Uneven milking intervals are adequate to achieve the benefits of increased milking frequency in early lactation.

Increasing milking frequency (MF) increases milk yield (MY) and farm profit, and optimal milking intervals (MI) prevent milk production decline. The objective of this experiment was to compare the MY effect of even and uneven 4 times daily (4×) MI in early lactation under increased MF. Fourteen multiparous and 6 primiparous cows were milked using unilateral frequent milking, with right udder halves milked 4× and left udder halves milked 2 times daily (2×) for 20 d in early lactation starting on d 5 postpartum. Ten (7 multiparous and 3 primiparous) cows were allocated evenly based on parity and assigned to either the even or the uneven MI groups distinguished by intervals of 9:3:9:3 h or 6:6:6:6 h. The left and right udder halves were milked at 0100 and 1300 h. The right udder glands were additionally milked at 0400 and 1600 h for the uneven MI group and at 0700 and 1900 h for the even MI group. Milk from each udder half was weighed and sampled for components on the final day of treatment and at 60, 120, 180, 240, and 300 d in milk. The overall effect of 4× milking on the right udder halves was a 5.96 ± 0.70 kg/d increase in MY on d 21 of unilateral frequent milking compared with the 2× udder halves. This elevated MY continued through 300 d in milk and averaged 1.56 ± 0.70 kg/d. Increased MF in early lactation increased the udder half difference in total yield throughout a 300-d lactation by 508 kg for milk, 25 kg for milk fat, and 15 kg for milk protein. Increased MF in early lactation increased milk component yields, but there were no differences between MI groups. The lack of treatment difference may be beneficial to farmers. The ability to achieve the same increased MY effect with uneven MI may optimize labor efficiency because early-lactation cows could be milked at the beginning and end of milking sessions. Farmers would not have to add additional milking sessions to achieve the enhanced MY response regardless of normal milking session length.

Technical note: The use of an accelerometer for measuring step activity and lying behaviors in dairy calves.

Calf behaviors such as step activity, lying bouts, and lying time may be an indicator of calf health and welfare. To reduce time-consuming visual observations, the use of behavioral monitoring systems have been developed to capture these data. Previous studies have validated lying behaviors using an accelerometer (HPG; HOBO Pendant G data logger, Onset Computer Corp., Bourne, MA) in calves. However, the HPG does not measure step activity. The objectives of this study were to (1) validate step activity, lying bouts, and lying time of AfiTag II (AT2; AfiTag II, Afimilk Ltd., Kibbutz Afikim, Israel) to observations from video, and (2) to compare the behavioral data from AT2 to the HPG. Calves (n=5) were group housed with an automatic calf feeder. Video cameras were installed at both sides of the pen, and observations were analyzed for 7h/calf. The AT2 and the HPG were both attached to the lateral side of the right rear leg of 5 calves, and data were recorded for 10 d. The full 10-d data set was used to examine correlations for lying bouts and lying time between AT2 and the HPG. The HPG was set at a 60-s sampling interval and the output was analyzed both unfiltered as well as utilizing a 1-min event filter to remove potentially erroneous readings. The AT2 recorded step activity, lying bouts, and lying time, and summarized these behaviors in 15-min periods. The AT2 recorded lying time in 3-min intervals, which were then automatically summarized in 15-min periods. The correlations of step activity, lying bouts, and lying time between video recordings and AT2 were 0.99. For the second objective, correlations between AT2 and the HPG were 0.99 for lying time and 0.93 for lying bouts. The 1-min event filter resulted in a 0.03 improvement in correlations for lying bouts between the HPG and AT2. The high correlation between video recordings and AT2 suggest that this device can be used to measure step activity, lying time, and lying bouts in unweaned dairy calves housed in groups.

Effect of feeding whole compared with cell-free colostrum on calf immune status: Vaccination response.

Vaccination contributes to improved herd health and production. Boosting immune development at a young age may have long-term effects by enhancing vaccine immune response and efficacy. In the bovine, colostrum is the sole source of maternal immunity, having a substantial effect on health status in the neonate. To date, colostral antibody concentration is used to evaluate colostrum quality. However, colostrum also contains proteins and cells, which may affect immune development and future responses to vaccines. To determine the effect of maternal colostral cells on immune development, 37 female Holstein and Jersey dairy calves were bottle-fed 4 quarts total of whole colostrum (WC) or cell-free colostrum (CFC) at birth. Calves were vaccinated with 2 series of multivalent vaccines. Series A consisted of vaccines given between 1 and 4mo of life. Series B consisted of vaccines given between 5 and 10mo of life. Calf peripheral blood samples were obtained before each vaccination series and monthly for 3mo after each vaccination series. Cellular blood parameters were determined by flow cytometry. Quantitative real-time PCR was used to determine cytokine gene expression in peripheral blood mononuclear cells before vaccination series B and once a month for 2mo after vaccination series B. Calves fed CFC had fewer numbers of B cells in mo 2 after vaccination series A when compared with WC-fed calves. Calves fed CFC had decreased gene expression levels of IL-2 in mo 1 and numbers of CD4(+)CD62L(+)CD45RO(-) and CD4(+)CD62L(+)CD45RO(+) T cells in mo 0 and 1 after vaccination series B as compared with WC-fed calves. Our findings indicate a greater response to vaccines up to 6 to 10mo post-WC feeding when compared with CFC. These data suggest that adoptive transfer of maternal colostral cells at birth has a long-term effect on development of the neonatal immune system.

