Expression of enzymes involved in the urea cycle and muscle and mammary gland development of Holstein × Gyr heifers in a rotational grazing system supplemented with increasing protein levels.

Studies evaluating the crude protein (CP) supplementation strategies across the year for grazing cattle and its association with the enzymes involved in the urea cycle and muscle and mammary gland developments are scarce. Thus, we aimed to evaluate the effect of supplementation with different levels of CP on the expression of genes involved in the urea cycle and muscle and mammary gland development of Holstein × Gyr crossbreed heifers grazing intensively managed Brachiaria decumbens throughout the year. Thirty-eight heifers with average initial BW of 172.5 ± 11.15 kg (mean ± SE) and 8.2 ± 0.54 mo of age were randomly assigned to 1 of 4 treatments: 3 protein supplements (SUP) fed at 5g/kg of body weight, plus a control group (CON, non-supplemented animals). The supplement CP levels evaluated were: 12, 24, and 36%. The study was divided into 4 seasons: rainy, dry, rainy-dry transition (RDT), and dry-rainy transition (DRT). On the penultimate day of each season, ultrasound images of the carcass and mammary gland were taken. Five animals from each treatment were randomly chosen on the last day of each season, and liver and muscle tissue biopsies were performed. The target genes were the mammalian target of rapamycin (mTOR) and adenosine monophosphate-activated protein kinase (AMPK) in the muscle samples. Carbamoyl phosphate synthetase (CPS), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS), arginosuccinate lyase (ASL), and arginase (ARG) were evaluated in the liver samples. Data were analyzed using PROC GLIMMIX of the SAS with repeated measures. We observed a greater rib eye area (cm2) and fat thickness (mm) in SUP animals than in non-supplemented animals. However, we did not observe differences among SUP levels for both variables. No effects of supplementation were detected on mammary gland development. Nevertheless, seasonal effects were observed, where the RDT and dry season had the most and least accumulated fat in the mammary gland. In muscle, we observed greater expression of AMPK in non-supplemented animals than SUP animals. On the other hand, no differences were observed in gene expression between SUP and non-supplemented animals and among SUP animals for mTOR. Season affected both AMPK and mTOR; heifers had a greater AMPK gene expression on rainy than RDT. For mTOR, we observed greater gene expression in RDT and DRT than in rainy. No differences were observed among RDT, dry, and DRT, and between dry and rainy seasons for mTOR. We observed greater CPS, ASL, and ARG gene expression in SUP animals than in non-supplemented animals. Among SUP animals, supplement CP linearly affected CPS. In conclusion, the supplementation strategy did not affect mammary gland development and mTOR expression in muscle tissue. However, we observed a seasonal effect on mammary gland development and AMPK and mTOR expression. The CP supplementation increased the rib eye area and fat thickness, directly affecting AMPK expression in the muscle. Moreover, the CP supplementation increased urea cycle enzyme expression, indicating greater urea production in the liver.

Protein requirements for pregnant dairy cows.

We aimed to estimate the protein requirements of pregnant Holstein × Gyr cows. A total of 61 Holstein × Gyr (HG) cows were used, with an average initial body weight (BW) of 480 ± 10.1 kg and age 5 ± 0.5 yr. Cows were divided into 3 groups: pregnant (n = 43), nonpregnant (n = 12), and baseline (n = 6). Baseline animals were slaughtered before starting the experiment to estimate the initial body composition of the remaining animals. Pregnant and nonpregnant cows received 2 diets: maintenance and ad libitum. Pregnant cows were slaughtered at 139, 199, 241, and 268 gestation days (GD). First, we used data only from nonpregnant cows to determine the requirements for maintenance and growth in adult cows. The requirements of metabolizable protein for maintenance (MPm; grams of empty metabolic BW [EBW0.75] per day) were estimated using a linear regression between the metabolizable protein intake (MPI, g/d) and average daily gain (g/d), and the MPm was defined as the intercept divided by the average metabolic BW. Net protein requirements for gain (NPg; g/d) were estimated by the first derivative of the allometric equation between final crude protein in the body (kg) and the final empty BW (EBW; kg). The efficiency of use of metabolizable protein for gain (k) was calculated from the regression between the retained protein (g EBW0.75/d) and the MPI (g EBW0.75/d), and k was the slope of this regression. The MPI was estimated by combining microbial protein synthesis (purine derivatives) with the digestible rumen undegradable protein [(total protein intake - rumen-degradable protein) × intestinal digestibility]. Second, an exponential model was used to fit the protein accumulation in the gestational components in the function of GD. The first derivative of that model was considered the net requirement for gestation (NPgest). The efficiency of protein utilization for gestation (kgest) was calculated by the iterative method using the equation Δ = MPI - (MPm + NPg/kg + NPgest/kgest), where kg is efficiency of protein utilization for gain. The iteration was performed aiming at a zero deviation between observed MPI and metabolizable protein (MP) estimated by the requirements determined herein. We obtained a value of 3.88 g EBW0.75/d for MPm. The estimation of NPg can be calculated according to the following equation: NPg = 0.716 × (EBWopen-0.308) × EBGc, where EBWopen is the EBW (kg) for nonpregnant animals and EBGc is the empty body gain (kg/d) corrected for the gestational component. The k was determined as 0.347. The NPgest requirements were determined as NPgest (g/d) = 0.0008722 × exp(0.01784 × GD) × (calf weight/35). The kgest was 0.625. It is important to highlight that different methods of MP estimates should not be mixed and that the proposed method requires the estimation of microbial protein (estimated via urinary estimates), which might limit practical application. In conclusion, new studies should be conducted to validate our results and the methodology adopted to determine protein requirements for pregnancy in dairy cows. Due to the pattern of protein accumulation in the gestational components, we suggest an exponential model to describe protein requirements for pregnancy for dairy cows.

