The interval from calving to commencement of luteal activity (CLA) was determined by progesterone measurements from milk samples obtained once a week until the 14th week post-partum in 513 German Holstein cows in first to third parity. Milk samples were analyzed by an "on-farm" device (eProCheck(®), Minitüb, Germany) and simultaneously by RIA. The objective of this study was to examine the effect of milk yield, protein content and body condition of a cow on the CLA post-partum. Milk progesterone concentrations of "on-farm" measurements correlated with measurements done by the RIA-method significantly (r=0.72; P<0.001). Within the analyzed herd the interval from calving until the first rise of progesterone averaged 5.6±2.4 weeks. The 100-days milk yield was not associated with CLA. Cows with a milk protein content at 1st milk recording of ≤3.5% revealed first luteal activity 1.3±0.3 weeks later than cows that had a content of >3.75% protein (P<0.01). Furthermore cows with assisted calving or dystocia presented significantly later CLA than cows which required no help during the calving process (P<0.05). The change in back fat thickness from 1st to 2nd milk recording had a significant influence on CLA (P<0.05). In conclusion the phenotypic impact of milk yield on fertility cannot be confirmed regarding to CLA. The negative energy balance after calving, caused by the high milk yields, is more detrimental for the cyclical activity as was shown by the parameters milk protein content and change in BFT.

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.

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.

Different factors determine the effectiveness of the use of sires in AI. Most important factors are the number of inseminated spermatozoa, the quality of spermatozoa, and the time of insemination. Especially in superovulated animals, the insemination scheme plays in important role to cover the whole ovulation period. The influence of 3 different dosages of spermatozoa (15 × 106, 5 × 106, and 1 × 106) on fertilization rate was examined in experiment A. In experiment B, one dosage of female and male spermatozoa of 3 different bulls was used for timed AI in 31 heifers. Timed AI in normal-cycling cattle [13 h after gonadotropin-releasing hormone (GnRH) application] with detected corpus luteum (Days 8 to 13 of cycle) was carried out after induction of luteolysis and induction of ovulation [GnRH application 60 h after prostaglandin F2α (PGF2) application]. Embryos and oocytes were flushed from the oviduct of 116 hemicastrated or slaughtered heifers on Day 4 after insemination. The ovulation rate in heifers was 95.4%. Eighty percent of the oocytes or embryos were recovered. The influence of the factors sire, ejaculate, and dosage were tested by GLM analyses of SAS® (SAS Institute Inc., Cary, NC, USA). There was no significant difference in the fertilization rate (93.3, 96.2, and 78.8%) and in the proportion of normally developed embryos (84.6, 80.7, and 75.8%) between groups. Significant differences were found in the mean number of accessory sperms/embryo and in the proportion of embryos with >10 accessory sperms/embryo or without accessory sperms; however, the proportion of intact embryos was similar. Using sexed semen in experiment B, similar results were obtained after flushing of the oviducts on Day 4 after insemination of hemicastrated or slaughtered animals. In total, an ovulation rate of 91.7%, a recovery rate of 70%, and a fertilization rate of 86.8% were obtained. There were no differences between female- and male-sorted spermatozoa and the control group. In experiment C, altogether 13 heifers were treated 8 times with FSH for 4 days starting between Day 8 to 12 of estrous cycle. Prostaglandin F2α was given 48 and 60 h after the first FSH injection. Insemination with sexed semen (n = 5 heifers) and with unsorted semen (n = 8; 15 × 106 and 1 × 106) was done at 55 and 71 h after induction of luteolysis. Flushing of the uterus was performed on Day 7. Using the time-oriented insemination after superovulation of animals, fertilization rates varied between 65 and 85%. There was no difference between groups regarding the number of transferable embryos (5.5, 4.9, and 4.8). The results demonstrate that the application of an approved insemination schedule may accomplish high fertilization rates after insemination with sexed or reduced dosages of spermatozoa in normal-cycling as well as superovulated cattle.

