Effects of continuous milking during a field trial on productivity, milk protein yield and health in dairy cows.

The objective of this field study with an automatic milking system was to evaluate the effects of omitting the dry period on health and productivity during the subsequent lactation in dairy cows. A total of 98 German Simmental cows of six Southern German farms were assigned randomly to two experimental groups: The first group was dried-off 56 days before calving (D for dried-off, n=49), and the second group was milked continuously during this period until calving (CM for continuous milking, n=49). From the latter a third group emerged, including cows that dried-off themselves spontaneously (DS for dried-off spontaneously, n=14). Blood serum values of glucose, ß-hydroxybutyrate (BHBA), non-esterified fatty acids (NEFA) and IGF-1 showed most pronounced fluctuations in D cows. Over the entire study period, the concentrations of BHBA and NEFA were markedly lower in the CM and DS groups. Furthermore, IGF-1 concentration was lowest for D cows and also decrease in back fat thickness was more pronounced. Mean concentration of milk protein was markedly higher in CM and DS cows (3.70% and 3.71%) compared with D cows (3.38%). Owing to the lower 305-day milk yield (-15.6%) and the lower total milk yield (-3.1%), the total amount of produced protein in the subsequent lactation was 2.5% (6.8 kg) lower, although the additional protein amount in CM cows from week -8 to calving was 35.7 kg. The greatest benefit resulted from positive effects on fertility and the lower incidence of diseases: CM cows had their first oestrus 1 week earlier compared with D cows, they also conceived earlier and showed a significantly lower risk of developing hypocalcaemia, ketosis and puerperal disorders. The present study showed that the costs of medical treatment and milk losses were twice as high in D cows, compared with CM and DS cows, and thus the reduced costs because of the more stable health outweighed the financial losses of milk yield by +18.49 € per cow and lactation.

Gene expression analysis of protein synthesis pathways in bovine mammary epithelial cells purified from milk during lactation and short-term restricted feeding.

The objective of the study was to investigate selected key regulatory pathways of milk protein biosynthesis in primary bovine mammary epithelial cells (MECs) of dairy cows during the first 155 days of lactation. In addition, cows were exposed to feed restriction for a short period (FR) during different stages of lactation (week 4 and 21 pp) to study adjustment processes of molecular protein biosynthesis to metabolic challenge. Morning milk samples from twenty-four Holstein-Friesian cows were collected throughout the experimental period (n = 10 per animal). MEC from raw milk were purified using an immunomagnetic separation technique and used for real-time quantitative PCR analyses. As was seen in transcript abundances of all major milk proteins, mRNA levels of E74-like factor 5 (ELF5), an enhancer of signal transducer and activator of transcription (STAT) action, concomitantly decreased towards mid-lactation. Expression of ELF5 as well as of all milk protein genes showed a similar increase during FR in early lactation. Occasional changes in expression could be seen in other Janus kinase (JAK)/STAT factors and in mammalian target of rapamycin (mTOR) pathway elements. Amino acid transfer and glucose transporter and the ß-casein expression were also partially affected. In conclusion, our findings suggest a pivotal role of the transcription factor ELF5 in milk protein mRNA expression with complementary JAK/STAT and mTOR signalling for the regulation of protein biosynthesis in the bovine mammary gland.

Development and validation of a sensitive enzyme immunoassay (EIA) for blood plasma cortisol in female cattle, buffaloes, and goats.