Association of housing and management practices with milk yield, milk composition, and fatty acid profile, predicted using Fourier transform mid-infrared spectroscopy, in farms with automated milking systems.

Milk fatty acid (FA) profile can be divided into (1) de novo (C4-C14) that are synthesized in the mammary gland; (2) preformed (≥C18) that are absorbed from blood and originate from mobilized adipose tissues or dietary fat; and (3) mixed (C16), which have both origins. Our objectives were to describe the FA profile, as predicted using Fourier transform mid-infrared spectroscopy, of bulk tank milk from automated milking system (AMS) farms and to assess the association of management and housing factors with the bulk tank milk composition and FA profile of those AMS farms. The data used were collected from 124 commercial Canadian Holstein dairy farms with AMS, located in the provinces of Ontario (n = 68) and Quebec (n = 56). The farms were visited once from April to September 2019, and information were collected on barn design and herd management practices. Information regarding individual cow milk yield (kg/d), days in milk, parity, and the number of milking cows were automatically collected by the AMS units on each farm. These data were extracted for the entire period that the bulk tank milk samples were monitored, from April 2019 to April 2020 in Quebec and from August 2019 to May 2020 in Ontario. Across herds, milk yield averaged (mean ± standard error) 35.9 ± 0.4 kg/d, with 3.97 ± 0.01% fat and 3.09 ± 0.01% protein, whereas FA profile averaged 26.2 ± 0.1, 33.1 ± 0.1, and 40.7 ± 0.2 g/100 g of FA for de novo, mixed, and preformed, respectively. The FA yield averaged 0.34 ± 0.01, 0.44 ± 0.01, and 0.54 ± 0.01 kg/d for de novo, mixed, and preformed, respectively. Multivariable regression models were used to associate herd-level housing factors and management practices with milk production, composition, and FA profile. Milk yield was positively associated with using a robot feed pusher (+2.1 kg/d) and the use of deep bedding (+2.6 kg/d). The use of a robot feed pusher, deep bedding, and greater stall raking frequency were positively associated with greater yield (kg/d) of de novo, mixed, preformed, and de novo + mixed FA. Use of deep bedding was negatively associated with concentration of fat, de novo FA, mixed FA, and de novo + mixed FA, expressed in grams per 100 g (%) of milk. A wider lying alley width (≥305 cm) was associated with a greater concentration (g/100 g of milk) of de novo and de novo + mixed FA. Greater frequency of partial mixed ration delivery (>2×/d vs. 1 and 2×/d) was positively associated with a greater proportion (g/100 g of FA) of de novo, mixed, and de novo + mixed FA and negatively associated with the proportion of preformed FA. Overall, these associations indicated that bulk tank FA profile can be used as a tool to monitor and adjust management and housing in AMS farms.

Short communication: Development and evaluation of equations to predict growth of Holstein dairy heifers in a tropical climate.

The objective of this study was to develop and validate equations for estimating growth of dairy heifers using measures of withers height, body weight (BW), and age. Measures of BW and withers height of 207 Holstein heifers raised in a tropical climate were taken from birth to calving, totaling to 2,047 observations. To be included in the database, the heifer had to have at least 4 measures recorded. After that, 4 models were built and evaluated as follows: (1) a linear model of BW as function of age (BW~A), (2) a linear model of the BW-to-height ratio as function of age (BW:H~A), (3) a quadratic model, adjusted for a defined plateau, to describe height as function of age (H~A), and (4) an exponential growth model of BW as function of height (BW~H). A cross-validation procedure was performed to evaluate accuracy and precision of the models. The linear relationship of BW~A and BW:H~A were estimated, respectively, by the equations: BW = 42.65 + 0.62 × A and BW:H = 0.70 + 0.0041 × A, where BW is in kilograms, BW:H = BW-to-height ratio (kg/cm), and A = age (d). Using the quadratic plateau for the model H~A, a critical "x" value of ~806 d and a height plateau of 138.6 cm were identified. Therefore, the following equations for estimating the height of animals younger and older than 806 d, respectively, were developed: H = 78.15 + 0.150 × A - 0.00009 × A2 and H = 78.15 + 0.150 × cvx - 0.00009 × cvx2, where H = height (cm) and cvx = 806 (critical "x" value; given in days). Additionally, the exponential model of W~H was estimated by the following equation: BW = 4.25 × exp(0.034 ×H), where BW is in kilograms and H = height (cm). A cross validation demonstrated that all equations had very high accuracy and precision. Overall, these models demonstrated that BW and BW-to-height ratio increase linearly as a function of age, while BW follows an exponential growth pattern as a function of height. Additionally, the H~A model predicted that heifers achieve a maximum height of 138.6 cm at 806 d of age.

Associations between dry period length and time to culling and pregnancy in the subsequent lactation.

