Using the natural abundance of nitrogen isotopes to identify cattle with greater efficiency in protein-limiting diets.

The difficulty in selecting cattle for higher feed and nitrogen use efficiency (NUE) is an important factor contributing to poor growth and reproductive performance in dry-tropics rangelands. Therefore, the objectives were to examine the cattle variation in retaining nitrogen in a protein-deficient diet and the natural abundance of stable isotopes in body tissues as a practical alternative for the detection of more efficient cattle. In experiment 1, feed efficiency parameters were determined in 89 Brahman steers fed a protein-limiting diet for 70 days, followed by 7 days in metabolism crates for total collection of urine and faeces and calculation of nitrogen retention and NUE. The diet-animal fractionation of nitrogen isotopes (Δ15N) was quantified in tail hair and plasma proteins using isotope-ratio MS. There was a large variation in growth performance, feed efficiency and nitrogen losses among steers. Quantifying Δ15N in tail hair (Δ15Ntail hair) resulted in stronger correlations with feed efficiency and nitrogen metabolism parameters than when quantified in plasma proteins. Δ15Ntail hair was positively correlated with nitrogen losses in urine (r = 0.31, P < 0.01) and faeces (r = 0.25, P = 0.04), leading to a negative correlation with NUE (r = -0.40, P < 0.01). The group of steers with lower Δ15Ntail hair had greater feed efficiency, lower nitrogen losses, and greater NUE. In experiment 2, for evaluation of isotope fraction as a predictor of reproductive performance, 630 Brahman-crossed cows were classified for reproductive performance for 2 years. From this group, 25 cows with poor reproductive performance and 25 cows with good reproductive performance were selected. Tail hair representing 7 months of growth were segmented and analysed for carbon (δ13C) and nitrogen (δ15N) isotope enrichment. Reproductive performance was not associated with diet selection, as there was no difference in tail hair δ13C between groups. However, more productive cows had lower (P < 0.05) tail hair δ15N during the dry season, indicating differences in N metabolism and possibly lower N losses. In addition, cows with better reproductive performance and, therefore, greater nutrient demands, had similar body condition scores and a tendency (P = 0.09) for higher live weight at the end of the trial. In conclusion, the findings of the present study confirm that nitrogen isotope fractionation in tail hair can be used as a predictor of nitrogen losses, NUE, and reproductive performance of Brahman cattle on low-protein diets.

Monensin and a blend of castor oil and cashew nut shell liquid used in a high-concentrate diet abruptly fed to Nellore cattle.

Monensin and functional oils (FO) were supplemented to a high-concentrate diet abruptly fed to 12 ruminally cannulated Zebu steers to study their effects on rumen fermentation, blood metabolites, and , , and relative population. A randomized complete block design with repeated measures over time within 2 experimental periods of 21 d each was used. Treatments were a control (CTR; with no additives), FO (included at 400 mg/kg), and monensin included at 30 mg/kg (M30) or 40 mg/kg (M40). All steers were fed the same high-concentrate basal diet, which consisted of 92.25% concentrate. The first 60 h after transition showed a treatment and hour interaction for ruminal propionate proportion ( = 0.028), and no change in acetate molar proportion ( = 0.633), rumen pH ( = 0.370), and time the rumen pH remained below 5.6 ( = 0.242) were observed. The acetate:propionate ratio decreased ( = 0.020) when monensin was fed in both concentrations (2.30 for the M30 treatment and 2.32 for the M40 treatment) compared with when the CTR was fed (2.85), without being different when the FO (2.71) treatment was fed. Only the M30 treatment did not show pH below 5.2 (P=0.047) over the 60 h after the abrupt transition. Within the entire period, DMI ( = 0.008) and mean ruminal pH ( = 0.040) as well as molar proportions of propionate ( = 0.034) and valerate ( = 0.031) had significant interactions between treatment and day. Total VFA concentration was greater ( = 0.017) for the M30 (117.36 m) and CTR treatments (115.77 m) compared with the M40 treatment (105.02 m), without being different for the FO treatment (111.55 m). Treatments did not change feed behavior parameters. Blood HCO ( = 0.006) and total carbon dioxide ( = 0.003) were greater for the M30 (27.8 and 29.3 mmol/L, respectively) and FO treatments (28.3 and 29.7 mmol/L, respectively) compared with the CTR treatment (25.7 and 26.9 mmol/L, respectively). ( < 0.0001) and ( < 0.0001) decreased their population throughout days, whereas ( = 0.026) increased its population. Independent of ciliated protozoa genera, the greatest ( < 0.0001) protozoa counts were observed for the CTR treatment (52.7 × 10/mL), intermediate for the FO treatment (35.3 x10/mL), and least for steers fed monensin in both concentrations (15 × 10/mL for the M30 treatment and 14 × 10/mL for the M40 treatment). Feed additives had different effects to reduce the subacute acidosis. The use of the FO and M40 treatments did not change most of the rumen fermentation variables, especially in the first week after abrupt transition, when the M30 treatment provided higher protection against acidosis.

