1. Introduction
The global population is expected to rise by 2 billion in the next three decades, increasing, in parallel, the demand for animal-derived products, with higher pressure on the food market to meet consumer requests [
1,
2]. In the past 60 years, this growth in the need for animal-derived foods has been met primarily by a steady increase in the number of animals reared and the nutritional value of the feeds, using higher levels of human-edible cereals and protein sources [
3].
Those solutions are no longer feasible. In fact, zootechnical systems, especially dairy and beef cattle farming, confront many sustainability challenges, such as human-induced greenhouse gas (GHG) emissions, in which both animal rearing and feed production are involved [
3,
4].
In response to these concerns, more efficient and sustainable dairy production systems need to be developed. Strategies to reduce the ruminal CH
4 production directly, enhance the overall production efficiency, and reduce the carbon footprint (CFP) of the feed used, may be considered. In fact, there is a positive relationship between production efficiency and environmental footprint, suggesting that strategies improving the productivity of dairy cows can lead to a simultaneous improvement in environmental impacts and profitability [
4]. Moreover, a lower CFP of the diets is related to a lower emission intensity, with reduced emissions per unit of milk [
5].
The use of alternative protein sources such as nonprotein nitrogen (NPN) to replace soybean meal (SBM) may be an effective strategy to address those challenges, mainly due to both the high environmental impact of SBM [
6,
7,
8] and the positive effect of NPN at the ruminal level [
9]. Traditionally, using alternative protein sources such as nonprotein nitrogen (NPN) to replace SBM was conducted primarily to reduce feed costs due to the high market prices of SBM and improve dietary protein utilization in dairy cows to enhance production efficiency [
9]. In recent years, this strategy is gaining interest in mitigating the environmental impacts of dairy products and improving dairy cows’ productivity and efficiency [
6,
7,
8].
Between the possible sources of NPN, feed grade urea was initially the most common in ruminants due to its low cost [
10]. However, feed grade urea is characterized by rapid hydrolysis in the rumen, with a consequent fast release of ammonia, exceeding the rate of carbohydrate fermentation. Consequently, this condition reduces the production, flow, and availability of microbial protein for milk production and reduces the nitrogen (N) utilization efficiency [
11]. Moreover, the rapid ruminal hydrolysis of urea increases N excretion through the urine and elevates blood NH
3 levels, with a potentially negative effect on cattle fertility [
12,
13].
Coating technologies are used to develop slow-release urea (SRU) products for controlling the urea degradation rate and release of NH
3 into the rumen, improving the efficiency of N utilization. The effects of SRU instead of SBM and feed grade urea have been reviewed in the literature, reporting positive effects on both beef [
14,
15] and dairy cattle [
8,
9,
10,
11,
12,
13,
14,
15,
16,
17]. Cherdthong et al. (2010) provided a narrative review of scientific literature that highlighted the potential efficacy of SRU in enhancing the efficiency of rumen N capture, microbial protein synthesis, and fiber digestion, with a consequent improvement in animals’ productivity and efficiency (cattle, buffalo, sheep, and goat) [
18]. Specifically, in dairy cows, the inclusion of SRU instead of SBM or other traditional protein sources resulted in improved production performances, namely higher milk yield, increased feed efficiency, and improved feed conversion rate, as a result of a healthier, more stable, and efficient rumen [
8,
9,
10,
11,
12,
13,
14,
15,
16,
17].
A new slow-release urea source based on a matrix of urea prills covered by a two-layer lipidic stratification was recently developed (Protigen, Phytotherapic Solutions, S.L. 08140 Caldes de Montbui, Barcelona, Spain). More information about the product can be found in
Supplementary Table S1.
We hypothesize that the partial substitution of soybean meal by the new sources of slow-release urea can be effectively used in dairy cattle due to its effect on rumen functionality, feed digestibility, production efficiency, and potentially lower environmental impact.
The present study aimed to evaluate the effects of the partial substitution of soybean meal (SBM) with a coated slow-release urea (SRU) source—Protigen—on the production performance, digestibility, and environmental impact, of high pedigree Holstein Frisian dairy cows.
2. Materials and Methods
2.1. Animal, Groups and Animal Care
The survey was conducted at the Del Santo farm located in Castelgerundo (Lodi, Italy), which well reflects the typical intensive dairy farm of the Po Valley area due to management and structural characteristics.
A total of 140 lactating Holstein Frisian cows were selected between the 200 lactating Holstein Frisian cows present on the farm at the beginning of the test and later enrolled in the trial. The animals were blocked by lactation number and days of lactation to create two balanced study groups with 70 cows each: (i) control (average lactation number of 2.30 ± 0.69; average days of lactation 53.86 ± 25.36); (ii) treatment (average lactation number of 2.31 ± 0.67; average days of lactation 51.86 ± 24.37).
The animals were reared in two separate groups in the same free housing barn, on a concrete floor with straw-bedded cubicles. All the cows were milked twice a day, in the morning at 07:00 and in the evening at 17:00, in a herringbone milking parlor that allows the simultaneous milking of 16 cows (8 + 8).
