2.1. Animals and Diets
The study was conducted at the Bonassai experimental farm of the Agricultural Research Agency of Sardinia (AGRIS Sardegna), located in the northwest of Sardinia (40° N, 32° E, 32 m a.s.l.), in Italy. The animal protocol described below was fully in compliance with the European Union (EU) and Italian regulations on animal welfare and experimentation, and it was approved by the veterinarians responsible of the ethic and welfare control in animal experimentation of AGRIS and the University of Sassari. All measurements were taken by personnel previously trained and authorized by the institutional authorities on ethical issues both from AGRIS and the University of Sassari.
The study consisted of two feeding experiments conducted on Sarda dairy sheep during mid (in March) and late (June–July) lactation. In experiment 1 (E1), four complete pelleted diets were tested on 20 ewes, and in experiment 2 (E2), other three complete pelleted diets were tested on 15 ewes. Each experiment consisted of a seven-day preliminary period, a fourteen-day adaptation period, and a nine-day experimental period. During the preliminary period, the ewes of each experiment grazed ryegrass-based pastures. were supplemented with a mixture made of equal proportions of the relative experimental pelleted diets for four days, and were then confined in pens for three days, during which time they received only the mixture of the experimental pelleted diets for three days. After the preliminary period, the ewes were allocated to homogeneous groups and put in individual metabolic cages for the adaptation and experimental periods.
In E1, the 20 mid-lactation ewes were allocated to four homogeneous groups of five animals each on the basis of their days in milk (DIM;
Table 1; mean ± s.d.), milk yield (MY), body weight (BW), body condition score (BCS), age, and parity. The ewes were fed the pelleted diets ad libitum in two daily meals. The same animals were then re-randomized and used in late lactation. In E2, 15 mid-lactation ewes were assigned to three homogeneous groups of five animals each, on the basis of their DIM (
Table 1), MY, BW, BCS, age, and parity. The same animals were then re-randomized and used in late lactation.
Throughout the period between the mid- and late-lactation measurements, the ewes were fed at pasture and machine-milked twice a day at 07:00 h and 15:00 h in a milking parlor. The animals were machine-milked twice a day at 07:00 h and 15:00 h inside the cages during the adaptation and experimental periods. All the animals had ad libitum access to water throughout the study.
The ingredients and the chemical composition of the diets used in E1 and E2 are summarized in
Table 2. On the basis of their main ingredient, the following diets were tested: CF = corn flakes, BM = barley meal, BP = beet pulp, and CC = corn cobs, in E1; and CM = corn meal, AA = dehydrated alfalfa, and SH = soybean hulls, in E2. All the diets contained dehydrated alfalfa as a common base and other ingredients (barley meal, corn flakes, corn meal, beet pulp, corn cobs, corn germ, corn gluten meal, soybean hulls, wheat middlings, minerals, and vitamins) were added in order to obtain different fiber (neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL)) and energy contents, while keeping CP concentrations similar.
The energy content of the diets was calculated as net energy for lactation (NE
L) on the basis of total digestible nutrient (TDN, % DM) [
15]:
whereas TDN was calculated as follows:
where dCPI = digestible CP intake (g/day), dNDFI = digestible NDF intake (g/day), dNFCI = digestible non-fiber carbohydrates (NFC) intake (g/day), dEEI = digestible ether extract intake (g/day), and DMI = dry matter intake (g/day). The data on intake of digestible nutrients measured in vivo and used in the above equations are reported in [
16]. In order to prevent acidosis in late lactation, due to a possible uneven feeding pattern associated with the high diurnal temperature typical of the late-lactation period (June–July), 10 g/day per head of sodium bicarbonate were added to all the diets.
2.3. Chemical Analyses
Feed samples were analyzed for DM; ash; CP; ether extract (EE, [
19]); ash-free fiber fractions as NDF, without the use of sodium sulfite, ADF, and ADL [
20]; CP fractions [
21]; and starch (polarimetric method, [
22]). The non-fiber carbohydrates (NFC) concentration was calculated as [100 − (NDF − NDIP) − CP − EE − ash], where NDIP = neutral detergent insoluble protein. Individual milk samples were analyzed for fat, true protein, and lactose using the infrared method (Milkoscan 4000, Foss Electric, Hillerød, Denmark) and urea by a colorimetric method (ChemSpec 150, Bentley Instruments Inc., Chaska, MN, USA; Broutin, 2000) calibrated by differential pH measurements (CL10-Eurochem, Rome, Italy).
2.4. Statistical Analysis
To target the first objective of the study (assessment of the effect of dietary energy on milk urea concentration), the results of the experiments were averaged by dietary group and physiological stage, and treatment means (n = 14) of the two experiments were pooled and used to study the relationships between dietary variables, nutrient intake, and MUC, as detailed below. First, values of MUC were regressed against dietary concentration and intake of nutrients, as well as PDIN balance, using a simple linear regression model:
where B
0 = intercept, C
0 = regression coefficient, Xi = independent variable, and ei = random error. Second, a stepwise regression analysis was performed to verify if any multiple regression model could fit better than simple regression models to predict MUC. All dietary variables already quoted were tested using
p < 0.15 as entry and stay probability thresholds. Because no variables were kept in the model, except for the content of dietary energy, no further attempts were made to test multiple regression models.
To pursue the second objective of the study (meta-analysis of the available literature on stall-fed dairy sheep) an extended dataset was made by adding to the current experiment results data from other experiments where dietary CP and energy intake and or their contents in sheep diet were related to milk urea. The search of relevant papers was done using Scopus with the keywords “sheep and nutrition and milk urea”, exploring all the scientific literature available to those engines in the time range of 1970–2019. Overall, the search resulted in 25 papers in the Scopus database, including primary and secondary documents, most of which were not relevant due to the focus on non-nutritional aspects or to the lack of accurate information on CP and energy intake (mostly grazing studies) or because urea was measured erratically or only in sheep plasma. Furthermore, all experiments that included diets containing tannins were discarded from the dataset because tannins are known for of their effects in modulating the use of dietary proteins. Low to moderate levels of tannins in the diet may actually reduce the protein degradation in the rumen and increase amino acid flow to the small intestine, while high levels can reduce voluntary feed intake and nutrient digestibility. At the end of this screening process, only six studies on stall-fed dairy sheep reported in the literature were selected [
6,
9,
23,
24,
25,
26], and their treatment means merged to those obtained in our study to form the extended dataset.
The relationships derived from the extended dataset were calculated using two statistical models: (1) simple linear regression models and (2) meta-analytical mixed models [
27], which included the regressors as fixed effects and the “study effect” (Exp) as random effect [
11]. In particular, the implemented model was as follows:
where A
0 = overall intercept, B
0 = overall regression coefficient, X
ij = independent variable, Exp
i = random effect of the study on the intercept, b
i = random effect of the study on the regression slope and e
ij = random error.