The objectives of this study were to obtain relevant blood flow indices of umbilical arteries (UmA) of porcine fetuses using a laparoscopic ultrasound probe and to relate these data with fetal size at early to mid gestation. Fetal parameters and flow indices, i.e., fetal length and area, fetal heart rate (FHR), systolic pulse duration (T1), interpulse duration (T2), T2/T1 ratio, peak systolic velocity (PSV), time averaged velocity (TAV), resistance index (RI) and pulsatility index (PI), were measured in 182 fetuses of 26 pregnant Landrace gilts on pregnancy day (PD) 36 (122 fetuses from 17 gilts), PD42 (19 fetuses from 3 gilts) and PD51 (42 fetuses from 6 gilts). Fetal heart rate was higher on PD36 than on PD42 (P<0.05). No differences (P>0.05) were obtained concerning systolic pulse duration, flow velocities and RI. On PD42, the PI was lower (P<0.05), while the interpulse duration (P=0.06) and T2/T1 ratio tended (P=0.08) to be higher on PD42 compared with PD36 and to PD51. To find differences in UmA blood flow parameters concerning fetal size, i.e., fetal length, fetuses were retrospectively grouped as follows: small (lower 25%), medium (mean 50%) and large (upper 25%), respectively. Although, fetuses differed in size (P<0.001) within and between days of pregnancy, FHR, PSV, TAV, RI and PI did not differ (P>0.05) among the size classes. Only systolic pulse duration tended to be longer (P=0.05) in large compared with small fetuses on PD36, and interpulse duration was lower in large fetuses on PD36 in comparison with PD51 (P<0.05). Though there was no link between fetal blood flow indices and fetal intrauterine growth retardation (IUGR), with further studies based on these flow indices, it might be possible to evaluate nutrient- or stress-related influences on fetal growth and development, particularly in the case of IUGR.

The milk yield of dairy cows has increased in the last decades. Furthermore, an increase in the severity of the negative energy balance (NEB) of dairy cows in the first weeks of the lactation period has been noted. It is discussed that decreasing fertility rates of dairy cows are directly attributed to this finding. Aim of this study was to analyze the mRNA expression pattern of selected candidate genes in the bovine oviduct in correlation with long-term effects of energy status. 24 multiparous Holstein-Friesian cows were included in this study. Negative energy balance was calculated weekly from food intake, milk yield and body weight. Furthermore, the amount of subcutaneous body fat mobilisation (BFM) was measured weekly as thickness lost since week 1 ante partum. Therefore, three groups (n=8) were accordingly defined: highest, medium or lowest BFM or NEB, respectively. In addition, the first luteal activity was recorded. Cows were slaughtered at least 10 weeks post partum (pp). Oviducts were collected directly after slaughter and classified according to distinct estrous cycle phases or metabolic classes. Total RNA was isolated from scraped oviductal mucosal cells and subjected to real-time RT-PCR amplification. The obtained mRNA expression data were correlated with the distinct energy status as long-term effects. About 30 candidate genes were selected which are associated with local reproductive events; e.g. growth factors, extracellular matrix components and enzymes involved in the prostaglandin synthesis. Statistical analysis showed if expression pattern were influenced by the factor estrous cycle, they were unaffected by the influence of the metabolic factors and vice versa. Mainly the mRNA expression of members of growth factor families like fibroblast growth factor (FGF) or vascular endothelial growth factor (VEGF) was affected by different BFM. FGF2 showed higher expression in animals with higher BFM in weeks 2 and 3 pp, but low expression directly after ovulation. VEGFA was positively correlated in its expression pattern with NEB. Matrix metalloproteinase 2 and its inhibitor (TIMP1) as well as the insulin-like growth factor 1 were expressed higher after ovulation compared with the luteal phase, but not influenced in their expression pattern by the energy status. Cyclooxygenase 2 (PTGS2) expression was neither influenced by the estrous cycle stage nor the energy status. PTGS1 and phospholipase A2G4B oviductal mRNA expression was higher in cows which showed early luteal activity compared with the cows having a delayed first cycle. However, PTGS1 expression was higher in the oviduct of cows with low energy balance or highest back fat thickness after week 9 pp. Enzymes for metabolizing hydroxybutyrate were expressed higher after ovulation, but their expression was not affected by the energy status. Progesterone receptor mRNA was expressed significantly higher after ovulation and in cows with early luteal activity. In contrast, estradiol receptor alpha expression was only influenced by the energy status. It seems that BFM and NEB can affect mRNA expression patterns of key factors for reproductive events even weeks after their occurrence. Since these factors are an integral part of the fine-tuned cross-talk in bovine reproduction, their expression changes due to energy imbalance may help understand the causes of poor fertility often associated with NEB. Supported by BMBF (Fugato-plus REMEDY).

