Investigation of biofilm production and icaA and icaD genes in Staphylococcus aureus isolated from heifers and cows with mastitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Index of thermal stress for cows (ITSC) under high solar radiation in tropical environments

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Comparative efficacy of exogenous eCG and progesterone on endogenous progesterone and pregnancy in Holstein cows submitted to timed artificial insemination

Coordenação de Apoio de Pessoal de Nível Superior (CAPES)

Heat stress induced alteration in bovine oocytes: functional and cellular aspects

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Manipulation of follicle development to ensure optimal oocyte quality and conception rates in cattle

Over the last several decades, a number of therapies have been developed that manipulate ovarian follicle growth to improve oocyte quality and conception rates in cattle. Various strategies have been proposed to improve the responses to reproductive biotechnologies following timed artificial insemination (TAI), superovulation (SOV) or ovum pickup (OPU) programmes. During TAI protocols, final follicular growth and size of the ovulatory follicle are key factors that may significantly influence oocyte quality, ovulation, the uterine environment and consequently pregnancy outcomes. Progesterone concentrations during SOV protocols influence follicular growth, oocyte quality and embryo quality; therefore, several adjustments to SOV protocols have been proposed depending on the animal category and breed. In addition, the success of in vitro embryo production is directly related to the number and quality of cumulus oocyte complexes harvested by OPU. Control of follicle development has a significant impact on the OPU outcome. This article discusses a number of key points related to the manipulation of ovarian follicular growth to maximize oocyte quality and improve conception rates following TAI and embryo transfer of in vivo-and in vitro-derived embryos in cattle.

Milk hygienic practices and occurrence of Staphylococcus aureus and Escherichia coli O157:H7 in small-scale dairy farms in Sao Paulo, Brazil

The objective of this study was to assess the relationship between somatic cell counts (SCC), the use of different milking practices, and the occurrence of Staphylococcus aureus and Escherichia coli O157:H7 in 42 small-scale dairy farms located in the state of Sao Paulo, Brazil. S. aureus and E. coli O157:H7 were isolated in the milk from dairy cows with low (< 200,000 cells/ml) and high SCC (>200,000 cells/ml), although no effect of SCC (p > 0.05) was observed on the incidence of the bacteria in raw milk. The use of disposable gloves during milking reduced S. aureus counts in milk (p < 0.05), but did not affect the occurrence of E. coli O157:H7. The other milking practices evaluated (closed milking system, use of pre- and post-dipping, mastitis diagnosis by strip cup test, and disinfection of teat cups) did not affect (p < 0.05) the occurrence of S. aureus or E. coli O157:H7 in raw milk. Results indicate the need for effective educational programs addressed to prevent the contamination of milk with S. aureus and E. coli O157:H7 in Brazilian small-scale dairy farms.

Oestradiol enhances plasma growth hormone and insulin-like growth factor-I concentrations and increased the expression of their receptors mRNAs in the liver of ovariectomized cows

Many metabolic hormones, growth hormone (GH), insulin-like growth factor-I (IGF-I) and insulin affect ovarian functions. However, whether ovarian steroid hormones affect metabolic hormones in cattle remains unknown. This study aimed to determine the effect of sex steroids on the plasma profiles of GH, IGF-I and insulin and their receptors in the liver and adipose tissues of dairy cows. Ovariectomized cows (n = 14) were randomly divided into four groups: control group (n = 3) was treated with saline on Day 0; oestradiol (E2) group (n = 3), with saline and 1 mg oestradiol benzoate (EB) on Day 0 and 5, respectively; progesterone (P4) group (n = 4) with two CIDRs (Pfizer Inc., Tokyo, Japan) from Day 0; and E2 + P4 group (n = 4) with two CIDRs on Day 0 that were removed on Day 6 and were immediately injected with 1 mg EB. The animals were euthanized after the experiment, and liver and adipose tissues samples were quantitatively analysed using real-time PCR for the expression of mRNA for the GH (GHR), IGF-I (IGFR-I) and insulin (IR) receptor mRNAs. Oestradiol benzoate significantly increased the number of peaks (p < 0.05), pulse amplitude (p < 0.05) and area under the curve (AUC; p < 0.01) for plasma GH; moreover, it increased plasma IGF-I concentration (p < 0.05), but it had no effect on the plasma insulin profile. P4 significantly decreased the AUC (p < 0.01), compared with the control group, whereas it did not affect the number of peaks and the amplitude of GH pulses. P4 + E2 did not affect the GH pulse profile. E2 increased the mRNA expression of GHR, IGFR-I and IR in the liver (p < 0.05), whereas both P4 and E2 + P4 did not change their expressions. Our results provide evidence that the metabolic and reproductive endocrine axes may regulate each other to ensure optimal reproductive and metabolic function.