A highly sensitive enzyme immunoassay (EIA) that used the second antibody coating technique and the cortisol-horseradish peroxidase conjugate as a label for determination of free and total cortisol in blood plasma of dairy animals (cows, buffaloes, and goats) was developed. For biological validation of the EIA, blood samples were collected from the animals at 48 and 24 h before and 0, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, and 132 h after dexamethasone administration. The EIA was performed directly with 20 µL of fresh plasma (for free cortisol) and also with 20 µL of heat-treated plasma (for total cortisol) after 1:5 dilutions with PBS. Cortisol standards ranging from 0.39 to 200 pg/well/20 µL were used, and the sensitivity of the EIA procedure was found to be 0.39 pg/well/20 µL, which corresponded to 0.02 ng/mL. In comparison with RIA the EIA was at least 4 times more sensitive and required 5 times less cortisol antiserum. In female cattle, buffaloes, and goats, the total, free, and bound plasma cortisol before dexamethasone administration was significantly (P < 0.05) higher than the total, free, and bound cortisol after dexamethasone administration. It can be concluded from these studies that the direct, sensitive EIA validated for estimating the free and total cortisol concentrations was sufficiently reliable and quick for studying the dynamics of cortisol distribution in blood plasma of dairy animals.

Expression of lymphangiogenic vascular endothelial growth factor family members in bovine corpus luteum.

The aim of this study was to evaluate mRNA expression, protein concentration and localization of the assumedly important lymphangiogenic factors VEGFC and VEGFD and the receptor FLT4 in bovine corpora lutea (CL) during different physiological stages. In experiment 1, CL were collected in a slaughterhouse and stages (days 1-2, 3-4, 5-7, 8-12, 13-16, >18) of oestrous cycle and month <3, 3-5, 6-7 and >8 of pregnancy. In experiment 2, prostaglandin F2α (PGF)-induced luteolysis was performed in 30 cows, which were injected with PGF analogue on day 8-12 (mid-luteal phase), and CL were collected before and 0.5, 2, 4, 12, 24, 48 and 64 h after PGF injection. The mRNA expression was characterized by RT-qPCR. All three factors were clearly expressed and showed significant changes during different groups and periods examined in both experiments. Protein concentrations of VEGFD and FLT4 measured by ELISA were not detectable in early cyclic CL but increased to higher plateau levels during pregnancy. After PGF-induced luteolysis FLT4 protein showed an increase within 2-24 h after the injection. FLT4 localization by immunohistochemistry in the cytoplasm of luteal cells was relatively weak in early CL. It increased in late CL and especially in CL during pregnancy. During pregnancy, a positive FLT4 staining in both the nucleus and cytoplasm of lymphatic endothelial cells in peripheral tissue was observed. In conclusion, our results lead to the assumption that lymphangiogenic factors are produced and regulated in CL and may be involved in mechanisms regulating CL function, especially during pregnancy.

Dexamethasone-induced eosinopenia is associated with lower progesterone production in cattle.

Eosinophilic cells accumulate in the capillaries of the bovine Graafian follicle shortly before ovulation and in the early developing corpus luteum (CL). Suppressing the migration of these eosinophilic cells by dexamethasone allowed us to evaluate their possible function in the CL development. Brown Swiss cows (n = 10) were randomly subdivided into two groups (n = 5). Every group was used once as control group and once as experimental group with two oestrous cycles between each treatment. Eighteen hours (h) after oestrus synchronization, dexamethasone or saline was given. Ovulation was induced 24 h later with gonadotropin-releasing hormone. Another injection of dexamethasone or saline was given 12 h later. Eosinophilic cells in the blood were counted daily until day 7 after the first dexamethasone injection. The collection of ovaries took place at days 1, 2 and 5. Gene expression, protein concentration and location of angiogenic factors, chemokines, insulin-like growth factor 1 (IGF1) and eosinophilic cells were studied. No eosinophilic cells were found in the CL of the treatment group. Blood progesterone decreased significantly in the dexamethasone group from day 8 to 17. The protein concentration of FGF2 increased significantly in CL tissue at day 2 and VEGFA decreased. Local IGF1 gene expression in the CL was not regulated. We assume from our data that the migration of eosinophilic cells into the early CL is not an essential, but an important stimulus for angiogenesis during early CL development in cattle.

Effects of induced energy deficiency on lactoferrin concentration in milk and the lactoferrin reaction of primary bovine mammary epithelial cells in vitro.