Effect of feeding whole compared with cell-free colostrum on calf immune status: The neonatal period.

Mortality and decreased weight gain resulting from infection and disease in dairy calves are problems within the dairy industry. The bovine neonate relies solely on colostrum to acquire antibodies through passive transfer. To date, colostrum quality is determined by the concentration of antibodies. However, proteins and cells in the colostrum might also enhance immune development in the neonate. To determine the effect of maternal colostral immune cells on calf health and immune status, maternal colostrum was fed either fresh or after lysis of cells by flash-freezing in liquid nitrogen. Thirty-seven female Holstein and Jersey dairy calves were fed 4 quarts total of whole colostrum (WC) or cell-free colostrum (CFC) at birth. Respiratory and fecal scores were measured from birth to d 45 of life. Calf peripheral blood samples were obtained before and after feeding colostrum as well as on d 1, 3, 7, 14, 21, and 28 of life. Peripheral blood mononuclear cells were collected and analyzed for cellular parameters by flow cytometry. Total respiratory scores were greater in CFC-fed calves compared with WC-fed calves on d 38 of life. There were fewer CD4+ T cells and CD4+CD62L+CD45RO- T cells on d 1 and fewer CD4+CD62L+CD45RO+ T cells on d 1 and 3 in CFC-fed calves compared with WC-fed calves. Compared with WC-fed calves, CFC-fed calves had a greater percentage of CD4+CD62L-CD45RO+ T cells on d 0.25, 1, 3, and 7, and a greater percentage of monocytes on d 7. Our data suggest that colostral cells adoptively transfer and enhance neonatal immunity during the first month of life.

Short communication: Characteristics of student success in an undergraduate physiology and anatomy course.

Several factors affect the success of students in college classes. The objective of this research was to determine what factors affect success of undergraduate students in an anatomy and physiology class. Data were collected from 602 students enrolled in the Agriculture and Life Sciences (ALS) 2304 Animal Physiology and Anatomy course from 2005 through 2012. The data set included 476 females (79.1%) and 126 males (20.9%). Time to complete exams was recorded for each student. For statistical analyses, students' majors were animal and poultry sciences (APSC), agricultural sciences, biochemistry, biological sciences, dairy science, and "other," which combined all other majors. All analyses were completed using the GLIMMIX procedure of SAS (SAS Institute Inc., Cary, NC). Gender, major, matriculation year, major by year interaction, gender by year interaction, and time to complete the exam affected final course grade. The significant gender effect was manifested in the final grade percentage of 75.9 ± 0.4 for female students compared with 72.3 ± 0.6 for male students. Junior males had final course grades comparable with those of females, but sophomore and senior males had lower final course grades than other combinations. Biology majors had a final grade of 82.4 ± 0.6 and this grade was greater than all other majors. Students classified as "other" had a final score of 74.4 ± 0.8, which was greater than agricultural science majors (69.5 ± 0.9). The APSC grade (72.6 ± 0.5) was higher than the agricultural science majors. Junior students had significantly greater final grades (76.1 ± 0.5) than sophomores (73.3 ± 0.6) and seniors (72.9 ± 0.9). All biology students had greater final grades than all other majors, but biochemistry juniors had greater final course grades than APSC, agricultural science, and dairy science juniors. "Other" seniors had greater final course grades than agricultural science seniors. The regression for time to complete the exam was curvilinear and suggests that highest exam scores were at about 90-min completion time. It may be that some male students need better preparation for anatomy and physiology and their educational preparation should mimic that of female students more in terms of advance-placement biology in high school. These results suggest that biology majors might be better prepared for animal anatomy and physiology than other students.

Short communication: effect of trans-10,cis-12 conjugated linoleic acid on activation of lipogenic transcription factors in bovine mammary epithelial cells.