The association between dry period length (DPL) and time to culling and pregnancy in the subsequent lactation may be important for the economically optimal length of the dry period. Therefore, this study aimed to (1) quantify the association between DPL and hazard of culling and pregnancy in the subsequent lactation; (2) develop continuous functions of DPL for the hazard ratios of culling and pregnancy; and (3) investigate the effect of a cause-specific hazards model and a subdistribution model to analyze competing events. The data used in this observational cohort study were from dairy herd improvement milk test lactation records from 40 states in the United States. After edits, there remained 1,108,515 records from 6,730 herds with the last days dry in 2014 or 2015. The records from 2 adjacent lactations (current, subsequent) were concatenated with the DPL of interest, 21 to 100 d, in between both lactations. We defined 8 DPL categories of 10 d each. Kaplan-Meier survival curves were used to show associations between DPL and time to culling or pregnancy for 3 lactation groups: lactation 1 and 2, lactation 2 and 3, and lactation 3 and greater. To control for confounding factors in Cox proportional models, we included 6 current lactation covariates and 3 time-dependent variables in the survival models. Hazard ratios of culling were estimated for 4 days in milk (DIM) categories from 1 to 450 DIM. Hazard ratios of pregnancy were estimated for 3 DIM categories from 61 to 300 DIM. Competing risk analysis of 8 disposal codes (i.e., farmer reported reasons) for culling and the culling event for pregnancy were conducted by a cause-specific hazards model and a subdistribution model. Hazard ratios were also estimated as quadratic polynomials of DPL. Compared with the reference DPL category of 51 to 60 d, hazard ratios of culling and pregnancy of the other 7 DPL categories ranged between 0.70 and 1.49, and 0.93 and 1.15, respectively. Short DPL were associated with lower risk of culling in the early lactation but not over the entire lactation. Short DPL were associated with greater hazard of pregnancy. Trends in hazard ratios over the ranges of the 8 DPL categories were not always consistent. Competing risk analysis with both models provided little differences in hazard ratios of culling and pregnancy. In conclusion, variations in DPL were associated with meaningful differences in the hazard ratios for culling and pregnancy and minor differences in the relative frequency of disposal codes. Subdistribution hazards models produced hazard ratios similar to cause-specific hazard models. The quadratic polynomials may be useful for decision support on customization of DPL for individual cows.

Piecewise modeling of the associations between dry period length and milk, fat, and protein yield changes in the subsequent lactation.

Our objective was to develop predictive models of 305-d mature-equivalent milk, fat, and protein yields in the subsequent lactation as continuous functions of the number of days dry (DD) in the current lactation. In this retrospective cohort study with field data, we obtained DHIA milk recording lactation records with the last DD in 2014 or 2015. Cows included had DD from 21 to 100 d. After editing, 1,030,141 records from cows in 7,044 herds remained. Three parity groups of adjacent (current, subsequent) lactations were constructed. We conducted all analyses by parity group and yield component. We first applied control models to pre-adjust the yields in the subsequent lactation for potentially confounding effects. Control models included the covariates mature-equivalent yield, days open, somatic cell score at 180 d pregnant, daily yield at 180 d pregnant, and a herd-season random effect, all observed in the current lactation. Days dry was not included. Second, we modeled residuals from control models with smooth piecewise regression models consisting of a simple linear, quadratic, and another simple linear equation depending on DD. Yield deviations were calculated as differences from predicted mature-equivalent yield at 50 DD. For validation, predictions of yield deviations from piecewise models by DD were compared with predictions from local regression for the DHIA field records and yield deviations reported in 38 experimental and field studies found in the literature. Control models reduced the average root mean squared prediction error by approximately 21%. Yield deviations were increasingly more negative for DD shorter than 50 d, indicating lower yields in the subsequent lactation. For short DD, the decrease in 305-d mature-equivalent milk yield ranged from 43 to 53 kg per DD. For mature-equivalent fat and protein yields, decreases were between 1.28 and 1.71 kg per DD, and 1.06 and 1.50 kg per DD, respectively. Yield deviations often were marginally positive and increasing for DD >50, so that the highest yield in the subsequent lactation was predicted for 100 DD. For long DD, the 305-d mature-equivalent milk yield increased at most 4.18 kg per DD. Patterns in deviations for fat and protein yield were similar to those for milk yield deviations. Predictions from piecewise models and local regressions were very similar, which supports the chosen functional form of the piecewise models. Yield deviations from field studies in the literature typically were decreasing when DD were longer, likely because of insufficient control for confounding effects. In conclusion, piecewise models of mature-equivalent milk, fat, and protein yield deviations as continuous functions of DD fit the observed data well and may be useful for decision support on the optimal dry period length for individual cows.

Comparison of microbial fermentation data from dual-flow continuous culture system and omasal sampling technique: A meta-analytical approach.

Although the omasal sampling technique (OST) has been successfully used to estimate ruminal fermentation and nutrient flow, alternatives to invasive animal trials should be pursued and evaluated. The objective of this study was to evaluate carbohydrate and N metabolisms using a meta-analytical approach to compare 2 methods: dual-flow continuous culture system (DFCCS) and OST. To be included, studies needed to report diet chemical composition and report at least 1 of the dependent variables of interest. A total of 155 articles were included, in which 97 used the DFCCS and 58 used the OST. The independent variables used were dietary nonfiber carbohydrate concentration, neutral detergent fiber (NDF) degradability, true crude protein (CP) degradability, and efficiency of microbial protein synthesis (EMPS). In addition, 12 dependent variables were used. Statistical analyses were performed using the Mixed procedure of SAS (SAS Institute Inc., Cary, NC). A random coefficients model was used considering study as a random effect and including the possibility of covariance between the slope and the intercept. The effect of method (DFCCS or OST) was included and tested in the estimates of the intercept, linear, and quadratic effects of the independent variable. There was no method effect when NDF degradability was regressed with total volatile fatty acids concentration, true CP degradability, and EMPS. Molar proportions of acetate and propionate were quadratically associated with NDF degradability. When NDF degradability was regressed with acetate and propionate there was a method effect, differing only in the intercept (ß0) estimate. True organic matter digestibility, bacterial N/total N, efficiency of N utilization, total volatile fatty acid concentration, and molar proportion of butyrate linearly increased as dietary nonfiber carbohydrate concentration increased, and none of these variables were affected by method. Concentration of ammonia N had a linear and positive association with true CP degradability. This was the only variable that had a method effect when regressed with true CP degradability, differing only in the estimate of the intercept (ß0). As EMPS increased, efficiency of N utilization also increased, and it was affected by method. Overall, the majority of DFCCS responses were similar to OST. When a method effect was observed, it was mainly on the estimate of the intercept, demonstrating that the magnitude of these responses was different. However, the relationships between independent and dependent variables were similar across methods.