A dietary energy restriction to 49% of total energy requirements was conducted with Red Holstein cows for three weeks in mid-lactation. At the last day of the restriction phase, primary bovine mammary epithelial cells (pbMEC) of eight restriction (RF) and seven control-fed (CF) cows were extracted out of one litre of milk and cultured. In their third passage, an immune challenge with the most prevalent, heat-inactivated mastitis pathogens Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was conducted. Lactoferrin (LF) was determined on gene expression and protein level. An enzyme-linked immunosorbent assay (ELISA) was developed to determine LF in milk samples taken twice weekly throughout the animal trial, beginning on day 20 pp (post-partum) until day 150 pp, in cell culture total protein and in cell culture supernatant. Milk LF increased throughout the lactation and decreased significantly during the induced energy deficiency in the RF group. At the beginning of realimentation, LF concentration increased immediately in the RF group and reached higher levels than before the induced deficit following the upward trend seen in the CF group. Cell culture data revealed higher levels (up to sevenfold up-regulation in gene expression) and significant higher LF protein concentration in the RF compared to the CF group cells. A further emphasized effect was found in E. coli compared to S. aureus exposed cells. The general elevated LF levels in the RF pbMEC group and the further increase owing to the immune challenge indicate an unexpected memory ability of milk-extracted mammary cells that were transposed into in vitro conditions and even displayed in the third passage of cultivation. The study confirms the suitability of the non-invasive milk-extracted pbMEC culture model to monitor the influence of feeding experiments on immunological situations in vivo.

Microfluidic high-throughput RT-qPCR measurements of the immune response of primary bovine mammary epithelial cells cultured from milk to mastitis pathogens.

Bovine mastitis, the inflammation of the udder, is a major problem for the dairy industry and for the welfare of the animals. To better understand this disease, and to implement two special techniques for studying mammary gland immunity in vitro, we measured the innate immune response of primary bovine mammary epithelial cells (pbMEC) from six Brown Swiss cows after stimulation with the heat-inactivated mastitis pathogens, Escherichia coli 1303 and Staphylococcus aureus 1027. The cells were extracted and cultivated from milk instead of udder tissue, which is usually done. The advantages of this technique are non-invasiveness and less contamination by fibroblasts. For the first time, pbMEC gene expression (GE) was measured with a microfluidic high-throughput real-time reverse transcription-quantitative PCR platform, the BioMark HD™ system from Fluidigm. In addition to the physiological analysis, the precision and suitability of this method was evaluated in a large data set. The mean coefficient of variance (± s.e.) between repeated chips was 4.3 ± 0.4% for highly expressed and 3.3 ± 0.4% for lowly expressed genes. Quantitative PCR (qPCR) replicate deviations were smaller than the cell culture replicate deviations, indicating that biological and cell culture differences could be distinguished from the background noise. Twenty-two genes (complement system, chemokines, inflammatory cytokines, antimicrobial peptides, acute phase response and toll-like receptor signalling) were differentially expressed (P < 0.05) with E. coli. The most upregulated gene was the acute phase protein serum amyloid A3 with 618-time fold. S. aureus slightly induced CCL5, IL10, TLR4 and S100A12 expression and failed to elicit a distinct overall innate immune response. We showed that, with this milk-derived pbMEC culture and the high-throughput qPCR technique, it is possible to obtain similar results in pbMEC expression as with conventional PCR and with satisfactory precision so that it can be applied in future GE studies in pbMEC.

Multiparous cows categorized by milk protein concentration and energy-corrected milk yield during early lactation--metabolism, productivity and effect of a short-term feed restriction.

The objective of this experiment was to study milk productivity, metabolic adaptation and effect of a short-term feed restriction (FR) on key performance indicators during early lactation in cows classified according to energy-corrected milk (ECM) yield and milk protein concentration. Twenty-three multiparous Holstein-Friesian cows were categorized in four groups according to respective averaged values on Days 23-25 postpartum: high ECM yield and high protein concentration; low ECM yield and low protein concentration; high ECM yield and low protein concentration and low ECM yield and high protein concentration. Dry matter intake was reduced to 68.3% for three subsequent days. Our results showed that short-time FR in early lactation succeeded in enhancing energy deficit of cows in all groups. Milk fat, milk protein and lactose concentrations as well as milk fat yield were not influenced by FR. Several hepatic genes encoding for enzymes involved in catabolism of amino acids, ß-oxidation, gluconeogenesis and ketogenesis as well as mRNA encoding for insulin receptor showed increased transcript abundances after FR, primarily in cows with high milk yield and low milk protein concentration.

