h after hCG administration. Altogether 454 COCs were aspirated from follicles with a diameter of more than 5 mm. Cumulus morphology in the majority of COCs recovered 2 h before and 10 h after hCG was compact (60.4 and 52.7%, respectively; P<0.05). At 22 h after hCG, COC morphology changed significantly from 10 h dramatically: 74% of COCs had an expanded cumulus (P<0.01). At 34 h after hCG, 100% of recovered COCs had an expanded cumulus. The percentage of oocytes with a mature meiotic configuration differed among COC morphologies and increased as the interval after hCG administration increased (P<0.05). The type of mitochondrial distribution in the oocytes (n=336) changed from homogeneous to heterogeneous as the interval after hCG administration increased (P<0.01) and was associated with the cumulus morphology. Representative mitochondrial distributions were found as follows: -2 h: fine homogeneous in compact and dispersed COCs; 10 h: granulated homogeneous in compact and dispersed COCs; 22 h: granulated homogeneous in expanded COCs; and 34 h: granulated heterogeneous and clustered heterogeneous in expanded COCs (P<0.01). The oxidative activity of mitochondria measured by fluorescence intensity (Em: 570 nm) per oocyte after Mitotracker CMTM Ros Orange labeling increased in the oocyte as the post-hCG interval increased (P<0.01) and depended on the type of mitochondrial distribution. Lowest oxidative activity of mitochondria was found in oocytes with fine homogeneous distribution (253.1+/-9.4 microA). The oxidative activity increased (334.4+/-10.3 microA) in oocytes with granulated homogeneous distribution of mitochondria, and reached highest level in oocytes with granulated heterogeneous (400.9+/-13.0 microA) and clustered heterogeneous distributions (492.8+/-13.9 microA) (P<0.01). Mitochondrial activity in oocytes coincided with apoptosis in surrounding cumulus cells which increased in a time-dependent manner during pre-ovulatory maturation in vivo (P<0.01). These results indicate that there is a relationship between meiotic progression, cumulus expansion and mitochondrial redistribution and their oxidative activity during final pre-ovulatory maturation in pig oocytes. It appears that increased levels of mitochondrial activities in oocytes are correlated to increased levels of apoptosis in surrounding cumulus cells, in which mitochondria may play a role.