Determination of macromineral requirements for preweaned dairy calves in tropical conditions.

International committees that have published nutrient requirements for dairy cattle have used data from mineral studies conducted in the 1920s to 1970s, and no study has reported data from animals less than 100 kg; therefore, there is a need to update mineral requirements for preweaned dairy calves. Thus, a meta-analysis was performed to estimate the mineral requirements of Ca, P, K, Mg, and Na for Holstein and Holstein × Gyr crossbred preweaned dairy calves using data from 5 studies developed at the Universidade Federal de Viçosa (Viçosa, MG, Brazil). A total of 210 calves were separated into 2 breeds: purebred Holstein calves (animals with a Holstein pedigree higher than 87.5%) and Holstein × Gyr crossbred calves (animals with a Holstein pedigree lower than 87.5%). The comparative slaughter technique was used to estimate animal body composition and empty body weight (EBW). Mineral requirements for maintenance were estimated by the regression between retained mineral and mineral intake, whereas mineral requirements for gain were obtained from the first derivative of the mineral content in the animal's body. In addition, breed effect was tested on the intercept and slope of the models. The effect of breed was not observed for all analyzed variables. Thus, net requirements for maintenance were 12.73, 11.81, 20.28, 3.50, and 6.37 mg/kg of EBW per day for Ca, P, K, Mg, and Na, respectively. Retention coefficients were 73.18, 65.20, 13.16, 29.55, and 24.28% for Ca, P, K, Mg, and Na, respectively. The following equations were determined to estimate net requirements for gain (NRG, g/d): NRG for Ca = 14.402 × EBW-0.139 × empty body gain (EBG); NRG for P = 5.849 × EBW-0.027 × EBG; NRG for K = 1.140 × EBW-0.048 × EBG; NRG for Mg = 0.603 × EBW-0.036 × EBG; and NRG for Na = 1.508 × EBW-0.045 × EBG. Due to the high variation between the data found in this study and in the available literature, we suggest that further studies should be conducted to evaluate the estimates of this study.

Effect of replacing calcium salts of palm oil with camelina seed at 2 dietary ether extract levels on digestion, ruminal fermentation, and nutrient flow in a dual-flow continuous culture system.

Camelina is a drought- and salt-tolerant oil seed, which in total ether extract (EE) contains up to 74% polyunsaturated fatty acids. The objective of this study was to assess the effects of replacing calcium salts of palm oil (Megalac, Church & Dwight Co. Inc., Princeton, NJ) with camelina seed (CS) on ruminal fermentation, digestion, and flows of fatty acids (FA) and AA in a dual-flow continuous culture system when supplemented at 5 or 8% dietary EE. Diets were randomly assigned to 8 fermentors in a 2 × 2 factorial arrangement of treatments in a replicated 4 × 4 Latin square design, with four 10-d experimental periods consisting of 7 d for diet adaptation and 3 d for sample collection. Treatments were (1) calcium salts of palm oil supplementation at 5% EE (MEG5); (2) calcium salts of palm oil supplementation at 8% EE (MEG8); (3) 7.7% CS supplementation at 5% EE (CS5); and (4) 17.7% CS supplementation at 8% EE (CS8). Diets contained 55% orchardgrass hay, and fermentors were fed 72 g of dry matter/d. On d 8, 9, and 10 of each period, digesta effluent samples were taken for ruminal NH3, volatile fatty acids, nitrogen metabolism analysis, and long-chain FA and AA flows. Statistical analysis was performed using the MIXED procedure (SAS Institute Inc., Cary, NC). We detected an interaction between FA source and dietary EE level for acetate, where MEG8 had the greatest molar proportion of acetate. Molar proportions of propionate were greater and total volatile fatty acids were lower on CS diets. Supplementation of CS decreased overall ruminal nutrient true digestibility, but dietary EE level did not affect it. Diets containing CS had greater biohydrogenation of 18:2 and 18:3; however, biohydrogenation of 18:1 was greater in MEG diets. Additionally, CS diets had greater ruminal concentrations of trans-10/11 18:1 and cis-9,trans-11 conjugated linoleic acid. Dietary EE level at 8% negatively affected flows of NH3-N (g/d), nonammonia N, and bacterial N as well as the overall AA outflow. However, treatments had minor effects on individual ruminal AA digestibility. The shift from acetate to propionate observed on diets containing CS may be advantageous from an energetic standpoint. Moreover, CS diets had greater ruminal outflow of trans-10/11 18:1 and cis-9,trans-11 conjugated linoleic acid than MEG diets, suggesting a better FA profile available for postruminal absorption. However, dietary EE at 8% was deleterious to overall N metabolism and AA outflow, indicating that CS can be fed at 5% EE without compromising N metabolism.

Effects of rumen-undegradable protein on intake, performance, and mammary gland development in prepubertal and pubertal dairy heifers.