Effects of cell culture techniques on gene expression and cholesterol efflux in primary bovine mammary epithelial cells derived from milk and tissue.

Primary bovine mammary epithelial cells (pbMEC) are often used in cell culture to study metabolic and inflammatory processes in the udder of dairy cows. The most common source is udder tissue from biopsy or after slaughter. However, it is also possible to culture them from milk, which is non-invasive, repeatable and yields less contamination with fibroblasts. Generally, not much is known about the influence of cell origin and cell culture techniques such as cryopreservation on pbMEC functionality. Cells were extracted from milk and udder tissue to evaluate if milk-derived pbMEC are a suitable alternative to tissue-derived pbMEC and to test what influence cryopreservation has. The cells were cultivated for three passages and stored in liquid nitrogen. The relative gene expression of the five target genes kappa-casein, lingual antimicrobial peptide (LAP), lactoferrin, lysozyme (LYZ1) and the prolactin receptor normalised with keratin 8 showed a tendency to decrease in the tissue cultures, but not in the milk-derived cultures, suggesting a greater influence of the cultivation process on tissue-derived cells, freezing lowered expression levels in both cultures. Overall expression of LAP and LYZ1 tended to be higher in milk cells. Cholesterol efflux was measured to compare passages one to seven in milk-derived cells. Passage number did not alter the efflux rate (p ≤ 0.05). We showed for the first time that the extraction of pbMEC from milk can be a suitable alternative to tissue extraction.

Immunological mechanisms to establish embryo tolerance in early bovine pregnancy.

A well-balanced immunological interaction between mother and the semi-allogenic embryo is of particular importance. The objective of the present study was to analyse mechanisms of immune tolerance in bovine pregnancy during peri-implantation. Simmental heifers inseminated with either cryopreserved spermatozoa or seminal plasma were killed 12, 15 or 18 days after oestrus. Uteri were flushed for the recovery of conceptuses and the ipsilateral intercaruncular endometrium was sampled for gene expression analysis. Indoleamine 2,3-dioxygenase (IDO) mRNA, coding for the initial enzyme of the kynurenine pathway, was 18-fold (P < 0.001) more abundant in the endometrium of Day 18 pregnant v. non-pregnant animals. Tandem mass spectrometry revealed a decrease of endometrial l-tryptophan (P = 0.0008), but an increase of l-kynurenine concentration (P = 0.005) from Day 12 to Day 18, suggesting increasing IDO activity (P < 0.03). An in vitro coculture model of endometrial cells showed an induction of IDO expression following interferon-τ exposure primarily in stroma cells, which was confirmed by in situ hybridisation localising IDO mRNA mainly in deep stroma cells. Immunohistochemical analysis revealed fewer CD45-positive leucocytes in the zona basalis of pregnant animals. Elevated IDO activity may reduce the presence of leucocytes in the pregnant endometrium, providing a possible mechanism for protecting the semi-allogenic conceptus from maternal rejection.

Changes in hoof health and animal hygiene in a dairy herd after covering concrete slatted floor with slatted rubber mats: a case study.