Best combination of pre-stimulation and latency period duration before cluster attachment for efficient oxytocin release and milk ejection in cows with low to high udder-filling levels

Experiments were designed to investigate the suitability of a combination of a short manual teat stimulation with a short latency period before teat cup attachment to induce and maintain oxytocin release and milk ejection without interruption. In Experiment 1, seven dairy cows in mid lactation were manually pre-stimulated for 15, 30 or 45 s, followed by either 30 s or 45 s of latency period. It was shown that all treatments induced a similar release of oxytocin without interruption until the end of milking. In particular, the latency period of up to 45 s did not cause a transient decrease of oxytocin concentration. In Experiment 2, milking characteristics were recorded in seven cows each in early, mid, and late lactation, respectively. Because the course of milk ejection depends mainly on the degree of udder filling, individual milkings were classified based on the actual degree of udder filling which differs between lactational stages but also between morning and evening milkings. All animals underwent twelve different udder preparation treatments, i.e. 15, 30, or 45 s of pre-stimulation followed by latency periods of 0, 30, 45, or 60 s. Milking characteristics were recorded. Total milk yield, main milking time and average milk flow rate did not differ between treatments if the degree of udder filling at the start of milking was >40% of the maximum storage capacity. However, if the udder filling was <40%, main milking time was decreased with the duration of a latency period up to 45 s, independent of duration of pre-stimulation. Average milk flow at an udder filling of <40% was highest after a pre-stimulation of 45 s followed by a latency period of another 45 s. In contrast, average milk flow reached its lowest values at a pre-stimulation of 15 s without additional latency period. However, average milk flow after a 15-s pre-stimulation increased with increasing latency period. In conclusion, a very short pre-stimulation when followed by a latency period up to 45 s before teat cup attachment remains a suitable alternative for continuous stimulation to induce milk ejection.

First report about the mode of action of combined butafosfan and cyanocobalamin on hepatic metabolism in nonketotic early lactating cows

The primary aim was to investigate the effect of combined butafosfan and cyanocobalamin on liver metabolism in early lactating cows through mRNA expression measurements of genes encoding 31 enzymes and transport proteins of major metabolic processes in the liver using 16 multiparous early lactating dairy cows. The treatments included i.v. injection of 10 mL/100 kg of body weight combined butafosfan and cyanocobalamin (TG, n = 8) on 3 d consecutively at 25 +/- 3 d in milk or injection with physiological saline solution similarly applied (CG, n = 8). Results include a higher daily milk production for TG cows (41.1 +/- 0.9 kg, mean +/- SEM) compared with CG cows (39.5 +/- 0.7 kg). In plasma, the concentration of inorganic phosphorus was lower in the TG cows (1.25 +/- 0.08 mmol/L) after the treatment than in the CG cows (1.33 +/- 0.07 mmol/L). The plasma beta-hydroxybutyrate concentration was 0.65 +/- 0.13 mmol/L for all cows before the treatment, and remained unaffected post treatment. The unique result was that in the liver, the mRNA abundance of acyl-coenzyme A synthetase long-chain family member 1, involved in fatty acid oxidation and biosynthesis, was lower across time points after the treatment for TG compared with CG cows (17.5 +/- 0.15 versus 18.1 +/- 0.24 cycle threshold, log(2), respectively). In conclusion, certain effects of combined butafosfan and cyanocobalamin were observed on mRNA abundance of a gene in the liver of nonketotic early lactating cows.