The objective of this study was to examine the effect of trans-10,cis-12 conjugated linoleic acid (t10c12CLA) on the activation of transcription factors that potentially regulate lipid synthesis in a bovine mammary epithelial cell line (MAC-T). Cells were transfected with luciferase reporter constructs containing sterol response element (SRE and SRE complex) for sterol regulatory element binding protein-1, peroxisome proliferator response element for peroxisome proliferator-activated receptor γ, or liver X receptor response element for liver X receptor. Different concentrations of t10c12CLA (0, 25, 50, 75, or 100µM) were applied to cells to determine the activation of transcription factors. The influence of t10c12CLA bond structure on transcription factor activation was also investigated by treating cells with different 18:1 fatty acid isomers (trans-10 18:1 or cis-12 18:1) at 100µM. Cells were harvested for luciferase assay after 24h of treatment. Compared with linoleic acid and cis-9,trans-11 CLA controls, the SRE reporters had significantly lower activity in t10c12CLA-treated cells at 50 and 75µM for SRE complex and SRE, respectively. Lower SRE and SRE complex activation was observed in t10c12CLA treatment at 25, 50, and 75µM compared with 0µM. The peroxisome proliferator response element and liver X receptor response element reporters did not respond differently between the t10c12CLA treatment and controls. Compared with t10c12CLA, both trans-10 18:1 and cis-12 18:1 increased the activities of SRE and SRE complex reporters by 1.3- to 4.2-fold. In conclusion, t10c12CLA has an inhibitory role in lipogenic transcription factor activation of SRE, and this negative effect is due to the conjugation of trans-10 and cis-12 double bonds in the fatty acid. Furthermore, we found no support for a regulatory role of response elements for peroxisome proliferator-activated receptor γ or liver X receptor in the t10c12CLA inhibition of mammary lipid synthesis.

Isoleucine, leucine, methionine, and threonine effects on mammalian target of rapamycin signaling in mammary tissue.

Improved representation of postabsorptive N metabolism in lactating dairy cows requires a better understanding of protein synthesis regulation in the mammary glands. This study aimed to determine the quantitative effects of Ile, Leu, Met, and Thr on the phosphorylation state of signaling proteins that regulate protein synthesis. The experiment used a composite design with a central point, 2 axial points per AA, and a complete 2(4) factorial. All of the other AA were provided at the concentrations in Dulbecco's modified Eagle's medium. The experiment was replicated with tissues from 5 lactating cows. Mammary tissue slices (0.12 ± 0.02 g) were incubated for 4h. Total and site-specific phosphorylated mammalian target of rapamycin (mTOR; Ser2448), eukaryotic elongation factor (eEF) 2 (Thr56), ribosomal protein S6 (Ser235/236), and eukaryotic initiation factor 2α (Ser51) were determined by western immunoblotting. Tissue concentrations of the 4 AA studied responded linearly to media supply. Addition of Ile, Leu, Met, or Thr had no effect on eukaryotic initiation factor 2α phosphorylation. Isoleucine and Thr positively affected mTOR phosphorylation. However, the 2 AA had an antagonistic relationship. Similarly, Ile linearly increased ribosomal protein S6 phosphorylation, and Thr inhibited the Ile effect. In addition, eEF2 phosphorylation was linearly decreased by Ile and Leu. Threonine curvilinearly decreased eEF2 phosphorylation, Ile and Leu negatively interacted on eEF2, and Thr tended to inhibit Leu effects on eEF2. This work demonstrated saturable responses and interactions between AA on activation of the mTOR pathway. Incorporation of these concepts into milk protein response models will help to improve milk and milk protein yield predictions and increase postabsorptive N efficiency and reduce N excretion by dairy cows.

Casein synthesis is independently and additively related to individual essential amino acid supply.

Specific AA affect rates of milk protein synthesis in the mammary glands of lactating cows. The objective of this study was to quantify the rate of αS1-casein synthesis in response to Ile, Leu, Met, and Thr supplementation, and to test the single-limiting AA theory for milk protein synthesis by exploring interactions among these AA. Effects of Ile, Leu, Met, and Thr were studied in vitro with a composite design containing a central point repeated 4 times, with 2 axial points per AA and a complete 2(4) factorial. Other AA were at the concentration in Dulbecco's modified Eagle medium/F12 medium (DMEM). The experiment was replicated with mammary tissue from 5 lactating cows. Mammary tissue slices (0.12 ± 0.02 g) were incubated for 4h at 37°C in 5 mL of treatment medium containing (2)H5-Phe. Caseins were precipitated from cell homogenate supernatants. Enrichment with (2)H5-Phe of the N[34]LLRFFVAPFPE αS1 peptide was determined by matrix-assisted laser desorption/ionization-tandem time-of-flight (MALDI-TOF-TOF), which was used to determine enrichment of Phe in the transfer (t)RNA pool and αS1-casein fractional synthesis rates (CFSR). Data were analyzed with a polynomial mixed model containing linear, quadratic, and 2-factor interactions for Ile, Leu, Met, and Thr, and cow and residual as random factors. Interactions were not significant at P<0.1 and were removed from the model. Increasing concentrations of Ile, Leu, Met, and Thr simultaneously increased CFSR curvilinearly with a predicted maximum response of 4.32 ± 0.84%/h at 63% of DMEM concentrations. The maximum response to each of the 4 AA was at 71, 49, 60, and 32% of the concentration in DMEM, for Ile, Leu, Met, and Thr, respectively. These values correspond to 270, 120, 440, and 140% the plasma concentrations of Ile, Leu, Met, and Thr observed in lactating cows fed to meet National Research Council requirements, respectively. The CFSR estimated at those maxima were similar among AA (3.6 ± 0.6%/h). Individual AA effects on CFSR did not correlate with mammalian target of rapamycin (mTOR) signaling. Independent responses of CFSR to individual essential AA observed in this study contradict the single-limiting AA theory assumed in current requirement systems. The saturable responses in CFSR to these 4 AA also highlight the inadequacy of using a fixed postabsorptive AA efficiency approach for determining AA requirements for milk protein synthesis.