Effects of ractopamine hydrochloride and dietary protein content on performance, carcass traits and meat quality of Nellore bulls.

Ractopamine hydrochloride (RH) alters protein metabolism and improves growth performance in Bos taurus cattle with high carcass fat. Our objective was to evaluate the effects of RH, dietary CP and RH×CP interaction on performance, blood metabolites, carcass characteristics and meat quality of young Nellore bulls. A total of 48 bulls were randomly assigned to four treatments in a 2×2 factorial arrangement. The factors were two levels of dietary CP (100% and 120% of metabolizable protein requirement, defined as CP100 and CP120, respectively), and two levels of RH (0 and 300 mg/animal·per day). Treated animal received RH for the final 35 days before slaughter. Animals were weighed at the beginning of the feedlot period (day 63), at the beginning of ractopamine supplementation (day 0), after 18 days of supplementation (day 18) and before slaughter (day 34). Animals were slaughtered and hot carcass weights recorded. After chilling, carcass data was collected and longissimus samples were obtained for determination of meat quality. The 9-11th rib section was removed for carcass composition analysis. Supplementation with RH increased ADG independently of dietary CP. There was a RH×CP interaction on dry matter intake (DMI), where RH reduced DMI at CP120, with no effect at CP100. Ractopamine improved feed efficiency, without RH×CP interaction. Ractopamine had no effect on plasma creatinine and urea concentration. Greater dietary CP tended to increase blood urea, and there was a RH×CP interaction for plasma total protein. Ractopamine supplementation increased plasma total protein at CP120, and had no effect at CP100. Ractopamine also decreased plasma glucose concentration at CP100, but had no effect at CP120. Ractopamine increased alkaline phosphatase activity at CP120 and had no effect at CP100. There was a tendency for RH to increase longissimus muscle area, independently of dietary CP. Ractopamine did not alter fat thickness; however, fat thickness was reduced by greater CP in the diet. Supplementation with RH decreased meat shear force, but only at day 0 of aging, having no effect after 7, 14 or 21 days. Greater dietary protein increased meat shear force after 0 and 7 days of aging, with no effect after 14 or 21 days. These results demonstrate for the first time the efficacy of ractopamine supplementation to improve gain and feed efficiency of intact Bos indicus males, with relatively low carcass fat content. Ractopamine effects were not further improved by increasing dietary protein content above requirements.

Zilpaterol hydrochloride improves feed efficiency and changes body composition in nonimplanted Nellore heifers.