The objective of this study was to evaluate the influence of different amounts of rumen-undegradable protein (RUP) on intake, N balance, performance, mammary gland development, carcass traits, and hormonal status of Holstein heifers at different physiological stages (PS). Sixteen prepubertal (PRE) heifers (initial BW = 106 ± 7.6 kg; age = 4.3 ± 0.46 mo) and 16 pubertal (PUB) heifers (initial BW = 224 ± 7.9 kg; age = 12.6 ± 0.45 mo) were used in an experiment over a period of 84 d. Four diets with increasing RUP contents (38, 44, 51, and 57% of dietary crude protein) and heifers at 2 PS (PRE or PUB) were used in a 4 × 2 factorial arrangement of treatments in a completely randomized design. Throughout the experiment, 2 digestibility trials were performed over 5 consecutive days (starting at d 36 and 78) involving feed and ort sampling and spot collections of feces and urine. At d 0 and 83, body ultrasound images were obtained for real-time carcass trait evaluation. The mammary gland was ultrasonically scanned at d 0 and every 3 wk during the experiment. Blood samples were taken at d 0 and 84 to determine serum concentrations of progesterone, estrogen, insulin-like growth factor I (IGF-I), and insulin. No interaction between PS and the level of RUP was found for any trait. Apparent digestibility of dry matter, organic matter, and neutral detergent fiber corrected for ash and protein was not affected by RUP level but was lower for PRE compared with PUB heifers. Sorting against neutral detergent fiber corrected for ash and protein (tendency only) and for crude protein was greater for PUB than PRE heifers. Pubertal heifers had greater average daily gain (905 vs. 505 g/d) and N retention (25.9 vs. 12.5 g/d) than PRE heifers. In addition, average daily gain and N retention were greatest at 51% RUP of dietary protein. Mammary ultrasonography indicated no effects of RUP amounts on mammary gland composition, whereas PRE heifers had greater pixel values than PUB, indicating higher contents of fat rather than protein in the mammary glands of PRE heifers. Serum progesterone and IGF-I concentration was affected only by PS, and PRE heifers had greater values of progesterone and IGF-I concentrations than PUB heifers. Serum insulin concentration was unaffected by PS but tended to be higher at 51% of RUP. In conclusion, an RUP level of 51% increases body weight, average daily gain, feed efficiency, and N retention in heifers regardless of the PS. In addition, PRE heifers have a lower sorting ability and reduced intake, total-tract digestibility, and N retention. They also have higher amounts of fat in their mammary glands, even at moderate growth rates.

Effects of methionine plus cysteine inclusion on performance and body composition of liquid-fed crossbred calves fed a commercial milk replacer and no starter feed.

This experiment aimed to evaluate the effects of supplying 4 different inclusion levels of Met + Cys to crossbred liquid-fed calves on animal performance and body composition. Thirty-six Holstein-Gyr male calves were separated into 2 age groups: 16 calves, slaughtered at an age of 30 d, representing the physiological phase from 8 to 30 d, and 20 calves, slaughtered at an age of 60 d, representing the physiological phase from 30 to 60 d. At 8 d of age, the animals were randomly distributed among the experimental treatments: 4 Met + Cys inclusion levels (Met + Cys: 8.0, 8.7, 9.4, and 10.2 g/d), provided by an AA supplement added to 1.0 kg (as fed) of commercial milk replacer containing soy protein concentrate and wheat protein isolate reconstituted at 13.8% (dry matter basis). The diet was supplied without allowing leftovers and no starter feed was provided. The experimental diets were supplied without allowing orts, so that the dry matter, crude protein, and ether extract intakes were the same for all animals, independent of Met + Cys level. Total weight gain, average daily gain, gain composition, and body composition were evaluated for both age groups separately. Digestibility of organic matter, crude protein, and ether extract was lower for 8 to 30 d than for 30 to 60 d. The effect of Met + Cys levels on the digestibility of nutrients was not observed; there also was no significant interaction between physiological phase and Met + Cys levels. For the 8 to 30 d group, no responses in performance were observed according to the different Met + Cys levels, which indicates that 8.0 g/d of Met + Cys met the requirements for this physiological phase. The 30 to 60 d group responded positively to higher Met + Cys inclusion in the diet. In conclusion, an optimal Met + Cys dietary level to ensure best performance and protein gain ranges from 8.41 to 9.81 g/d.

Meta-analysis of the energy and protein requirements of hair sheep raised in the tropical region of Brazil.

The objective of this study was to estimate, through mathematical models, energy and protein requirements for maintenance and gain of hair sheep raised in the tropical region of Brazil. To determine the equation parameters, a meta-analysis of seven independent experiments of nutrient requirements was performed, comprising a total of 243 experimental units (animals), which were conducted under tropical conditions, using hair sheep in growing and finishing phases and endowed of the following quantitative data for each animal: body weight (BW), empty body weight (EBW), average daily gain (ADG), empty body gain (EBG), heat production (HP), metabolizable energy intake (MEI), retained energy (RE), metabolizable protein intake (MPI) and body protein content. The regression equations generated were as follows: for Net Energy for maintenance, (NEm ): LogHP(MJEBW-0.75day-1)=-0.6090(±0.07470)+0.5149(±0.07216)×MEI(MJEBW-0.75day-1); for Net Energy for gain, (NEg ): LogRE(MJEBW-0.75day-1)=0.03084(±0.05334)+0.8455(±0.04355)×LogEBG(kg/day); for Metabolizable Protein for maintenance,(MPm ): MPI(g/day)  = 24.8470 (±7.3646) + 560.28 (±99.6582) × EBG(kg/day) ; for Net Protein for gain, (NPg ): NPg(kg/day)=0.1941×EBW(kg)-0.1058. The NEm requirement was 0.246 MJ EBW-0.75  day-1 . The metabolizable energy for maintenance requirement was 0.391 MJ EBW-0.75  day-1 . Considering an ADG of 100 g, the NEg requirement ranged from 0.496 to 1.701 MJ/day for animals with BW ranging from 10 to 40 kg respectively. The efficiencies of use of the metabolizable energy for maintenance and gain were 0.63 and 0.36 respectively. The MPm requirement was 3.097 g EBW-0.75  day-1 . Considering an ADG of 100 g, the NPg requirement ranged from 12.4 to 10.5 g/day for animals with BW ranging from 10 to 40 kg respectively. The total metabolizable energy and protein requirements were lower than those reported by the NRC and AFRC systems. Thus, our results support the hypothesis that nutrient requirements of hair sheep raised in tropical regions differ from wool sheep raised in temperate regions. Therefore, the use of the equations designed in this study is recommended.

Foetal development of skeletal muscle in bovines as a function of maternal nutrition, foetal sex and gestational age.

