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.

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.

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.

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.

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.

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.

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.

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.

Quantitative characterization of prostaglandins in the uterus of early pregnant cattle.

Prostaglandins (PGs) are important regulators of reproductive processes including early embryonic development. We analyzed the most relevant PG in bovine uteri at different preimplantation pregnancy stages when compared with non-pregnant controls. Additionally, endometrium and trophoblast tissues were examined regarding specific enzymes and receptors involved in PG generation and function. Simmental heifers were artificially inseminated or received seminal plasma only. At days 12, 15, or 18, post-estrus uteri were flushed for PG determination by liquid chromatography-tandem mass spectrometry. Endometrium and trophoblast tissues were sampled for RNA extraction and quantitative real-time PCR analysis. At all days and points of time examined, the concentration of 6-keto PGF(1alpha) (stable metabolite of PGI(2)) was predominant followed by PGF(2alpha)>PGE(2)>PGD(2) approximately TXB(2) (stable metabolite of TXA(2)). At days 15 and 18, PG increased from overall low levels at day 12, with a much more pronounced increase during pregnancy. The PGF(2alpha)/PGE(2) ratio was not influenced by status. The highest PG concentration was measured at day 15 with 6-keto PGF(1alpha) (6.4 ng/ml) followed by PGF(2alpha) (1.1 ng/ml) and PGE(2) (0.3 ng/ml). Minor changes in endometrial PG biosynthesis enzymes occurred due to pregnancy. Trophoblasts revealed high transcript abundance of general and specific PG synthases contributing to uterine PG. As PGI(2) and PGF(2alpha) receptors were abundantly expressed by the trophoblast, abundant amounts of PGI(2) and PGF(2alpha) in the uterine lumen point towards an essential role of PG for the developing embryo. High amounts of PG other than PGE(2) in the preimplantation uterus may be essential rather than detrimental for successful reproduction.

Regulatory changes of apoptotic factors in the bovine corpus luteum after induced luteolysis.

The corpus luteum (CL) offers the opportunity to study not only proliferative, but also regressive processes. During luteolysis of the CL a sudden death of luteal and endothelial cells seems to be involved (apoptosis). The aim of this study was to examen the mRNA expression of factors known to be involved in apoptotic processes: monocyte chemoattractant protein-1 (MCP-1), factors of the extrinsic and intrinsic apoptotic pathways, caspase3, -6, -7 and interferone gamma (IFNgamma). Luteolysis was induced by injection of 500 microg Cloprostenol during mid-luteal phase. The CLs were collected at 0.5, 2, 4, 12, 24, 48, and 64 hr after PGF2alpha-injection. Control CLs (Days 8-12) were collected at the slaugtherhouse. Real-time RT-PCR determined the mRNA expressions. Western blot analysis of poly(ADP-ribose) polymerase (PARP-1) and IFNgamma as well as protein measurement of tumor necrosis factor alpha (TNFalpha) by EIA were performed. The mRNA levels of MCP-1, IFNgamma and most factors of the extrinsic pathway were significantly increased between 0.5 and 2 hr. The factors of the intrinsic pathway were mostly later up-regulated at 24-48 hr after PGF2alpha. Caspase6 and 3 revealed a significant increase from 2 and 12 hr, respectively, whereas caspase7 was significantly up-regulated after 24 hr. The protein level of TNFalpha increased significantly to a maximum level at 12 hr. The Western blot revealed an increasing level of an 89 kDa fragment of PARP-1 from 12 to 24 hr, which is specific for apoptosis. We assume that the extrinsic pathway is more important for the onset of luteolysis, because of its earlier and higher increase during induced luteolysis.

Detectability of testosterone esters and estradiol benzoate in bovine hair and plasma following pour-on treatment.