The objective was to investigate the effect of changing the flooring in the alleys of a barn from slatted concrete to slatted rubber mats on hoof disorders and animal hygiene in 44 loose-housed Brown Swiss dairy cows. Cows were examined for disorders of the hind hooves (hemorrhages, white line fissures, ulcers, heel horn erosion, and digital dermatitis) and for skin lesions. The dirtiness of the animals and of the floor was recorded. Climatic (temperature, humidity) and ammonia gas conditions were measured. Evaluations were carried out when the cows were housed on a concrete slatted floor and after 4 and 10 mo on soft flooring (slatted rubber mats, 29-mm thick). The anatomical portion of claw (medial, lateral), number of lactations (parity), and days in milk were included as covariates in the statistical model. Changing the flooring from slatted concrete to slatted rubber mats increased the score for white line fissures [1.0 ± 0.3 (concrete) vs. 2.5 ± 0.4 (10 mo rubber mats)] and influenced air humidity (i.e., the difference in the absolute humidity between the inside and outside of the barn increased from 1.5 ± 0.1 to 1.7 ± 0.2g/m(3)), whereas the other hoof disorders, skin lesions (score of 8.7 ± 0.3), the dirtiness of the animals (score of 5.9 ± 0.3), and the floor (score of 2.1 ± 0.1), and ammonia gas concentration (2.6 ± 0.3mg/kg) were not affected (overall scores or measures; mean ± SE). Lateral claws were more affected (except for heel horn erosion) than medial claws (estimated effects between 1.3 ± 0.2 and 3.0 ± 0.6). Parity influenced hoof disorders (except for hemorrhages) and skin lesions (estimated effects between -0.6 ± 0.3 and 0.5 ± 0.2). Days in milk influenced hoof disorders, but had no effect on skin lesions and on the dirtiness of the animal. Irrespective of floor type, the slots (2.6 ± 0.1) were dirtier than the slats (1.6 ± 0.1). In conclusion, covering slatted concrete flooring with slatted rubber mats partially impaired hoof health but did not influence skin lesions or the dirtiness of the cows or the floor. Similar results were found for climatic conditions, as ammonia gas concentration was not affected, but absolute humidity increased in the barn when rubber mats were present.

Influence of anabolic combinations of an androgen plus an estrogen on biochemical pathways in bovine uterine endometrium and ovary.

The application of anabolic steroids in food producing animals is forbidden in the EU since 1988, but the abuse of such drugs is a potential problem. The existing test systems are based on known compounds and can be eluded by newly emerging substances. The examination of physiological effects of anabolic hormones on different tissues to indirectly detect misuse might overcome this problem. Two studies were conducted with post-pubertal 24-months old Nguni heifers and pre-pubertal female 2-4 weeks old Holstein Friesian calves, respectively. The animals of the accordant treatment groups were administered combinations of estrogenic and androgenic compounds. The measurement of the gene expression pattern was undertaken with RT-qPCR. Target genes of different functional groups (receptors, angiogenesis, steroid synthesis, proliferation, apoptosis, nutrient metabolism and others) have been quantified. Several biochemical pathways were shown to be influenced by anabolic treatment. Both studies identified significant regulations in steroid and growth factor receptors (AR, ERß, LHR, FSHR, Flt-1, PR, IGF-1R, Alk-6), angiogenic and tissue remodeling factors (VEGFs, FGFs, BMPs, ANGPT-2, MMPs, TIMP-2, CTSB), steroid synthesis (S5A1, HSD17, CYP19A1), proliferation (TNFα, IGF-1, IGFBPs, p53, c-fos; CEBPD, c-kit), apoptosis (CASP3, FasL, p53) and others (C7, INHA, STAR). Several genes were regulated to opposite directions in post-pubertal compared to pre-pubertal animals. PCA for Nguni heifers demonstrated a distinct separation between the control and the treatment group. In conclusion, anabolics modify hormone sensitivity and steroid synthesis, and they induce proliferative effects in the whole reproductive tract (uterus and ovary) as well as anti-angiogenic effects in the ovary. However, the extent will depend on the developmental stage of the animals.

The potential of bovine vaginal smear for biomarker development to trace the misuse of anabolic agents.