To determine the effects of maternal nutrition on modifications of foetal development of the skeletal muscle and possible increase in the potential of skeletal muscle growth in cattle, gestating cows were either fed 190% NRC recommendations (overnourished; ON) or 100% NRC recommendation (control; CO). Interaction between maternal nutrition (MN) and the foetal sex (FS) was also investigated. Foetuses were necropsied at four different time points throughout gestation (139, 199, 241 and 268 days of gestation) to assess the mRNA expression of myogenic, adipogenic and fibrogenic markers in skeletal muscle. Phenotypic indicators of the development of skeletal muscle fibres, intramuscular lipogenesis and collagen development were also evaluated. Modifications in mRNA expression of skeletal muscle of foetuses were observed in function of MN and FS despite the lack of effect of MN and FS on foetal weight at necropsy. Maternal ON increased the mRNA expression of the myogenic marker Cadherin-associated protein, beta 1 (CTNNB1) and adipogenic markers Peroxissome proliferator-activated receptor gamma (PPARG) and Zinc finger protein 423 (ZNF423) at midgestation. However, no differences on foetal skeletal muscle development were observed between treatments at late gestation indicating that a compensatory development may have occurred on CO foetuses making the effect of MN on skeletal muscle development not significant at late gestation. Moreover, our data have shown an evidence of sexual dimorphism during foetal stage with a greater skeletal muscle development in male than in female foetuses. In conclusion, providing a higher nutritional level to pregnant cows changes the trajectory of the development of skeletal muscle during midgestation, but apparently does not change the potential of post-natal growth of muscle mass of the offspring, as no differences in skeletal muscle development were observed in late gestation.

Performance strategies affect mammary gland development in prepubertal heifers.

In Brazil, the majority of dairy cattle are Holstein × Gyr (H×G). It is unknown whether excessive energy intake negatively affects their mammary development to the same extent as in purebred Holsteins. We hypothesized that mammary development of H×G heifers can be affected by dietary energy supply. We evaluated the effect of different average daily gains (ADG) achieved by feeding different amounts of a standard diet during the growing period on biometric measurements, development of mammary parenchyma (PAR) and mammary fat pad (MFP), and blood hormones. At the outset of this 84-d experiment, H×G heifers (n = 18) weighed 102.2 ± 3.4 kg and were 3 to 4 mo of age. Heifers were randomly assigned to 1 of 3 ADG programs using a completely randomized design. Treatments were high gain (HG; n = 6), where heifers were fed to gain 1 kg/d; low gain (LG; n = 6), where heifers were fed to gain 0.5 kg/d; and maintenance (MA; n = 6), where heifers were fed to gain a minimal amount of weight per day. Heifers were fed varying amounts of a single TMR to support desired BW gains. Over the 84 d, periodic biometric and blood hormone measurements were obtained. On d 84, all heifers were slaughtered and carcass and mammary samples were collected. At the end, HG heifers weighed the most (181 ± 7.5 kg), followed by LG (146 ± 7.5 kg) and MA (107 ± 7.5 kg) heifers. The ADG were near expected values and averaged 0.907, 0.500, and 0.105 ± 0.03 kg/d for HG, LG, and MA, respectively. In addition, body lengths, heart girths, and withers heights were affected by dietary treatment, with MA heifers generally being the smallest and HG heifers generally being the largest. Body condition scores differed by treatment and were highest in HG and lowest in MA heifers; in vivo subcutaneous fat thickness measurement and direct analysis of carcass composition supported this. The HG heifers had the heaviest MFP, followed by LG and then MA heifers. Amount of PAR was highest in LG heifers and was the same for HG and MA heifers. The percentage of udder mass occupied by PAR was lowest in HG heifers, differing from LG and MA heifers. Composition of MFP was not evaluated. Regarding PAR composition, no differences in ash or DM were found. On the other hand, CP concentration of PAR for HG heifers was lower than that for LG heifers, which was lower than that for MA heifers. Regarding the fat content, HG treatment was higher than LG and MA treatment, which did not differ from each other. In PAR, differences in relative abundance of genes related to both stimulation and inhibition of mammary growth were observed to depend on dietary treatment, sampling day, or both. The same can be said for most of the blood hormones that were measured in this experiment. In this experiment, high ADG achieved by feeding different amounts of a standard diet during the growing period negatively affected mammary development.

Energy and protein requirements of crossbred (Holstein × Gyr) growing bulls.

The objective of this study was to estimate the energy and protein requirements of crossbred (Holstein × Gyr) growing bulls. Twenty-four 10-mo-old bulls [initial body weight (BW) = 184 ± 23.4 kg] were used in a comparative slaughter trial. Six bulls were slaughtered at the beginning of the experiment as the reference group, to estimate initial empty body weight (EBW) and energy and protein contents of the remaining animals. The remaining bulls were assigned to a completely randomized design with 3 levels of dry matter intake and 6 replicates. The levels of dry matter intake were 1.2% of BW, 1.8% of BW, and ad libitum to target orts equal to 5% of the total amount that was fed. The remaining bulls were slaughtered at the end of the experiment. The bulls were fed a diet consisting of 59.6% corn silage and 40.4% concentrate on a dry matter basis. The equation that determined the relationship between EBW and BW was EBW = (0.861 ± 0.0031) × BW. The relationship between empty body gain (EBG) and average daily gain (ADG) was demonstrated by the following equation: EBG = (0.934 ± 0.0111) × ADG. Net energy for maintenance (NEM) was 74.8 ± 2.89 kcal/kg of EBW0.75 per day, and metabolizable energy for maintenance (MEM) was 120.8 kcal/kg of EBW0.75 per day. The detected efficiency of use of metabolizable energy for maintenance (km) was 61.9%. The equation used to estimate net energy for gain (NEG) was as follows: NEG = (0.049 ± 0.0011) × EBW0.75 × EBG0.729 ± 0.0532. The efficiency of use of metabolizable energy for gain (kg) was 35.7%. The metabolizable protein for maintenance (MPM) was 3.05 g/kg of BW0.75. The equation used to estimate net protein requirements for gain (NPG) = (87.138 ± 65.1378 × EBG) + [(40.436 ± 21.3640) × NEG]. The efficiency of use of metabolizable protein for gain (k) was 35.7%. We concluded that the estimates of energy and protein requirements presented herein are more appropriate than the National Research Council dairy cattle model and the Brazilian BR-CORTE system to balance the diets of crossbred (Holstein × Gyr) growing bulls.