This research aimed to evaluate the effects of zilpaterol hydrochloride (ZH; MSD Animal Health, São Paulo, SP, Brazil) on the performance, carcass traits, serum metabolites, body composition, and gain composition of nonimplanted Nellore heifers. Nellore heifers ( = 72; average BW = 267 ± 16 kg; average 18 mo of age) were maintained in a feedlot system for 118 d. Heifers were separated into 2 groups: Control and ZH. The ZH group received ZH (8.3 mg/kg diet DM) for 30 d with 3 d of withdrawal before slaughter. Heifers were allotted to 18 pens, 9 pens per treatment, and assigned to a randomized block design. The animals were weighed, blood samples were collected, and subgroups of heifers were slaughtered at the beginning of supplementation and after 20 and 33 d to evaluate performance, blood metabolites, empty BW (EBW), and EBW composition. Hot carcass and kidney-pelvic fat weights were recorded at slaughter. At 24 h postmortem, carcasses were fabricated and the 9-10-11th rib (HH) section was removed from the primal rib to analyze moisture, protein, ash, and ether extract (EE) content in empty body (EB) and gain composition. Heifers fed ZH had gains in HCW that were 19.7 kg greater than controls, reflecting the 30% increase ( < 0.01) in ADG. There was no change in DMI, resulting in a 20% greater G:F ratio ( < 0.01) for heifers fed ZH. Heifers supplemented with ZH had carcass dressing percentages that were 3% greater than controls ( < 0.01), and there was also a 19% reduction in kidney-pelvic fat ( = 0.05) in ZH-treated heifers. Zilpaterol increased serum creatinine ( < 0.01), tended to increase ( = 0.06) serum triacylglycerol, decreased serum NEFA ( = 0.04), and tended to decrease ( = 0.06) serum glucose. The EBW composition was changed after 20 d of ZH supplementation ( = 0.02), with ZH increasing the moisture, ash, and protein contents, whereas carcass fat was decreased by ZH by 14%. Consequently, the carcass CP:EE ratio after 20 d was increased ( = 0.03) by 24% with ZH supplementation. There was no change on EBW composition after 30 d of ZH supplementation ( = 0.17). Regarding carcass gain composition, ZH increased EBW gain ( = 0.02) by 842 g/d from d 0 to d 30, EB protein gain by 221 g/d ( = 0.05) from d 0 to d 20, and by 180 g/d ( = 0.01) from d 0 to d 33. In conclusion, ZH supplementation in nonimplanted Nellore heifers altered the composition of body weight gain, promoting greater lean tissue deposition and improving feed efficiency.

Evaluation of methods of DNA extraction from Staphylococcus aureus in milk for use in real-time PCR.

The aim of this study was to evaluate the repeatability and performance of 4 methods of extracting DNA from Staphylococcus aureus (SAU) and the gene encoding bovine mitochondrial cytochrome B (BMCB) in milk samples from cows with subclinical mastitis for use in amplification by real-time polymerase chain reaction. Two milk samples were obtained from cows naturally infected with S. aureus and subjected to the following extraction methods: Qiagen DNA extraction kit; Axyprep DNA extraction kit; in silica column boil and in silica column method. After extraction in duplicate, eluates were subjected to purification and precipitation to determine purity (A260/A280 ratio) and concentration (µg/µL) by spectrophotometry and amplification by real-time polymerase chain reaction of target genes (SAU and BMCB). There was no effect of the DNA extraction method on DNA concentration and threshold cycle for BMCB and SAU. The purity ratio (A260/A280 ) was higher when using Qiagen DNA extraction (1.76 ± 0.136) compared to the other methods tested. Our results indicate that the DNA extraction kit from Qiagen produces samples of the highest purity ratio compared to other methods.

Effect of fiber digestibility and conservation method on feed intake and the ruminal ecosystem of growing steers.