In the European Union the use of anabolic hormones in meat production is forbidden since 1988 and this ban of anabolic agents in animal production is strictly controlled. New hormone cocktails passing the detection systems are attractive for the practice and so new approaches to discover their illegal use have to be developed steadily. Verifying physiological effects caused by anabolic steroids will be a new way to develop potential monitoring systems. One promising matrix in female animals will be vaginal smear containing vaginal epithelial cells, because the vaginal epithelium is a primary steroid hormone responsive organ. In this study we quantified the gene expression in vaginal smear of sexually mature cattle in order to observe physiological effects. Further we aimed to establish a new screening method by testing the effect of a combination of certain anabolic steroid hormones on physiological regulations of mRNA expression of selected genes. In an animal trial Nguni heifers were treated with the anabolic combination trenbolone acetate plus estradiol. Vaginal smear samples were taken at 4 different time points. Gene expression of 27 candidate genes, selected by screening the actual literature for steroidal effects on vaginal epithelial cells, were estimated using quantitative real-time RT-PCR. There were different expression changes observed at different time points. It could be shown that the applied anabolic combination significantly influenced the expression of the steroid receptor ERα, the keratinization factor CK8, the proinflammatory interleukins IL-1α and IL-1ß, the growth factors FGF7, EGF, EGFR, IGF-1R, TGFα and LTF, the oncogen c-jun and other factors like actinß and ubiquitin 3. Using biostatistical tools like principal components analysis or hierarchical cluster analysis, the potential to develop a gene expression pattern for targeting the illegal use of growth promoters could be demonstrated.

Effects of the prebiotics inulin and lactulose on intestinal immunology and hematology of preruminant calves.

Prebiotics are suggested as an alternative to antibiotics in animal rearing. Fermentable substances such as inulin or lactulose have been proposed to stimulate the immune system and health by modulation of the intestinal flora and its fermentation products. In this study, effects of inulin and lactulose on the intestinal health and hematology of calves have been investigated. Both prebiotics significantly decreased thrombocyte counts in peripheral blood. Only inulin was able to increase hemoglobin concentration and hematocrit. Total leukocyte count was decreased by lactulose while both prebiotics tended to lower monocyte proportions. mRNA expression of inflammation-related markers in the intestine was also affected by both prebiotics hinting at a decreased inflammatory status. This may be due to a possible decrease in intestinal pathogen load that remains to be verified. Only mRNA amounts of interleukin 8 were increased by lactulose in mesenteric lymph nodes. In the ileum, expression of a proliferation marker was increased by inulin while an apoptosis-related gene was increased by both prebiotics. The results of this study show a clear effect of prebiotics on certain parameters associated with animal health and performance that remain to be studied in detail in future investigations.

Effects of long-term feeding of genetically modified corn (event MON810) on the performance of lactating dairy cows.

A long-term study over 25 months was conducted to evaluate the effects of genetically modified corn on performance of lactating dairy cows. Thirty-six dairy cows were assigned to two feeding groups and fed with diets based on whole-crop silage, kernels and whole-crop cobs from Bt-corn (Bt-MON810) or its isogenic not genetically modified counterpart (CON) as main components. The study included two consecutive lactations. There were no differences in the chemical composition and estimated net energy content of Bt-MON810 and CON corn components and diets. CON feed samples were negative for the presence of Cry1Ab protein, while in Bt-MON810 feed samples the Cry1Ab protein was detected. Cows fed Bt-MON810 corn had a daily Cry1Ab protein intake of 6.0 mg in the first lactation and 6.1 mg in the second lactation of the trial. Dry matter intake (DMI) was 18.8 and 20.7 kg/cow per day in the first and the second lactation of the trial, with no treatment differences. Similarly, milk yield (23.8 and 29.0 kg/cow per day in the first and the second lactation of the trial) was not affected by dietary treatment. There were no consistent effects of feeding MON810 or its isogenic CON on milk composition or body condition. Thus, the present long-term study demonstrated the compositional and nutritional equivalence of Bt-MON810 and its isogenic CON.

Effects of continuous milking during the dry period or once daily milking in the first 4 weeks of lactation on metabolism and productivity of dairy cows.