Coordinated gene expression in adipose tissue and liver differs between cows with high or low NEFA concentrations in early lactation

Dairy cows with high and low plasma non-esterified fatty acid (NEFA) concentrations in early lactation were compared for plasma parameters and mRNA expression of genes in liver and subcutaneous adipose tissue. The study involved 16 multiparous dairy cows with a plasma NEFA concentration of >500 mumol/l [n = 8, high NEFA (HNEFA)] and <140 mumol/l [n = 8, low NEFA (LNEFA)] in the first week post-partum (pp). Blood samples, adipose and liver tissues were collected on day 1 (+1d) and at week 3 pp (+3wk). Blood plasma was assayed for concentrations of metabolites and hormones. Subcutaneous adipose and liver tissues were analysed for mRNA abundance by real-time qRT-PCR encoding parameters related to lipid metabolism. Results showed that mean daily milk yield and milk fat quantity were higher in HNEFA than in LNEFA cows (p < 0.01), and the NEB was more negative in HNEFA than in LNEFA in +3wk too (p < 0.05). HNEFA cows had slightly lower (p < 0.1) insulin concentrations than LNEFA cows across the study period, and the body condition score decreased more from +1d to +3wk in HNEFA than in LNEFA (p = 0.09). The mRNA abundance of genes in the liver related to fatty acid oxidation (carnitine palmitoyltransferase 2 and very long chain acyl-coenzyme A dehydrogenase) and ketogenesis (3-hydroxy-3-methylglutaryl-coenzyme A synthase 2) were lower in HNEFA than in LNEFA cows. No differences between the two groups were observed for mRNA expression of genes in adipose tissue. The number of calculated significant correlation coefficients (moderately strong) between parameters in the liver and in adipose tissue was nearly similar on +1d, and higher for HNEFA compared with LNEFA cows in +3wk. In conclusion, dairy cows with high compared with low plasma NEFA concentrations in early lactation show differentially synchronized mRNA expression of genes in adipose tissue and liver in +3wk that suggests a different orchestrated homeorhetic regulation of lipid metabolism.

Short-term feed intake is regulated by macronutrient oxidation in lactating Holstein cows

In addition to plasma metabolites and hormones participating as humoral signals in the control of feed intake, oxidative metabolic processes in peripheral organs also generate signals to terminate feeding. Although the degree of oxidation over longer periods is relatively constant, recent work suggests that the periprandial pattern of fuel oxidation is involved in regulating feeding behavior in the bovine. However, the association between periprandial oxidative metabolism and feed intake of dairy cows has not yet been studied. Therefore, the aim of this study was to elucidate possible associations existing between single feed intake events and whole-body net fat and net carbohydrate oxidation as well as their relation to plasma metabolite concentrations. To this end, 4 late-lactating cows equipped with jugular catheters were kept in respiratory chambers with continuous and simultaneous recording of gas exchange and feed intake. Animals were fed ad libitum (AL) for 24h and then feed restricted (RE) to 50% of the previous AL intake for a further 24h. Blood samples were collected hourly to analyze β-hydroxybutyrate (BHBA), glucose, nonesterified fatty acids (NEFA), insulin, and acylated ghrelin concentrations. Cross-correlation analysis revealed an offset ranging between 30 and 42 min between the maximum of a feed intake event and the lowest level of postprandial net fat oxidation (FOX(net)) and the maximum level of postprandial net carbohydrate oxidation (COX(net)), respectively. During the AL period, FOX(net) did not increase above -0.2g/min, whereas COX(net) did not decrease below 6g/min before the start of the next feed intake event. A strong inverse cross-correlation was obtained between COX(net) and plasma glucose concentration. Direct cross-correlations were observed between COXnet and insulin, between heat production and BHBA, between insulin and glucose, and between BHBA and ghrelin. We found no cross-correlation between FOX(net) and NEFA. During RE, FOX(net) increased with an exponential slope, exceeded the threshold of -0.2g/min as indicated by increasing plasma NEFA concentrations, and approached a maximum rate of 0.1g/min, whereas COX(net) decayed in an exponential manner, approaching a minimal COX(net) rate of about 2.5 g/min in all cows. Our novel findings suggest that, in late-lactating cows, postprandial increases in metabolic oxidative processes seem to signal suppression of feed intake, whereas preprandially an accelerated FOX(net) rate and a decelerated COX(net) rate initiate feed intake.

Understanding diversity of hepatic metabolism and related adaptations in the early lactating dairy cow.