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±9mg/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 β-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.

Anti-Muellerian hormone (AMH) is a glycoprotein that is expressed only in the gonads. While secretion of AMH from the testicular Sertoli cells is essential for male fetal sex differentiation during embryonic development, in adult females it is produced only from granulosa cells of small and preantral follicles. Therefore it is used as an endocrine marker for this pool of small gonadotropin responsive follicles, which are also termed the ovarian follicular reserve in humans. Woman with high AMH plasma levels (great ovarian follicular reserve) can get lower FSH doses for and have a better ovarian response after a stimulatory treatment. The aim of our study was to determine AMH levels in equine plasma and to describe their relationship to follicular growth in mares. After weekly repeated ultrasound guided follicles aspiration sessions, we chose 4 mares, out of 12 oocyte donor mares, which had constantly a high number of follicles between the aspiration sessions (11.1 +/- 1.1 follicles) and 4 mares, which showed constantly low numbers of new grown follicles (6.2 +/- 0.5 follicles). Plasma AMH levels were measured with an ELISA (DSL-10-144400; Beckman Coulter, USA) in blood samples, which were taken from each mare at five consecutive follicle aspiration sessions. Mares with many new grown follicles between consecutive follicle aspirations had significant higher plasma AMH levels (1.2 +/- 0.05 ng/ml) compared to mares with a lower number of new grown follicles (0.6 +/- 0.04 ng/ml; p<0.001). Individual AMH levels remained at a constant level during the observed period in each mare. While we found a significant positive correlation between AMH levels and the number of weekly new grown follicles (r=0.67), the number of recovered oocytes per aspiration session showed no relationship to plasma AMH levels. In conclusion, plasma AMH levels can be used to predict the number of available follicles in a follicle aspiration program in mares and therefore it could be also an important marker to calculate the ovarian follicular reserve in mares with regard to a superovulation program in the future.

The implantation of the blastocyst into the endometrium is an indispensable premise for successful embryonic development. This process is regulated by maternal and embryonic signals that influence gene expression at the translational level, among other processes. Recently, we have shown that proteolytical cleavage of the prototypical 25-kDa, mRNA cap-binding protein eIF4E produces a stable variant with a molecular mass of approximately 23 kDa exclusively in the porcine endometrium during implantation. This is accompanied by dephosphorylation and reduction of the abundant repressor 4E-BP1. Here, we investigate the distribution of the truncated eIF4E and of 4E-BP1 in the porcine uterine tissue, their binding in native samples, and we analyzed eIF4E-, eIF4G-, and 4E-BP1-specific proteolytic activities. Our results show that in pigs, the truncated eIF4E is located in the endometrial luminal epithelium during implantation. Neither glandulary tissue nor stroma expressed any truncated eIF4E. The reduced abundance of 4E-BP1 during implantation is mainly the result of decay in the glandular epithelia. Moreover, steroid replacements, in vitro protease assays, and cell lysate fractionation showed that eIF4E cleavage and 4E-BP1 decay both depended on the ovarian steroid hormones estradiol and progestrone, but these effects are the result of different proteolytic activities. Although eIF4G cleavage also depends on calcium, stimulation by these steroids could not be established. We propose that the translation initiation process in the endometrium is differently regulated by the truncated eIF4E, utilizing different abundances of 4E-BP1 and binding dynamic of eIF4E/4E-BP1 in distinct forms of implantation.

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.