Phenotypic responses of chickens to long-term, bidirectional selection for juvenile body weight--historical perspective.

A long-term selection experiment for high (HWS) and low (LWS) BW at 8 wk of age (BW8) was conducted in White Plymouth Rock chickens. Over 54 generations of selection, responses to bidirectional selection were profound. Increase in BW8 in line HWS was linear, and there was a significant quadratic response in line LWS for BW at both 4 and 8 wk of age. Although there is no indication that line HWS has come close to approaching a selection limit in more than 50 generations, selection limits occurred in line LWS chickens at generation 48 for females and generation 50 for males. Evidence also exists that one or more beneficial mutations have occurred in line HWS, aiding in progressive increases in BW8 over generations. Analyses of ratios of BW at 4 wk of age with those at 8 wk of age (ratio 4/8) revealed that LWS females grew proportionately faster through 4 wk of age than LWS males or HWS chickens. Comparisons of the selected lines with contemporary lines in which selection had been relaxed (discontinued) indicated that, in line HWS, the relaxed lines generally regressed toward original (preselection) values, suggesting that the linear response to single-trait selection was at least partially due to continued genetic variance. In LWS chickens, a series of plateaus in selection response occurred, but relaxed contemporary lines still regressed toward preselection values for BW8. In spite of the length of this selection experiment (54 generations), genetic variance and beneficial mutations have allowed continued, linear response to selection for increased BW8. Response to selection for decreased BW8 has been tempered by physiological barriers that have decreased survival of young chicks or the ability of females to reproduce. These findings are discussed in a historical perspective.

Hormones, metabolites, and reproduction in Holsteins, Jerseys, and their crosses.

Holsteins (HH), Jerseys (JJ), and their crosses in first (n=157) and second (n=107) lactation were used to determine if reproduction, progesterone (P4), insulin-like growth factor 1 (IGF-1), insulin, nonesterified fatty acids (NEFA), and milk production differed between genetic groups. Thirty-four cows were Holstein-Jersey (HJ) crosses, 46 were Jersey-Holstein (JH) crosses, 48 were purebred Holsteins (HH), and 29 were purebred Jerseys (JJ) in first lactation, whereas the second-lactation animals included 23 HJ, 35 JH, 35 HH, and 14 JJ. Blood samples were collected weekly for the first 10 wk postpartum. Analyses were conducted using the MIXED, chi-square, and GLIMMIX procedures (SAS Institute Inc., Cary, NC). Seasons of calving were cold (November to May) and hot (June to October) and were combined with year to form 8 year-seasons. Days open and number of services were affected by genetic group. The HH were open 169±8 d, which was greater than HJ (143±9 d), JJ (132±10 d), and JH (127±8 d). The HH had 2.4±0.1 services per pregnancy, which was greater than JH (1.9±0.1), but not different from HJ (2.1±0.2) or JJ (2.1±0.2). Concentrations of NEFA were greater in lactation 2 (0.52±0.02 mEq/L) than in lactation 1 (0.45±0.02 mEq/L) and decreased over the 10-wk period. Concentrations of NEFA were greater in the cold season except in yr 3. Insulin in lactation 1 (0.81±0.03 ng/mL) was greater than in lactation 2 (0.72±0.03 ng/mL); insulin decreased to wk 2 then gradually increased. The HJ had the greatest insulin concentrations (0.87±0.04 ng/mL) and the JJ had the lowest (0.66±0.04 ng/mL), and IGF-1 gradually increased over the 10-wk period. Milk production (actual yield in the first 305 d, not adjusted for fat and protein) was affected by genetic group, lactation number, year-season, and wk 1 insulin. The HH produced 10,348±207 kg of milk, which was greater than the HJ (9,129±230 kg), the JH (9,384±190 kg), and the JJ (7,080±240 kg). Milk production in lactation 2 (9,676±163 kg) was greater than that in lactation 1 (8,294±160 kg). The JJ (10.3±4.7%) had the highest frequency of mastitis. The chance of getting mastitis for HH (1.1±0.9%) differed from that for HJ (9.4±4.1%), JH (8.1±3.4%), and JJ (10.3±4.7%). Genetic group affected hormones and metabolites, which may partially explain differences in reproductive measures and milk yield.