The onset of lactation in dairy cows represents a major metabolic challenge that involves large adaptations in glucose, fatty acid, and mineral metabolism to support lactation and to avoid metabolic dysfunction. The complex system of adaptation can differ considerably between cows, and may have a genetic base. In the present review, the variation in adaptive reactions in dairy cows is discussed. In these studies, the liver being a key metabolic regulator for understanding the variation in adaptive performance of the dairy cow was the main focus of research. Liver function was evaluated through gene expression measurements; to explain the associated phenotypic variability and to identify descriptors for metabolic robustness in dairy cows. Hence, the identified genes involved act as a connecting link between the genotype encoded on the DNA and the phenotypic expression of the target factors at a protein level. The integration of phenotypic data, including gene expression profiles, and genomic data will facilitate a better characterization of the complex interplay between these levels, and will improve the genetic understanding necessary to unravel a certain trait or multi-trait such as metabolic robustness in dairy cows.

Clinical outcomes and molecular genotyping of Staphylococcus aureus isolated from milk samples of dairy primiparous Mediterranean buffaloes (Bubalus bubalis)

Staphylococcus aureus is one of the most important pathogens causing mastitis in dairy cows and in Mediterranean buffaloes. Genotype B (GTB) is contagious in dairy cows and may occur in up to 87% of cows of a dairy herd. It was the aim of this study to evaluate genotypes present, clinical outcomes, and prevalence of Staph. aureus in milk samples of primiparous Mediterranean dairy buffaloes. Two hundred composite milk samples originating from 40 primiparous buffaloes were collected from May to June 2012, at d 10, 30, 60, 90, and 150 d in milk (DIM) to perform somatic cell counts and bacteriological cultures. Daily milk yields were recorded. Before parturition until 40 to 50 DIM, all primiparous animals were housed separated from the pluriparous animals. Milking was performed in the same milking parlor, but the primiparous animals were milked first. After 50 DIM, the primiparous were mixed with the pluriparous animals, including the milking procedure. Individual quarter samples were collected from each animal, and aliquots of 1 mL were mixed and used for molecular identification and genotyping of Staph. aureus. The identification of Staph. aureus was performed verifying the presence of nuc gene by nuc gene PCR. All the nuc-positive isolates were subjected to genotype analysis by means of PCR amplification of the 16S-23S rRNA intergenic spacer region and analyzed by a miniaturized electrophoresis system. Of all 200 composite samples, 41 (20.5%) were positive for Staph. aureus, and no genotype other than GTB was identified. The prevalence of samples positive for Staph. aureus was 0% at 10 DIM and increased to a maximum of 22/40 (55%) at 90 DIM. During the period of interest, 14 buffaloes tested positive for Staph. aureus once, 6 were positive twice, and 5 were positive 3 times, whereas 15 animals were negative at every sampling. At 90 and 150 DIM, 7 (17.5%) and 3 buffaloes (7.5%), respectively, showed clinical mastitis (CM), and only 1 (2.5%) showed CM at both samplings. At 60, 90, and 150 DIM, 1 buffalo was found with subclinical mastitis at each sampling. At 30, 60, 90, and 150 DIM, 2.5 (1/40), 22.5 (9/40), 35 (14/40), and 10% (4/40) were considered affected by intramammary infection, respectively. Buffaloes with CM caused by Staph. aureus had statistically significantly higher mean somatic cell count values (6.06 ± 0.29, Log10 cells/mL ± standard deviation) and statistically significantly lower mean daily milk yields (7.15 ± 1.49, liters/animal per day) than healthy animals (4.69 ± 0.23 and 13.87 ± 2.64, respectively), buffaloes with IMI (4.82 ± 0.23 and 11.16 ± 1.80, respectively), or with subclinical mastitis (5.47 ± 0.10 and 10.33 ± 0.68, respectively). Based on our knowledge, this is the first time that Staph. aureus GTB has been identified in milk samples of dairy Mediterranean buffaloes.