The abuse of synthetic esters of natural steroids such as testosterone and estradiol in cattle fattening and sports is hard to detect via routine urine testing. The esters are rapidly hydrolysed in vivo into substances which are also endogenously present in urine. An interesting alternative can be provided by the analysis of the administered synthetic steroids themselves, i.e., the analysis of intact steroid esters in hair by liquid chromatography tandem mass spectrometry (LC/MS/MS). However, retrospective estimation of the application date following a non-compliant finding is hindered by the complexity of the kinetics of the incorporation of steroid esters in hair. In this study, the incorporation of intact steroid esters in hair following pour-on treatment has been studied and critically compared with results from intramuscular treatment. To this end animals were pour-on treated with a hormone cocktail containing testosterone cypionate, testosterone decanoate and estradiol benzoate in different carriers. The animals were either treated using injection and pour-on application once or three times having 1 week between treatments using injection and pour-on application. Animals were slaughtered from 10-12 weeks after the last treatment. Both hair and blood plasma samples were collected and analysed by LC/MS/MS. From the results, it is concluded that after single treatment the levels of steroid esters in hair drop to CCbeta levels (5-20 microg/kg) after 5-7 weeks. When treatment is repeated two times, the CCbeta levels are reached after 9-11 weeks. Furthermore, in plasma, no steroid esters were detected; not even at the low microgramme per litre level but--in contrast with the pour-on application--after i.m. injection, significant increase of 17beta-testosterone and 17beta-estradiol were observed. These observations suggest that transport of steroid esters after pour-on application is not only performed by blood but also by alternative fluids in the animal so probably the steroid esters are already hydrolysed and epimerized before entering the blood.

Analysis of key molecules of the innate immune system in mammary epithelial cells isolated from marker-assisted and conventionally selected cattle.

Mastitis is the most prevalent infectious disease in dairy herds. Breeding programs considering mastitis susceptibility were adopted as approaches to improve udder health status. In recent decades, conventional selection criteria based on phenotypic characteristics such as somatic cell score in milk have been widely used to select animals. Recently, approaches to incorporate molecular information have become feasible because of the detection of quantitative trait loci (QTL) affecting mastitis resistance. The aims of the study were to explore molecular mechanisms underlying mastitis resistance and the genetic mechanisms underlying a QTL on Bos taurus chromosome 18 found to influence udder health. Primary cell cultures of mammary epithelial cells from heifers that were selected for high or low susceptibility to mastitis were established. Selection based on estimated pedigree breeding value or on the basis of marker-assisted selection using QTL information was implemented. The mRNA expression of 10 key molecules of the innate immune system was measured using quantitative real-time PCR after 1, 6, and 24 h of challenge with heat-inactivated mastitis pathogens (Escherichia coli and Staphylococcus aureus) and expression levels in the high and low susceptibility groups were compared according to selection criteria. In the marker-assisted selection groups, mRNA expression in cells isolated from less-susceptible animals was significantly elevated for toll-like receptor 2, tumor necrosis factor-alpha, IL-1beta, IL-6, IL-8, RANTES (regulated upon activation, normal t-cell expressed and secreted), complement factor C3, and lactoferrin. In the estimated pedigree breeding value groups, mRNA expression was significantly elevated only for V-rel reticuloendotheliosis viral oncogene homolog A, IL-1 beta, and RANTES. These observations provide first insights into genetically determined divergent reactions to pathogens in the bovine mammary gland and indicate that the application of QTL information could be a successful tool for the selection of animals resistant to mastitis.

Expression and localization of gap junctional connexins 26 and 43 in bovine periovulatory follicles and in corpus luteum during different functional stages of oestrous cycle and pregnancy.

The aim of this study was to characterize the regulation of connexins (Cx26 and Cx43) in the bovine ovary (experiment 1-3). Experiment 1: ovaries containing preovulatory follicles or corpora lutea (CL) were collected at 0, 4, 10, 20, 25 (follicles) and 60 h (CL) relative to injection of GnRH. Experiment 2: CL were assigned to the following stages: days 1-2, 3-4, 5-7, 8-12, 13-16, >18 (after regression) of oestrous cycle and of early and late pregnancy (<4 and >4 months). Experiment 3: induced luteolysis, cows on days 8-12 were injected with PGF2alpha analogue (Cloprostenol), and CL were collected by transvaginal ovariectomy before and 0.5, 2, 4, 12, 24, 48 and 64 h after PGF2alpha injection. Real-time RT-PCR was applied to investigate mRNA expression and immunofluorescence was utilized for protein localization. Cx26 mRNA increased rapidly 4 h after GnRH injection (during LH surge) and decreased afterwards during the whole experimental period. Cx43 mRNA expression decreased continuously after GnRH application. Cx26 mRNA in CL increased significantly in the second part of oestrous cycle and after regression. In contrast, the highest mRNA expression for Cx43 in CL was detected during the early luteal phase. After induced luteolysis the mRNA expression of Cx26 increased significantly at 24 h. As shown by immunofluorescence, Cx26 was predominantly localized in the connective tissue and blood vessels of bovine CL, whereas Cx43 was present in the luteal cells and blood vessels. This resulted in a strong increase of Cx26 expression during the late luteal phase and after luteal regression. Subsequently, Cx43 expression was distinctly decreased after luteal regression. These data suggest that Cx26 and Cx43 are involved in the local cellular mechanisms participating in tissue remodelling during the critical time around periovulation as well as during CL formation (angiogenesis), function and regression in the bovine ovary.