The effects of experimentally induced Escherichia coli mastitis and flunixin meglumine administration on activity measures, feed intake, and milk parameters.

The use of flunixin meglumine (FM), a nonsteroidal antiinflammatory drug, during experimentally induced Escherichia coli mastitis was evaluated. Twenty-four primiparous and multiparous lactating dairy cows were challenged with 1×10(2) cfu of E. coli 727 in 1 uninfected quarter. Of the 24 E. coli-challenged animals, 12 were administered FM [ECF; 100mg (2 cc)/45.5 kg of body weight) at the onset of clinical mastitis signs. The remaining 12 challenged cows were untreated (EC). An additional 11 cows were infused with 1 mL of sterile phosphate-buffered saline and served as the nonchallenged control (CTL) group. Activity measures, dry matter intake (DMI), milk production, milk bacterial counts from challenged mammary glands, and somatic cell score (SCS) were collected on all animals. Activity measurements were collected using both a behavior-monitoring system and data loggers. Activity was summarized by day (behavior-monitoring system) and in 3-h time periods (data loggers). An examination of animal activity indicated that EC and ECF cows stood more and lay less as compared with the CTL animals in the first 6h after FM administration. When DMI was analyzed, CTL and ECF animals had greater DMI than the EC animals on d 1 postchallenge. However, by d 2 postchallenge, DMI for ECF and EC cows was significantly less than for the CTL cows. The ECF cows had greater milk yield than did EC animals by d 3 and 4 postchallenge, and no significant difference in yield was observed between the ECF and CTL animals. No differences in SCS were observed between the parity groups. Yet, bacterial counts in milk were greater in multiparous animals compared with the primiparous cows. Therefore, it can be concluded that E. coli mastitis does alter animal activity and may have a negative effect on animal well-being. However, the improvement in DMI and milk production for ECF animals provides evidence for using a nonsteroidal antiinflammatory drug as supportive therapy in alleviating the adverse effects associated with E. coli mastitis.

Cow and herd variation in milk urea nitrogen concentrations in lactating dairy cattle.

Milk urea nitrogen (MUN) is correlated with N balance, N intake, and dietary N content, and thus is a good indicator of proper feeding management with respect to protein. It is commonly used to monitor feeding programs to achieve environmental goals; however, genetic diversity also exists among cows. It was hypothesized that phenotypic diversity among cows could bias feed management decisions when monitoring tools do not consider genetic diversity associated with MUN. The objective of the work was to evaluate the effect of cow and herd variation on MUN. Data from 2 previously published research trials and a field trial were subjected to multivariate regression analyses using a mixed model. Analyses of the research trial data showed that MUN concentrations could be predicted equally well from diet composition, milk yield, and milk components regardless of whether dry matter intake was included in the regression model. This indicated that cow and herd variation could be accurately estimated from field trial data when feed intake was not known. Milk urea N was correlated with dietary protein and neutral detergent fiber content, milk yield, milk protein content, and days in milk for both data sets. Cow was a highly significant determinant of MUN regardless of the data set used, and herd trended to significance for the field trial data. When all other variables were held constant, a percentage unit change in dietary protein concentration resulted in a 1.1mg/dL change in MUN. Least squares means estimates of MUN concentrations across herds ranged from a low of 13.6 mg/dL to a high of 17.3 mg/dL. If the observed MUN for the high herd were caused solely by high crude protein feeding, then the herd would have to reduce dietary protein to a concentration of 12.8% of dry matter to achieve a MUN concentration of 12 mg/dL, likely resulting in lost milk production. If the observed phenotypic variation is due to genetic differences among cows, genetic choices could result in herds that exceed target values for MUN when adhering to best management practices, which is consistent with the trend for differences in MUN among herds.

Reproductive soundness is higher in chickens selected for low as compared with high antibody response.

White Leghorn chickens were selected for 36 generations for high (HAS) or low (LAS) antibody response to SRBC 5 d after an intravenous challenge. The aim of this study was to investigate possible changes in reproductive soundness resulting from that selection. Age and BW at onset of lay (first egg), along with weight of the first egg, were recorded on 45 hens from each line. Intensity of lay was measured as the number of ovulations within a 15-d period over 15 sequential intervals (total 225 d). Three cycles of fertility also were assessed, coinciding with early, middle, and late production stages. For fertility of males and females within a line to be independently evaluated, roosters and hens were mated by artificial insemination to an unrelated control line of White Plymouth Rocks. Twenty roosters from each antibody line were considered, as well as the 45 hens. Pooled semen from the control line was used for mating the hens from the antibody lines. Hens from the LAS line commenced lay at a younger age (11.67±3.53 d; P<0.001), lighter BW (-169.46±40.20 g; P<0.001), and with greater intensity (2.68±0.25%; P=0.001) than those from the HAS line. Any differences in intensity thereafter were trivial between lines (P=0.42), with intensity decreasing sharply toward the end of the 7-mo production period in both lines. Length of fertility differed between hens of the antibody lines during the first cycle (3.35±0.85 d; P=0.002) and between roosters during the first (3.58±1.06 d; P=0.02) and second (3.38±1.07 d; P=0.03) cycles, with chickens from the LAS line having the longer length of fertility in both sexes. A correlated response in reproductive soundness to divergent selection for antibody response was observed. This may in part be due to differences in resource allocations, with particular impact on duration of fertility.