Fiber digestibility is an important factor regulating DMI in ruminants. Additionally, the ensiling process can also affect digestibility and chemical composition of the forage. The objective of this study was to investigate effects of sugarcane NDF digestibility (NDFD) and conservation method on intake, rumen kinetics, and the ruminal ecosystem of steers. Eight ruminally cannulated Nellore steers (275±22 kg BW) were used in a replicated 4×4 Latin square design with a 2×2 factorial arrangement of treatments. Two sugarcane genotypes divergent for stalk NDFD were used: IAC86-2480 with high NDFD and SP91-1049 with low NDFD. Experimental diets were formulated with 40% sugarcane, either freshly cut or as silage, and 60% concentrate on a DM basis. Each experimental period lasted for 14 d, with the last 4 d used for determination of intake, ruminal evacuation, and ruminal fluid collection. The effect of fiber digestibility on DM and NDF intake was dependent on the forage conservation method (P=0.01). High NDFD increased (P<0.01) DMI only when sugarcane was offered as silage, having no effect (P=0.41) on DMI when offered as freshly cut. Conservation method had no effect on total ruminal mass, with only a tendency (P<0.10) for greater NDF and indigestible NDF ruminal mass in steers fed the low-NDFD genotype. The NDF turnover and passage rates were greater (P<0.05) for the genotype with high NDFD but only when offered as silage. Liquid turnover rate in the rumen was greater (P=0.02) for diets containing silage, with no effect of genotype (P=0.87). There was no effect of NDFD genotype on ruminal pH (P=0.77); however, diets containing sugarcane as silage increased (P<0.01) ruminal pH. Total concentration of short chain fatty acids (P=0.05) and proportions of propionate (P=0.01) were greater for diets containing fresh sugarcane. Diets with fresh sugarcane increased the ruminal population of Streptococcus bovis (P<0.01) and Ruminococcus albus (P=0.03). The relative population of R. albus was also greater (P=0.04) for diets containing the sugarcane genotype with high NDFD. Feeding diets containing the sugarcane genotype with high NDFD increased Fibrobacter succinogenes population but only when sugarcane was fed as freshly cut (P=0.02). Using sugarcane genotypes with high NDFD can increase intake and benefit fiber-degrading bacteria in the rumen.

Detection and enumeration of Staphylococcus aureus from bovine milk samples by real-time polymerase chain reaction.

The aim of this study was to develop and evaluate a real-time quantitative PCR (qPCR)-based method to detect and quantify Staphylococcus aureus in bronopol-preserved milk samples from subclinical intramammary infections (IMI). Serial dilutions of milk artificially inoculated with Staph. aureus ATCC 29213 were used to establish a standard curve (cfu/mL) of the qPCR assay targeting the Staph. aureus thermonuclease-encoding gene nuc according to the strain plate count. The analytical sensitivity, specificity, and repeatability of the qPCR assay were determined. A total of 60 milk samples, collected from mammary quarters without abnormal appearance and with positive isolation of Staph. aureus, were submitted to both the qPCR protocol and Staph. aureus plate counting and results from both methods were compared. Staphylococcus aureus from bronopol-preserved, subclinical IMI milk samples were not accurately enumerated by qPCR compared with plate counting of the nonpreserved, raw milk sample. The detection limit of the qPCR protocol of inoculated Staph. aureus ATCC 29213 in bronopol-preserved milk samples was 1.04 × 10(1) cfu/mL. The qPCR protocol can be a high-throughput and rapid diagnostic assay to accurately detect Staph. aureus IMI from bronopol-preserved milk samples compared with a traditional culturing method. However, the proposed qPCR protocol is not accurate for counting of Staph. aureus in bronopol-preserved milk samples from naturally infected mammary glands.

Effect of chronic infusion of leptin and nutrition on sexual maturation of zebu heifers.

The amount of fat in the carcass has been proposed as a regulator of initiation of puberty in cattle. To test if changes in energy intake and in circulating leptin concentration are each capable of altering age, BW, and body composition at puberty, 36 prepubertal Nellore heifers, 18 to 20 mo old, 275.8 ± 17.2kg BW, and BCS of 5 ± 0.5 (1 to 9 scale), were randomly assigned to each of 3 treatments (n = 12): High (high energy diet), Low (low energy diet), and LL [low energy diet + ovine leptin (oLeptin)]. Diets were formulated to promote BW gain of 0.4 kg/d (groups Low and LL) or 1.2 kg/d (High group). After 14 d of adjustment to diet, heifers in LL group received subcutaneous injections of oLeptin at 4.8 µg/kg BW twice a day for 56 d. Groups High and Low received similar injections of 2 mL saline solution. Age at puberty was considered to be the age on first detection of a corpus luteum, confirmed by plasma concentrations of progesterone of >1 ng/mL. Heifers were slaughtered on the second day after first corpus luteum detection. Expression of leptin gene was quantified by real-time PCR using ribosomal protein-L19 (RP-L19) as a control gene. Leptin administration increased (P = 0.04) leptin serum concentration but had no effect (P > 0.05) on age, BW, or BCS at puberty. High energy intake increased (P < 0.01) leptin concentration, accelerated (P = 0.02) puberty, and increased (P < 0.01) BCS at puberty, without altering (P = 0.17) BW at puberty. High energy intake also accelerated (P = 0.04) follicular development. Leptin administration caused a significant (P < 0.05) but transient increase in follicular development, which was similar to the transient increase in leptin serum concentration. Results from leptin gene expression demonstrated that high energy intake increased (P < 0.01) and leptin administration decreased (P < 0.01) leptin expression in 3 adipose tissues. The observed decrease in leptin gene expression after administration of leptin could explain the reduction in leptin serum concentration after 30 d of treatment and consequently the failure of leptin to accelerate puberty. Our findings did not support the hypothesis that reduced serum concentration of leptin is an important hindrance for puberty onset in malnourished zebu heifers. Although exogenous administration of leptin temporarily enhanced rate of follicular growth, it did not accelerate puberty.