The objective was to compare the effects of 3 management systems in high-yielding dairy cows on metabolic profiles and milk production. Thirty-six multiparous Brown Swiss cows were randomly assigned to 1 of 3 treatment groups (n=12 cows/group): the control (C) group, in which cows were dried off 56 d before calving and milked twice daily throughout next lactation (305 d); the once daily milking (ODM) group, in which cows were dried off 56 d before calving and milked once daily for the first 4 wk of lactation and twice daily for the remaining lactation; and the continuous milking (CM) group, in which cows were milked twice daily until calving and also during the subsequent lactation. Serum glucose concentrations decreased between wk 1 and 4 exclusively in C cows. Serum concentrations of NEFA and BHBA in the first 4 wk of lactation were highest in C cows compared with ODM and CM cows. Decreased backfat thickness during early lactation and reduction of body condition score were markedly more pronounced in C cows compared with ODM and CM cows. Mean lactational milk yield of C cows [11,310+/-601 kg of energy-corrected milk (ECM)/305 d] was approximately 16% higher compared with ODM cows (9,531+/-477 kg of ECM/305 d) and CM cows (9,447+/-310 kg of ECM/305 d). The lactation curve of CM cows compared with C cows was characterized by a similar time of peak yield (wk 3), a reduced peak yield, and no obvious differences in persistency. Mean percentage of milk protein was significantly higher for CM cows (3.91%) compared with C cows (3.52%). In contrast, once daily milking was accompanied by a reduced and significantly delayed peak yield (wk 8) compared with the control treatment, whereas persistency was better and milk protein (3.79%) was higher in ODM cows than in C cows. In conclusion, continuous milking and once daily milking, targeting the interval before or after calving, respectively, substantially reduced the metabolic challenge of fresh cows and improved milk protein percentage. Continuous milking and once daily milking increased milk protein percentage markedly; furthermore, once daily milking during the first 4 wk of lactation improved the lactation curve.

Divergent development of testosterone secretion in male zebu (Bos indicus) and crossbred cattle (Bos indicus x Bos taurus) and buffaloes (Bubalus bubalis) during growth.

Delayed pubertal development and low fertility of Bos indicus x Bos taurus crossbred male cattle and domestic buffaloes is hardly understood hence, a sensitive enzymeimmunoassay (EIA) was developed using the second antibody-coating technique and testosterone-3-O-carboxymethyloxime-horseradish peroxidase conjugate as a label for determination of testosterone in blood plasma. The EIA was validated by standard criteria. Blood samples were collected by venipuncture from growing male cattle (Karan Fries and Sahiwal) and buffalo (Murrah) and testosterone was estimated using the EIA procedure. Plasma testosterone concentrations increased significantly (P < 0.05) with advancing age. Testosterone concentrations were significantly (P < 0.01) higher in Sahiwal males in comparison to Karan Fries males. The low testosterone levels in crossbred than Sahiwal could imply that crossbred males have either not stabilized genetically or not adapted well in Indian climatic conditions resulting in poor libido and poor semen quality. The low testosterone levels in Murrah buffalo males may be the possible reason for delayed maturity in this species. The direct, sensitive EIA validated for estimating the plasma testosterone concentration was reliable for studying the testosterone profile in blood plasma of males. The results suggest that there could be a requirement for higher testosterone secretion by males during early stages of growth for attaining early sexual maturity.

Nuclear magnetic resonance and mass spectrometry-based milk metabolomics in dairy cows during early and late lactation.

Milk production in dairy cows has dramatically increased over the past few decades. The selection for higher milk yield affects the partitioning of available nutrients, with more energy being allocated to milk synthesis and less to physiological processes essential to fertility and fitness. In this study, the abundance of numerous milk metabolites in early and late lactation was systematically investigated, with an emphasis on metabolites related to energy metabolism. The aim of the study was the identification and correlation of milk constituents to the metabolic status of the cows. To investigate the influence of lactation stage on physiological and metabolic variables, 2 breeds of different productivity were selected for investigation by high-resolution nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. We could reliably quantify 44 different milk metabolites. The results show that biomarkers such as acetone and beta-hydroxybutyrate are clearly correlated to the metabolic status of the individual cows during early lactation. Based on these data, the selection of cows that cope well with the metabolic stress of early lactation should become an option.