Application of sensitive enzymeimmunoassay for determination of testosterone in blood plasma of yaks (Poephagus grunniens L.).

As an alternative to radioimmunoassays, a simple, highly sensitive and quick enzymeimmunoassay (EIA) for determination of testosterone in blood plasma of yaks on microtitreplates using second antibody coating technique and testosterone-horseradish peroxidase as a label has been developed for the first time. The wells of the microtitreplate were coated with affinity-purified goat immunoglobulin (antirabbit IgG) that binds the hormone specific antibody. The EIA was carried out directly in 20 mICROl of plasma after 1:10 dilution with assay buffer. The testosterone standard curve ranged from 0.2 to 200 pg/well. The sensitivity of the assay was 0.20 pg/well. Testosterone standard curve in buffer showed parallelism with serially diluted yak plasma containing high endogenous testosterone. Intra- and inter-assay coefficients of variation (CV) determined using pooled plasma was found 5.24 and 8.54%, respectively. Recovery of known concentrations of added testosterone in charcoal stripped plasma was linear (r=0.98). For biological validation of testosterone enzymeimmunoassay, the blood samples were collected from yak cows at -2h before and thereafter at 2h interval until 24h. after gonadotropin releasing hormone (GnRH) administration. There was a rapid increase (p<0.01) of luteinizing hormone (LH) and testosterone 2 and 6h after GnRH administration. Plasma testosterone concentration in normal adult yak bulls was found to be 0.52+/-0.09 ng/ml. In conclusion, the EIA developed in this study is simple, highly sensitive, valid and sufficiently reliable method for estimation of testosterone directly in yak plasma.

Effects of tryptophan supplementation on plasma tryptophan and related hormone levels in heifers and dairy cows.

This study was conducted to investigate the effects of rumen-protected tryptophan (125 g tryptophan per day) in heifers and dairy cows. Blood samples from dairy cows and heifers were collected for 24h in 3-h intervals on the day before tryptophan supplementation, on day 2, 5 and 7 of tryptophan supplementation, and in heifers additionally on d 14 after tryptophan supplementation was ceased. Plasma tryptophan, melatonin, serotonin, and prolactin concentrations were determined. Tryptophan plasma concentrations on d 5 were augmented at day (11:00 h) and nighttime (02:00 h), (P<0.05) in response to tryptophan supplementation in heifers by 119% and in dairy cows by 47%, respectively, as compared with d 0. Melatonin increased (P<0.05) in response to tryptophan supplementation in heifers, but not in cows. The effect of tryptophan supplementation on plasma tryptophan and melatonin was reversible as demonstrated in heifers on d 14 after cessation of tryptophan supplementation. Serotonin and prolactin in plasma did not respond to tryptophan supplementation. However, milk yield during morning milking increased significantly in tryptophan supplemented cows on d 1, 3 and 4 as compared to the day before tryptophan supplementation. Additional blood samples were taken during afternoon milking in cows at 1-min intervals for the analyses of oxytocin and prolactin on the day before the start and on d 7 of tryptophan supplementation. Milk flow curves were recorded during milking. No effect of tryptophan supplementation on the milking related release of oxytocin and prolactin and on any characteristic of milk flow was observed. In conclusion, tryptophan supplementation caused increased plasma tryptophan in cows and heifers and plasma melatonin in heifers. However, plasma serotonin, prolactin and oxytocin release in cows remained unchanged by tryptophan supplementation. Milk yield at morning milking increased slightly and transiently in response to tryptophan supplementation.

What happens with cow behavior when replacing concrete slatted floor by rubber coating: a case study.