Intramammary infusion of leptin decreases proliferation of mammary epithelial cells in prepubertal heifers.

High energy intake and excessive body fatness impair mammogenesis in prepubertal ruminants. High energy intake and excessive fatness also increase serum leptin. Our objective was to determine if an infusion of leptin decreases proliferation of mammary epithelial cells of prepubertal heifers in vivo. Ovine leptin at 100 microg/ quarter per d with or without 10 microg of insulin-like growth factor (IGF)-I was infused via the teat canal into mammary glands of prepubertal dairy heifers; contralateral quarters were used as controls. After 7 d of treatment, bromodeoxyuridine was infused intravenously and heifers were slaughtered approximately 2 h later. Tissue from 3 regions of the mammary parenchyma was collected and immunostained for bromodeoxyuridine (BrdU), proliferating cell nuclear antigen (Ki-67), and caspase-3. Leptin decreased the number of mammary epithelial cells in the S-phase of the cell cycle by 48% in IGF-I-treated quarters and by 19% in saline-treated quarters. Leptin did not alter the number of mammary epithelial cells within the cell cycle, as indicated by Ki-67 labeling. Caspase-3 immunostaining within the mammary parenchyma was very low in these heifers, but leptin significantly increased labeling in saline-treated quarters. Leptin enhanced SOCS-3 expression in IGF-I-treated quarters but did not alter SOCS-1 or SOCS-5 expression. We conclude that a high concentration of leptin in the bovine mammary gland reduces proliferation of mammary epithelial cells. The reduced proliferation is accompanied by an increase in SOCS-3 expression, suggesting a possible mechanism for leptin inhibition of IGF-I action. Whether leptin might be a physiological regulator of mammogenesis remains to be determined.

Short communication: intramammary infusion of IGF-I increases bromodeoxyuridine labeling in mammary epithelial cells of prepubertal heifers.

When dairy heifers are fed to gain more than 900 g of body weight/d, they have less mammary parenchymal DNA at puberty but more insulin-like growth factor-I (IGF-I) in serum. This negative relationship between serum IGF-I concentration and mammary epithelial cell proliferation is in disagreement with the extensively reported role of IGF-I as a stimulator of mammary epithelial cell proliferation. Despite the large body of evidence suggesting that an increase in IGF-I concentration should lead to an increase in mammary epithelial cell proliferation of prepubertal heifers, it had not been previously tested. Our objective was to determine if intramammary infusions of IGF-I would stimulate mammogenesis in prepubertal heifers in vivo. After 7 d of treatment, bromodeoxyuridine was infused intravenously and heifers were slaughtered 3 h later. Samples from 3 regions of the mammary parenchyma were collected, fixed, sliced, and incubated with bromodeoxyuridine monoclonal antibody to identify cells in the S-phase of the cell cycle. Intramammary infusion of IGF-I increased the percentage of epithelial cells in the S-phase by 52% (6.4 vs. 4.2%, +/- 0.3%). Proliferation was similar in all 3 parenchymal regions, and the response to IGF-I was similar in each region. We conclude that local IGF-I increases proliferation of mammary parenchymal epithelial cells in prepubertal heifers.