Mycoplasma bovis infections in Swiss dairy cattle: a clinical investigation

Mycoplasma bovis causes mastitis in dairy cows and is associated with pneumonia and polyarthritis in cattle. The present investigation included a retrospective case–control study to identify potential herd-level risk factors for M. bovis associated disease, and a prospective cohort study to evaluate the course of clinical disease in M. bovis infected dairy cattle herds in Switzerland. Eighteen herds with confirmed M. bovis cases were visited twice within an average interval of 75 d. One control herd with no history of clinical mycoplasmosis, matched for herd size, was randomly selected within a 10 km range for each case herd. Animal health data, production data, information on milking and feeding-management, housing and presence of potential stress- factors were collected. Composite quarter milk samples were aseptically collected from all lactating cows and 5% of all animals within each herd were sampled by nasal swabs. Organ samples of culled diseased cows were collected when logistically possible. All samples were analyzed by real-time polymerase chain reaction (PCR). In case herds, incidence risk of pneumonia, arthritis and clinical mastitis prior to the first visit and incidence rates of clinical mastitis and clinical pneumonia between the two visits was estimated. Logistic regression was used to identify potential herd-level risk factors for M. bovis infection. In case herds, incidence risk of M. bovis mastitis prior to the first visit ranged from 2 to 15%, whereas 2 to 35% of the cows suffered from clinical pneumonia within the 12 months prior to the first herd visit. The incidence rates of mycoplasmal mastitis and clinical pneumonia between the two herd visits were low in case herds (0–0.1 per animal year at risk and 0.1-0.6 per animal year at risk, respectively). In the retrospective-case-control study high mean milk production, appropriate stimulation until milk-let-down, fore-stripping, animal movements (cattle shows and trade), presence of stress-factors, and use of a specific brand of milking equipment, were identified as potential herd-level risk factors. The prospective cohort study revealed a decreased incidence of clinical disease within three months and prolonged colonization of the nasal cavity by M. bovis in young stock.

Monensin supplementation of lactating cows fed tropical grasses and cane molasses or grain

Effects of monensin (Mon) on performance of Holstein-Friesian cows fed tropical grasses and cane molasses (M) or cereal grain were examined in three experiments. In experiment I (incomplete 4 x 4 Latin square), three rumen-fistulated cows [188 I I days in milk (DIM)] were fed mixed diets based on rhodes grass (Chloris gayana cv. Callide) bay where M was substituted for wheat grain (W) at rates of 0 (MO), 125 (M 125) or 250 (M250) g/kg dry matter (DM). A fourth diet contained M250 plus 0.02 g Mon/kg DM (M250 + Mon). Substituting M for W tended (P < 0.10) to decrease the ratio of rumen molar proportions of acetate+butyrate (Bu):propionate (Pr) (4.3 versus 3.8 and 4.0 for M0, M125 and M250, respectively). There were no treatment effects (P> 0.10) on intake, organic matter digestibility, milk production or liveweight (LW) change. In experiment 2, 48 cows (173 &PLUSMN; 28.3 DIM) grazing kikuyu (Pennisetum clandestinum cv. common) pastures and supplemented with maize silage and a grain-based concentrate were offered either M (2.6 kg DM/(cow day)) or barley grain (B) (2.7 kg DM/(cow day)). Within each supplement type, half were fed 0 or 320 mg of Mon/(cow day). There were Mon x supplement interactions (Mon x S; P < 0.05) on the rumen molar proportion of Pr and Bu at 15:00 h, with B + Mon having the highest value for Pr (0.259 mmol/mmol) and lowest value for Bu (0.121 mmol/mmol). A Mon x S effect (P < 0.05) on milk fat content was noted with Mon causing a lower value regardless of energy source (31 and 36 g/l versus 40 and 38 g/l for B + Mon, M + Mon, B - Mon and M - Mon, respectively). As a main effect, M as opposed to B, reduced yields of milk (P < 0.05; 16.21/(cow day) versus 18.01/(cow day)) and protein (P < 0.05; 479 g/(cow day) versus 538 g/(cow day)). Monensin reduced milk fat yield (P < 0.05; 669 g/(cow day) versus 562 g/(cow day)), raised milk protein concentration (P < 0.05; 31 g/l versus 29 g/l) and caused LW gain rather than loss (P < 0.05; +0.06 kg/(cow day) versus -0.30 kg/(cow day)). No treatment effects on pasture intake were noted. In experiment 3, 48 cows (91 &PLUSMN; 16.1 DIM) grazing kikuyu pasture and supplemented with grain-based concentrate, sugar cane silage and 2.7 kg DM(cow day) of M were supplemented with either 0 or 320 mg Mon/(cow day). Monensin reduced (P < 0.05) milk fat content (33 g/l versus 30 g/l) and tended (P < 0.10) to reduce milk protein content (29 g/l versus 28 g/l). No effects of Mon on other milk production parameters, LW change or pasture intake were noted. Feeding monensin to mid-lactation Holstein-Friesian cows offered diets based on tropical grasses, and cane molasses or grain, improves rumen fermentation efficiency, thereby improving energy efficiency resulting in higher LW gain. Monensin had no effect on milk yield, but reduced milk fat concentration.