An enhanced productive life cycle and improved animal welfare are aims pursued in dairy husbandry. This study assesses experimental observations on floor-associated behavior during the stepwise replacement of concrete slatted flooring by rubber mats. For this purpose, estrus (mounting) and hygiene behavior (licking while standing on 3 legs and caudal licking) within a herd of 50 loose-housed Brown Swiss dairy cows were analyzed by video observation before and after floor reconstruction. Still photographs and pedometers were used to asses step length and number of steps, representing walking behavior. Compared with the concrete floor surface, rubber coating led to an increase in step length (58 +/- 1 vs. 70 +/- 1 cm; n = 35) and in steps per day (4,226 +/- 450 vs. 5,611 +/- 495; mean +/- SEM; n = 9). Mounting was higher on the flooring covered with rubber mats (23 vs. 112). Collapsing or slipping during mounting only occurred on concrete slatted flooring (in 19 out of 23 mounting actions). Licking while standing on 3 legs and caudal licking increased up to 4-fold (105 vs. 511 observations). In conclusion, improvements were found in behavior when rubber-coated slatted floor surfaces were used in dairy cattle housing in transition from concrete flooring. Disorders in estrus and hygiene behavior were associated with the flooring of the barn and were relatively easy to investigate within the framework of farm welfare assessments.

Staphylococcus aureus and Escherichia coli cause deviating expression profiles of cytokines and lactoferrin messenger ribonucleic acid in mammary epithelial cells.

Pathogens invading the mammary gland cause a complex signaling network that activates the early immune defense and leads to an outcome of inflammation symptoms. To examine the importance of mammary epithelial cells in these regulations and interactions resulting in a pathogen-related course of mastitis, we characterized the mRNA expression profile of key molecules of the innate immune system by quantitative real-time PCR. Mammary gland epithelial cells isolated on d 42 of lactation from 28 first-lactation Holstein dairy cows were cultured separately under standardized conditions and treated for 1, 6, and 24 h with heat-inactivated gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria. Both pathogens increased mRNA expression patterns of proteins involved in pathogen recognition such as Toll-like receptors and nuclear factor-kappa B, whereas gram-negatives acted as a stronger stimulus. Furthermore, this could be confirmed by the expression profile of the proinflammatory cytokines tumor necrosis factor alpha, IL-1 beta, IL-6, and chemokines such as IL-8 and RANTES (regulated upon activation, normal T-cell expressed and secreted). Remarkably, at a low level of mRNA expression after 1 h of treatment these cytokines and chemokines were expressed at a significantly higher level in Staphyloccocus aureus than in Escherichia coli affected cells. Lactoferrin showed a deviating expression pattern to pathogen stimulation (i.e., at the 1-h measuring point Escherichia coli induced a higher mRNA expression, whereas the highest level was reached after 24 h of stimulation with Staphylococcus aureus). Complement factor 3 was the only measured factor that responded equally to both microorganisms. Our data emphasize the role of mammary epithelial cells in the immune defense of the udder and confirm their contribution to pathogen-related different courses of mastitis.

Doppler sonography of the uterine arteries during a superovulatory regime in cattle. Uterine blood flow in superovulated cattle.

Transrectal color Doppler sonography was used to investigate the effects of a gonadotropin treatment to induce superovulation on uterine blood flow and its relationship with steroid hormone levels, ovarian response and embryo yield in dairy cows. The estrous cycle of 42 cows was synchronized by using PGF(2alpha) during diestrus and GnRH 48 h later (Day 0). Cows were examined on the day of eCG (2750 IU)-administration (Day 10), 3 days after eCG (Day 13) and 7 days after artificial insemination (Day 22), including the determination of total estrogens (E) and progesterone (P(4)) in peripheral plasma. Eight days after insemination (Day 23) the uterus was flushed and the number of total ova and embryos as well as transferable embryos was determined. The ovarian response was defined by the number of follicles>5.0mm in diameter on Day 13 and the number of corpora lutea on Day 22. Uterine blood flow was reflected by the blood flow volume (BFV) and the pulsatility index (PI) in the uterine arteries. Both variables showed distinct changes throughout the superovulatory cycle: BFV increased by 94% and PI decreased by 30% between Days 10 and 22 (P<0.0001). On Day 13, BFV but not PI correlated with follicle numbers (r=0.35; P<0.05); no correlation was found with E and P(4) (P>0.05). On Day 22, BFV correlated positively and PI correlated negatively with the number of corpora lutea (r=0.45 and r=-0.37; P<0.05) and P(4) (r=0.39 and r=-0.30; P<0.05). The number of transferable embryos was solely related to BFV measured on Day 13 (r=0.32; P<0.05). Our results show for the first time that in cows a superovulatory treatment is associated with a marked increase in BFV and a marked decrease in PI in the uterine arteries, concurrent with the development of multiple follicles and corpora lutea. However, transrectal color Doppler sonography of the uterine arteries does not facilitate the prediction of embryo yields following superovulatory treatment.