Technical note: Mammary gland ultrasonography to evaluate mammary parenchymal composition in prepubertal heifers.

Bovine mammary gland development studies are often terminal or involve invasive biopsy procedures. Therefore, noninvasive means of assessing mammary development should be considered as alternative methods in live animals. The objective was to test if mammary ultrasonography can be used as a noninvasive way to estimate mammary parenchyma (PAR) composition in prepubertal dairy heifers with different average daily body weight gains. In the 84 d preceding, the ultrasound exam heifers were maintained in 1 of 3 treatment groups. Individual heifers were fed a high gain (1 kg/d; n = 6), low gain (0.5 kg/d, n = 6), or maintenance (n = 6) treatment diet. To achieve desired body weight gains, heifers were fed differing amounts of the same silage-based diet. Mammary glands of 18 crossbred heifers Holstein:Gyr underwent a single mammary ultrasound exam immediately before heifer slaughter, which took place when heifers weighed 142.0 ± 8.0 kg and were 200 d old. The 4 mammary glands of each heifer were evaluated using a real-time B-mode ultrasound machine equipped with a 6.5-MHz micro-convex transducer. Digital images (8-bit) of glands were obtained and PAR was identified within gland. Average pixel values per unit of PAR area were determined for each gland and analyzed at the level of heifer. Pixel results were interpreted on the basis that lower average pixel values reflect PAR with relatively high amounts of protein as opposed to fat. To help validate that the pixel value within PAR is associated with composition of PAR, pixel findings were compared with histological [number of adipocytes in PAR (Nad) and epithelial area in PAR (Ep)] and biochemical [percent crude protein in PAR (%CP), percent ether extract in PAR (%EE), PAR weight (WPAR), and mammary fat pad weight (WFAT)] composition of PAR in these same heifers. Within PAR, %EE and WFAT were positively correlated with pixel values, whereas %CP, Ep, and Nad were negatively correlated. Parenchyma weight did not correlate with pixel values. Regression analyses (fixed effect log-pixel value; random effect treatment) were used to estimate Nad, Ep, %CP, %EE, WPAR, and WFAT. Sensitivity analysis of regression equations revealed that accuracy of tested equations ranged from 0.77 to 0.93 and precision ranged from 0.56 to 0.82. Concordance correlation coefficients of the equations ranged from 0.41 to 0.76. In conclusion, ultrasonography of PAR can accurately measure and predict PAR composition in prepubertal dairy heifers growing at various rates of gain.

Determination of energy and protein requirements for crossbred Holstein × Gyr preweaned dairy calves.

The objective was to quantify the energy and protein nutritional requirements of Holstein × Gyr crossbred preweaned dairy calves until 64 d of age. Thirty-nine Holstein × Gyr crossbred male calves with an average initial live weight (mean ± SEM; for all next values) of 36 ± 1.0 kg were used. Five calves were slaughtered at 4 d of life to estimate the animals' initial body composition (reference group). The remaining 34 calves were distributed in a completely randomized design in a 3 × 2 factorial arrangement consisting of 3 levels of milk (2, 4, or 8 L/d) and 2 levels of starter feed (presence or absence in diet). At 15 and 45 d of life, 4 animals from each treatment were subjected to digestibility trials with total collection of feces (for 72 h) and urine (for 24 h). At 64 d of age, all animals were slaughtered, their gastro-intestinal tract was washed to determine the empty body weight (EBW; kg), and their body tissues were sampled for subsequent analyses. The net energy requirement for maintenance was estimated using an exponential regression between metabolizable energy intake and heat production (both in Mcal/EBW0.75 per d) and was 74.3 ± 5.7 kcal/EBW0.75 per d, and was not affected by inclusion of starter feed in the diet. The metabolizable energy requirement for maintenance was determined at the point of zero energy retention in the body and was 105.2 ± 5.8 kcal/EBW0.75 per d. The net energy for gain was estimated using the EBW and the empty body gain (EBG; kg/d) as 0.0882 ± 0.0028 × EBW0.75 × EBG0.9050±0.0706. The metabolizable energy efficiency for gain (kg) of the milk was 57.4 ± 3.45%, and the kg of the starter feed was 39.3 ± 2.09%. The metabolizable protein requirement for maintenance was 3.52 ± 0.34 g/BW0.75 per d. The net protein required for each kilogram gained was estimated as 119.1 ± 32.9 × EBW0.0663±0.059. The metabolizable protein efficiency for gain was 77 ± 8.5% and was not affected by inclusion of starter feed in the diet. In conclusion, the energy efficiency for gain of milk is higher than that of starter and the net protein required per unit protein gain increases with empty body weight.

Intestinal development of bovine foetuses during gestation is affected by foetal sex and maternal nutrition.