Influence of cysteine in conjunction with growth factors on the development of in vitro-produced bovine embryos.

Cysteine supplementation to in vitro maturation (IVM) media of bovine oocytes increases cellular glutathione production. Beneficial effects of growth factors for improving the rate of blastocyst development have been reported, but combined effects are unknown. This study was conducted to determine the additive effect of cysteine with epidermal growth factor (EGF) and/or insulin-like growth factor-I (IGF-I) on embryo development. Bovine oocytes from slaughterhouse ovaries were matured in TCM-199 (control), with or without the addition of 0.6 mm cysteine (C) at 0 or 12 h of maturation. After in vitro fertilization, embryos were allocated to culture treatments containing synthetic oviductal fluid medium. Culture treatments included fetal calf serum (FCS, 4%) alone; IGF-I (100 ng/ml); EGF (10 ng/ml); and IGF-I + EGF (100 + 10 ng/ml). Although rates for blastocysts development were not different among treatments, an increased proportion of embryos attaining morula formation was achieved when cysteine was added to the maturation media (12 h C IGF-I + EGF, 41.4%; 0 h C EGF, 40.0%) as compared to control (FCS: 34.6%). When cysteine treatments were combined, percent cleavage was greater for IGF-I + EGF (70.8%) compared to FCS (61.2%). The abundance of mRNA from the apoptotic genes, Bax and Bcl-2, and the oxidative stress genes, copper (Cu)-zinc (Zn) superoxide dismutase (SOD) and manganese (Mn) SOD in embryos was assessed. No treatment effect was observed on the expression of these genes. In conclusion, supplementation of cysteine during IVM of oocytes, in conjunction with growth factors could effectively be used as a replacement for FCS.

Interactions of energy and predicted metabolizable protein in determining nitrogen efficiency in the lactating dairy cow.

Lactating cows are relatively inefficient in converting dietary N to milk N compared with the efficiency of N use for growth in simple-stomached animals. The majority of productive N losses occur in the postabsorptive system. The aim of the study was to test whether predicted metabolizable protein (MP) and dietary energy exerted independent effects on milk protein synthesis and postabsorptive N efficiency. If true, postabsorptive N efficiency would be expected to be greater when animals are fed high-energy diets. Forty mid-lactation cows (32 multiparous Holstein and 8 primiparous Holstein x Jersey crossbreds) were used in a complete randomized design with a 2 x 2 factorial arrangement of diets. Cows were assigned to 1 of 4 dietary treatments: high-energy, high-protein (HE/HP); high-energy, low-protein (HE/LP); low-energy, high-protein (LE/HP); and low-energy, low-protein (LE/LP). Energy concentrations were 1.55 (HE/HP and HE/LP) or 1.44 (LE/HP and LE/LP) Mcal of net energy for lactation (NE(L))/kg of dry matter (DM). Changes in predicted MP were achieved by feeding diets with 6.6 (HE/HP and LE/HP) or 4.6% (HE/LP and LE/LP) ruminally undegradable protein (DM basis). Ruminally degradable protein was held constant at 10.1% of DM. All cows were fed the HE/HP diet from d 1 to 21 followed by the respective treatments from d 22 to 43 (n=10). Milk protein yield was reduced as dietary energy was reduced. Milk yield followed a similar pattern as milk protein yield. There was a trend for decreased milk yield as crude protein was reduced. There were no interactions between dietary energy and protein for either milk or protein yield. Plasma amino acid concentrations were not affected by treatment. Milk urea N was affected by energy and protein with a significant interaction (HE/HP=17.2, HE/LP=12.2, LE/HP=21.0, LE/LP=12.2 mg/dL). Nitrogen efficiency calculated from predicted MP supply was affected by energy and protein supplies with no apparent interaction and ranged from a low of 31% (LE/HP) to a high of 43% (HE/LP). The National Research Council model would predict N efficiency more accurately if a representation of the effects of energy on N efficiency were included in the postabsorptive system.

Effective nitrogen preservation during urine collection from Holstein heifers fed diets with high or low protein content.