Short communication: relationship between body growth and mammary development in dairy heifers.

Our objective was to determine if prepubertal rate of body weight (BW) gain, independent of diet, was related to mammary development of dairy heifers. Data from two studies recently conducted at Michigan State University were used to identify factors, within a dietary treatment group, that would account for variation in first lactation milk production or amount of mammary parenchymal DNA at the time of puberty. Factors analyzed for variation in milk production during first lactation were: postpartum BW, prepubertal BW gain, gestational BW gain, postpartum BW gain, body condition score (BCS) at breeding, and BCS at calving. Factors analyzed for variation in mammary parenchymal DNA at puberty were: BW at slaughter, age at puberty, prepubertal BW gain and body protein and body fat content at slaughter. For both analyses, prepubertal BW gain did not account for any of the variation in mammary development. The only significant covariate for the milk production model (r2 = 0.44) was BCS at breeding. Similarly, the only significant covariate in the parenchymal DNA model (r2 = 0.22) was body fat content at slaughter. These results suggest that, within a dietary treatment, heifers that grow faster do not have impaired mammary development, and increased body fatness may be a better predictor of impaired mammary development than BW gain.

Effect of dietary protein on prepubertal mammary development in rapidly growing dairy heifers.

The objective was to determine whether increased dietary protein would enhance mammary development in prepubertal heifers fed for rapid body growth (1.2 kg/d). Fifty-four Holstein heifers (weighing approximately 134 kg) were assigned to one of three treatments. Heifers were fed a total mixed ration with metabolizable energy at 2.85 Mcal/kg and metabolizable protein at low, standard, or high concentrations (37, 41, or 44 g/Mcal of metabolizable energy, respectively) from 3.5 mo of age until slaughter at approximately 46 d after puberty. Heifers fed low, standard, and high protein gained 1130, 1170, and 1180 g/d, respectively. Dietary protein did not affect age or weight of heifers at puberty or slaughter, withers height gain, or carcass composition. Average mammary parenchymal DNA content for heifers on diets of low, standard, and high protein was 595, 619, and 670 mg/100 kg of body weight, respectively, and was not significantly different. However, for heifers that attained puberty early, those fed low protein had 33% less parenchymal DNA than those fed high protein, even though their body growth and carcass composition were not compromised. We conclude that dietary protein does not have a major effect on mammary development of rapidly grown prepubertal heifers, provided the diet contains adequate protein for normal body growth. But we suggest that feeding low-protein diets increases the risk of impaired mammary development when heifers are fed for rapid growth and attain puberty early and that the new National Research Council guidelines for protein relative to energy seem adequate for optimal mammary development.

Evidence for a local effect of leptin in bovine mammary gland.

On average, high-energy diets promoting body growth rates above 1 kg/d before puberty impair mammary development by 15 to 20% in cattle. We hypothesized that leptin, a protein produced by adipocytes, mediates the inhibitory effect of high-energy diets on mammary development. Therefore, our objectives were to determine the effect of leptin on mammary epithelial cell proliferation, and the distribution of mRNA for two leptin receptor isoforms in prepubertal bovine mammary glands and other peripheral tissues. Addition of leptin to culture media containing either 5 ng/ml of insulin-like growth factor-I (IGF-I) or 1% fetal bovine serum decreased DNA synthesis of a bovine mammary epithelial cell line (MAC-T) in a dose-dependent manner. The minimal doses of leptin that decreased IGF-I- and fetal bovine serum-stimulated cell proliferation were 64 and 1 ng/ml, respectively. In addition, we determined that MAC-T cells and isolated bovine mammary epithelial cells express the long form of leptin receptor (Ob-Rb) mRNA. Ob-Rb mRNA was detected in all bovine tissues examined. In contrast with reports on other species, mRNA expression of the short form of leptin receptor (Ob-Ra) was detected only in bovine liver, pituitary body, and spleen. These results support the concept that leptin mediates the inhibitory effect of high-energy diets on mammary development.