We aimed to evaluate the effects of maternal nutrition (MN) and foetal sex on the intestinal development of bovine foetuses throughout different days of gestation (DG). Forty-four multiparous, dry Holstein × Gyr cows with average initial body weight of 480 ± 10 kg were fed the same diet of either restricted feeding at 1.15% of body weight (CO, n = 24) or fed ad libitum (overnourished, ON, n = 20). Six cows from CO group and five cows from ON group were slaughtered at 139, 199, 241 and 268 DG, and foetuses were necropsied to evaluate the intestinal development. The mass, length and density of foetal intestines were not affected by MN (p ≥ 0.260). An interaction between MN and DG was observed for the villi length of jejunum (p = 0.006) and ileum (p < 0.001). Villi length of jejunum and ileum was higher (p < 0.10) in foetuses from ON-fed cows than in foetuses from CO-fed cows at 139 DG. However, at 199 DG, the villi length of jejunum and ileum of foetuses from CO-fed cows was higher than in foetuses from ON-fed cows. Despite these differences, MN did not affect the villi length of jejunum and ileum at 268 DG (p > 0.10). Female foetuses had greater small intestine mass (p = 0.093), large intestine mass (p = 0.022), small intestine mass in proportion to body mass (p = 0.017) and large intestine mass in proportion to body mass (p < 0.001) than male foetuses. Female foetuses had also longer small intestine (p = 0.077) and greater small intestine density (p = 0.021) and villi length of jejunum (p = 0.001) and ileum (p = 0.010) than males. We conclude that MN affects the pathway for the development of foetal villi length throughout the gestation in bovine foetuses without changing the final villi length. Female foetuses had higher intestinal mass, density and villi length than males during the foetal phase in bovines.

Technical note: Assessment of the oxygen pulse and heart rate method using respiration chambers and comparative slaughter for measuring heat production of cattle.

The objective of this study was to assess the oxygen pulse and heart rate (O2P-HR) technique using the respiration chamber (RC) and comparative slaughter (CS) methods for measuring the heat production (HP) of crossbred (Holstein × Gyr) yearling bulls. Twenty-four bulls were used. Six bulls were slaughtered at the beginning of the experiment as a reference group to estimate the initial empty body weight (BW) and energy content of the remaining animals. The remaining bulls were assigned to a completely randomized design with 3 levels of dry matter intake, with 6 replicates. The levels of dry matter intake were 1.2% of BW, 1.8% of BW and ad libitum, with target orts of 5%. The bulls were fed a diet consisting of 59.6% corn silage and 40.4% concentrate on a dry matter basis. The HP (kcal/BW0.75) was measured using 3 techniques, first using O2P-HR, followed by the RC and CS methods. The HP did not differ among assessed techniques, averaging 162.7kcal/BW0.75. The intercepts of the linear regressions (mean ± SE) were 64.82±25.515 (H0: intercept=0; P=0.024), 33.77±13.418 (H0: intercept=0), and 50.02±27.495 (H0: intercept=0) for O2P-HR versus RC, CS versus RC, and O2P-HR versus CS, respectively. The slopes of the linear regressions were 0.59±0.153 (H0: slope=1), 0.88±0.081 (H0: slope=1), and 0.62±0.155 (H0: slope=1) for O2P-HR versus RC, CS versus RC, and O2P-HR versus CS, respectively. The coefficients of determination were 0.52, 0.90, and 0.52 for O2P-HR versus RC, CS versus RC, and O2P-HR versus CS, respectively. The concordance correlation coefficients, 0.70 and 0.68, were moderate for O2P-HR versus RC and O2P-HR versus CS, respectively, but high, 0.90, for CS versus RC. The between-animal coefficient of variation was greater for the O2P-HR method (16.6%) compared with RC (7.7%) or CS (6.7%). We conclude that there was an agreement among the HP measurements detected using the assessed methods and that O2P-HR is able to predict HP in cattle with great accuracy but only moderate precision. Therefore, the O2P-HR method may have limitations in terms of assessing HP in low numbers of replications due to greater between-animal coefficient of variation than either the RC or CS methods.

Effect of maternal nutrition and days of gestation on pituitary gland and gonadal gene expression in cattle.

This study investigated effects of maternal overnutrition on gonadal development and pituitary-gonadal gene expression in cattle fetuses at mid- and late-gestation. Twenty-seven multiparous dry cows were fed either high (ad libitum, H) or moderate (M) intake of the same diet. Twelve cows from H (n=6) and M (n=6) intake carrying females fetuses were euthanized at 199 and 268d of gestation (DG; n=3 for H or M on each DG). Fifteen cows from H (n=6) and M intake (n=9) carrying male fetuses were euthanized at 139, 199, and 241 DG (n=2 for H and n=3 for M on each DG). Fetal gonads and pituitary gland were sampled for gene expression and histological analyses. Sex-specific responses to maternal intake were observed. Primordial and total follicle numbers were lower in fetal ovaries from H than in M intake cows. These results were the reverse for preantral and antral follicles. Volumetric proportion and diameter of seminiferous cord were lower in fetal testis of H than M intake cows. The expression level of FSHB was greater in pituitary gland of the female fetus from H compared with M intake cows, irrespective of DG, whereas LHB gene expression did not differ. In males, FSHB and LHB gene expression levels were similar between maternal intake groups. Fetal ovarian expression of P450 aromatase, StAR, BMPR2, TGFBR1, GDF9, FSHR, Bax, and CASP3 genes were higher in H than in M intake cows, irrespective of DG. Fetal testicular expression of StAR, HSD17B3, IGF1, IGF2, and IGF1R genes was higher in M than in H intake cows. The differences in gene expression for steroidogenesis, folliculogenesis, and apoptosis may explain the distinct pattern of follicular growth between offspring of M and H intake cows. By contrast, the lower volumetric proportion, diameter, and length of seminiferous cord may relate to decreased gene expression in fetal testis from H intake cows. In conclusion, maternal H intake seems to affect fetal ovarian follicular growth and number of follicles, which may affect the size of ovarian reserve in their offspring. In male fetus, maternal H intake seems to disturb testicular development and may have implications on sperm production. The underlying mechanism of differential gene expression and the effect on offspring reproductive potential should be the focus of further research, especially considering larger sample size, reducing the chance for type I errors.