Six Holstein heifers (body weight=535-625 kg) fed a total mixed ration containing either high protein (13.4%) or low protein (9.0%) were used to evaluate the effect of 3 urine collection methods (chilled, acidified before collection, or acidified after 6h of collection) on urinary N preservation. In a 2-period crossover design, 16-d diet adjustment stages preceded five 24-h collections. Urinary catheters were inserted 1 d before the collection periods. Urine collection tubes were configured to split urine to 3 collection containers: 1 acidified with 6 N HCl before collection at a rate calculated to reduce pH to below 2, 1 acidified every 6h during collection to pH below 2, and 1 located in a large cooler of ice. Collection method did not affect urinary concentration of N or urine urea-N (9.2+/-0.9 g/L and 6.58+/-0.9 g/L, respectively) or urinary excretion of N or urea-N (82+/-3.8 g/d and 59.5+/-3.8 g/d, respectively). These 3 collection methods are equally effective in preserving N during urine collection, but the "chilled immediately" approach may be useful for studies focused on ammonia emission. Urinary and fecal N excretion were significantly different across collection days; fecal N was more highly variable than urinary N. Intake and apparent N digestibility decreased during the collection week, and excretion of urinary and fecal N increased, particularly on d 5. (Stable rectal temperatures suggested no urinary infections.) Improvements in total collection methodology will support continued progress in the understanding of livestock N utilization and post-excretion changes in manure N.

Regulation of protein synthesis in mammary glands of lactating dairy cows by starch and amino acids.

The objective of this study was to evaluate local molecular adaptations proposed to regulate protein synthesis in the mammary glands. It was hypothesized that AA and energy-yielding substrates independently regulate AA metabolism and protein synthesis in mammary glands by a combination of systemic and local mechanisms. Six primiparous mid-lactation Holstein cows with ruminal cannulas were randomly assigned to 4 treatment sequences in a replicated incomplete 4 x 4 Latin square design experiment. Treatments were abomasal infusions of casein and starch in a 2 x 2 factorial arrangement. All animals received the same basal diet (17.6% crude protein and 6.61 MJ of net energy for lactation/kg of DM) throughout the study. Cows were restricted to 70% of ad libitum intake and abomasally infused for 36 h with water, casein (0.86 kg/d), starch (2 kg/d), or a combination (2 kg/d starch+0.86 kg/d casein) using peristaltic pumps. Milk yields and composition were assessed throughout the study. Arterial and venous plasma samples were collected every 20 min during the last 8h of infusion to assess mammary uptake. Mammary biopsy samples were collected at the end of each infusion and assessed for the phosphorylation state of selected intracellular signaling molecules that regulate protein synthesis. Animals infused with casein had increased arterial concentrations of AA, increased mammary extraction of AA from plasma, either no change or a trend for reduced mammary AA clearance rates, and no change in milk protein yield. Animals infused with starch had increased milk and milk protein yields, increased mammary plasma flow, reduced arterial concentrations of AA, and increased mammary clearance rates and net uptake of some AA. Infusions of starch increased plasma concentrations of glucose, insulin, and insulin-like growth factor-I. Starch infusions increased phosphorylation of ribosomal protein S6 and endothelial nitric oxide synthase, consistent with changes in milk protein yields and plasma flow, respectively. Phosphorylation of the mammalian target of rapamycin was increased in response to starch only when casein was also infused. Thus, cell signaling molecules involved in the regulation of protein synthesis differentially responded to these nutritional stimuli. The hypothesized independent effects of casein and starch on animal metabolism and cell signaling were not observed, presumably because of the lack of a milk protein response to infused casein.

Effects of milk replacer formulation on measures of mammary growth and composition in Holstein heifers.

Overfeeding prepubertal heifers may impair mammary parenchymal growth and reduce milk production, but evidence suggests that increased intake of a high-protein milk replacer before weaning may be beneficial. This study was designed to evaluate effects of milk replacer (MR) composition on mass and composition of mammary parenchyma and fat pad, growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis gene expression, and putative mammary epithelial stem cells. Specifically, we hypothesized that positive effects of faster rates of gain during the preweaning period alter the development, persistence, or activity of populations of putative mammary epithelial stem cells, possibly through involvement of GH/IGF-I axis molecules. Twenty-four newborn heifers were fed 1 of 4 MR diets (n = 6/diet): control [20% crude protein (CP), 21% fat MR fed at 441 g of dry matter (DM)/d], high protein, low fat (28% CP, 20% fat MR fed at 951 g of DM/d), high protein, high fat (27% CP, 28% fat MR fed at 951 g of DM/d), and high protein, high fat+ (27% CP, 28% fat MR fed at 1,431 g of DM/d). Water and starter (20% CP, 1.43% fat) were offered ad libitum. Animals were killed on d 65 and mammary tissue was subjected to biochemical, molecular, and histological examination. No differences in mammary parenchymal mass or composition, with or without adjusting for empty body weight, were detected. Mass was increased and composition of the mammary fat pad was altered by nutrient intake. No diet differences in putative mammary epithelial stem cell abundance or abundance of transcripts for genes of the GH/IGF-I axis were detected. In this study, growth of the mammary epithelium, size of the mammary epithelial stem cell population, and components of the GH/IGF-I axis did not depend on diet. However, an underlying positive correlation between telomerase, a marker of mammary stem cells, and growth of the mammary parenchyma was detected. Implications of diet-induced effects on mammary fat pad and possible effects on subsequent development and function remain to be determined.