Enhanced proapoptotic gene expression of XAF1, CASP8 and TNFSF10 in the bovine endometrium during early pregnancy is not correlated with augmented apoptosis.

Bovine trophoblast cells release interferon-tau (IFNT), a type I IFN, as the pregnancy recognition signal. Since type I IFNs exert growth inhibitory and proapoptotic actions, the effect of the conceptus on components of the apoptosis pathways was determined in the bovine endometrium during the periimplantation period. Uteri of Simmental heifers were flushed post mortem at days 12, 15, and 18 of cycle or pregnancy for the recovery of conceptuses and the sampling of ipsilateral endometrial tissue at slaughter for quantitative RT-PCR, immunohistochemistry, caspase activity and TUNEL assays. Endometrium samples of pregnant animals revealed increased transcript levels for the proapoptotic genes XAF1 (day 15: 2.9-fold; day 18: 15.1-fold; p=0.005) and CASP8 (day 18: 2.4-fold; p=0.007). The mRNA expression increased significantly with the day of the cycle for the proapoptotic genes FASLG, TNFSF10, TNF and TNFSF1A (p=0.004, p=0.006, p=0.001 and p=0.007) and the antiapoptotic gene BIRC4 (p=0.03). We detected high amounts of FASLG transcripts in day 18 conceptuses (16-fold higher than day 18 endometria). This finding was validated at the protein level by immunohistochemistry. To further analyse the endometrial activation of the caspase cascade, the activities of initiator caspase 8 and effector caspases 3/7 were determined luminometrically. No difference between pregnant and cyclic animals was found for either caspase activity. Additionally, a TUNEL assay showed no increase of apoptotic cells in the pregnant endometrium. In conclusion, although the bovine conceptus induces the expression of proapoptotic genes, neither an activation of a caspase cascade nor an increase of apoptotic cells was noticed. These results suggest inhibitory mechanisms preventing endometrial cells from programmed cell death.

PGFM (13,14-dihydro-15-keto-PGF(2alpha)) in pregnant and pseudo-pregnant Iberian lynx: a new noninvasive pregnancy marker for felid species.

In mammals, uterine and placental prostaglandin F(2alpha) is involved in the regulation of reproduction-related processes such as embryonic development, initiation of parturition, and resumption of ovarian activity. Prostaglandin F(2alpha) (PGF(2alpha)) is rapidly metabolized to its plasma metabolite PGFM (13,14-dihydro-15-keto-PGF(2alpha)), which has also been detected in urine. Therefore, the current study aimed to develop and validate an efficient, quick, and inexpensive enzyme immunoassay (EIA) for PGFM estimation in urine of the Iberian lynx (Lynx pardinus) for pregnancy monitoring and for differentiation between pregnancy and pseudo-pregnancy. Urine samples collected from captive Iberian lynx (11 pregnant and 4 pseudo-pregnant cycles) were subjected directly to a PGFM EIA. The assay was validated for parallelism, precision, and stability of urinary PGFM. In addition, high-performance liquid chromatography (HPLC) immunograms and liquid chromatography-mass spectrometry (LCMS) were performed to identify PGFM within urine samples. Urinary PGFM levels before mating and after parturition were about 1.5 ng/mL. After Day 20 postmating, both pregnant and pseudo-pregnant females showed slight increase of hormone levels; in pseudo-pregnant females, this elevation did not exceed 7 ng/mL. A significant increase in pregnant females was observed after Day 45 postmating; urinary PGFM increased from 10 ng/mL at Day 45 toward a peak of 46.0+/-19.3 ng/mL around parturition. First results show that PGFM is detectable in feces as well and follows similar courses as shown for urine. In conclusion, the presented and validated PGFM assay is an easy and reliable method for noninvasive pregnancy diagnosis in the Iberian lynx (and probably other felids) if applied approximately 20 d prior parturition in pure urine or fecal extracts. High PGFM levels in urine or fecal samples may allow a pregnancy diagnosis without knowledge of mating time, making the PGFM test applicable to free-ranging animals.