Effects of body weight and nutrition on histological mammary development in Holstein heifers.

Our objective was to determine the effects of rate of gain and body weight (BW) on development of the mammary parenchyma. Mammary tissue samples were collected from heifers (n = 72) reared on 1 of 2 dietary treatments (restricted, 650 g/d of daily gain; or elevated, 950 g/d of daily gain) and slaughtered at 100, 150, 200, 250, 300, or 350 kg of BW. Mammary samples were excised, preserved, prepared for histology, and stained with hematoxylin and eosin. Digital images of tissue sections were captured for analysis. Tissue areas occupied by the interlobular and intralobular stroma, epithelium, and lumen were measured (mum(2)). The numbers of epithelial and luminal structures per image were tabulated to measure the complexity of ductal development. Mean percentages of mammary parenchyma occupied by the interlobular stroma, epithelium, lumen, and intralobular stroma were 29, 20, 7, and 43%, respectively. Percentage of area occupied by the intralobular stroma was affected by BW and was lower for 100-kg heifers compared with heifers 200 kg and heavier (33 +/- 4 vs. 46 +/- 4), but the percentage of area occupied by other tissue elements did not differ by BW or treatment, nor was there an interaction. However, the numbers of both epithelial (8.3 +/- 4 vs. 47 +/- 4) and luminal-containing (6 +/- 4 vs. 38 +/- 4) structures per image increased markedly between 100 and 350 kg of BW, irrespective of diet. For heifers slaughtered between 100 and 350 kg of BW, alterations in the rate of gain between 650 and 950 g/d, accomplished by feeding varying amounts of the same diet, had no significant effect on tissue characteristics or the pattern of mammary parenchymal development. These data emphasize the importance of BW and age in determining developmental characteristics of the heifer mammary parenchyma and suggest that the rate of gain per se has a minimal impact on histological development, and thus do not support the hypothesis that rate of gain has a direct negative impact on ductal development.

Dietary calcium has little effect on mineral balance and bone mineral metabolism through twenty weeks of lactation in Holstein cows.

Calcium and P balance and mobilization from bone were evaluated through 20 wk of lactation to determine the timing and extent of net resorption of bone mineral and mineral balance in lactating dairy cows. Eighteen Holstein cows were blocked by parity and calving date and randomly assigned to 1 of 3 dietary treatments: high (1.03%, HI), medium (0.78%, MED), or low (0.52%, LOW) dietary Ca. Dietary P was 0.34% in all diets. Cows consumed treatment diets from calving to 140 DIM. Total collection of milk, urine, and feces was conducted 2 wk before expected calving and in wk 2, 5, 8, 11, and 20 of lactation. Blood samples were collected at 14 and 10 d before expected calving and 0, 1, 3, 5, 10, 14, 21, 28, 35, 56, 70, 84, 98, and 140 d after calving. Blood samples were analyzed for Ca, P, and parathyroid hormone concentration. Serum concentrations of osteocalcin (OC), a marker of bone formation, and deoxypyridinoline (DPD), a marker of bone resorption, were measured to assess bone mobilization. Rib bone biopsies were conducted within 10 d postcalving and during wk 11 and 20 of lactation. Dietary Ca concentration affected Ca balance, with cows consuming the HI Ca diet in positive Ca balance for all weeks with the exception of wk 11. Interestingly, all cows across all treatments had a negative Ca balance at wk 11, possibly the result of timed estrous synchronization that occurred during wk 11. At wk 20, Ca balances were 61.2, 29.9, and 8.1 g/d for the HI, MED, and LOW diets, respectively. Phosphorus balances across all treatments and weeks were negative. Bone Ca content on a fat-free ash weight basis was least in cows consuming the MED diet, but bone P was not different. Serum Ca and P were not affected by treatment. Dietary Ca concentration did not affect P balance in the weeks examined, but there was a clear effect of parity on balance, markers of bone metabolism, and bone P. Primiparous cows had greater serum OC and DPD concentrations than multiparous cows. Regardless of dietary treatment, serum OC concentration peaked around d 35 of lactation. Simultaneously, DPD concentration began to decrease, which may indicate a switch from net bone resorption to formation after d 35. However, this was not reflected in balance measures. This information may help refine dietary mineral recommendations for lactating dairy cows and suggests that dietary P requirements are independent of dietary Ca.