The study lasted for 140 days.
2.2. Diets and Feeding Management
The two groups received two isoenergetic and isonitrogenous diets that differed for the protein sources used (
Table 1). The control diet was based on soybean meal (SBM) as the main protein source and did not include any sources of slow-release urea (SRU). In the treatment diet, part of SBM (1.33% as fed, from 6.54 to 5.21%) was replaced, with 0.22% as fed (100 g/head/day) of SRU (Protigen). The SRU product used (Protigen) had a content of 250% of crude protein.
The two diets were formulated to meet or exceed the requirements for all nutrients [
19].
The diets were administered ad libitum in the form of a total mixed ration (TMR) and distributed once a day in the morning through the use of a mixer wagon (Grizzly 71.26/2, capacity of 26 cubic meters mixing system with 2 vertical augers, Sgariboldi, Codogno, 2685 (LO), Italy), equipped with a balance, and designed to weigh both the inclusion of the individual ingredients and the unloaded TMR. Water was available ad libitum.
2.3. Parameters Recorded
2.3.1. Production Performances: Milk Yield, Energy Corrected Milk (ECM), Milk Quality, Feed Intake, Feed Conversion Rate, Body Condition Score, Reproductive Performances
The daily milk yield (L/head/day) was recorded for each cow in the two groups. The milk yield was stored using a program, similar to the DairyComp programs available, developed specifically for the farm several years ago from a farm computer system company (Cremona, Italy). The feed intake for the two groups was evaluated daily by weighing the feed administered and then the residue in the manger 24 h later. The weekly average feed intake was calculated for both groups. The FCR was calculated, comparing the daily average feed intake with the daily average milk yield per group. Then the weekly FCR average was calculated for both groups.
Milk quality analyses were performed monthly. Milk samples were analyzed for fat, protein, lactose, urea, and somatic cell counts. Milk analyses were performed by the Lombardy Regional Breeders Association (ARAL) laboratory with the Milkoscan TM FT 6500 Plus instrument (Foss, Hillerød, Denmark) that employs the Fourier Transform Infrared Spectroscopy (FTIR) measuring principle. The milk urea content was evaluated using a specific kit (Urea Assay Kit Rapid K-URAMR, Megazyme, Astori Tecnica s.n.c. Poncarale (BS) 25020).
Monthly, the energy corrected milk (ECM) was evaluated by comparing the values of fat and protein obtained from the analyses and average milk production of the same week. The ECM was calculated following the equation proposed by Tyrrel and Reid (1965) [
20]:
The BCS was assessed monthly by the farm veterinary staff on all cows involved in the trial, as proposed by Edmonson et al. (1989) [
21] and Ferguson et al. (1994) [
22], through a visual and tactile evaluation of body fat reserves using a 5-point scale with 0.25-point increments (1—very thin cow; 5—excessively fat cow) where 3 represents the average body condition. The evaluation focused on the rump and loin.
Reproductive performance was also evaluated in the two groups considering the days open and number of services for pregnancy as the main indicators of fertility.
All the cows were checked daily for health status by the farm veterinary staff.
2.3.2. Characteristics of the Dies, Feces, and Digestibility of the Feeds
The characteristics of both the diet and feces were monitored twice per month (start and end of each month) using a portable NIR instrument (Polispec, IT Photonics, Fara Vicentino 36030 (VI), Italy). The monthly averages were then calculated. The characteristics of the TMR were analyzed in fresh feed with the portable NIR instrument while considering the entire bunk. Specifically, every time three measurements were gauged with the portable instrument along the entire length of the feed bunk (beginning, middle, and end of the manger). Similarly, the characteristics of the feces were analyzed for each group in a pool of fecal material collected the day after each feed analysis. The pool of fecal material was collected directly by a rectal grab in 20 cows per group. Samples of feces from the same group were then pooled together and mixed to create a single sample for each group. The pooled sample was analyzed with the portable NIR instrument.
The portable NIR instrument directly analyzed the two substrates (feed and feces) for dry matter, crude protein, crude fats, acid detergent fiber (ADF), neutral detergent fiber (NDF), acid detergent lignin (ADL), starch, and ash. The content of hemicelluloses was obtained from the difference between NDF and ADF. The content of cellulose was obtained from the difference between ADF and ADL. Sugars and pectin were obtained by the calculation: 100 –(ash + fats + proteins + NDF + starch).
The digestibility was evaluated through the following formula:
where:
2.3.3. Environmental Impact: Diet Carbon Footprint (CFP)
The CFP of the two diets was calculated to evaluate the effect of partial replacement on the traditional SBM with an SRU source on greenhouse gas emissions.
The contribution of each feed’s raw material to the feed’s CFP was estimated by multiplying the inclusion level of the raw material and the CFP per kilogram of dry matter of raw material (g CO
2-eq/kg). The CFP of each feed’s raw material was obtained from both the feed database created by Salami et al. (2021) [
8], which includes CFP values from the Dutch FeedPrint and Plurimix software as well as the AgriFootprint databases (2014). The CFP for each raw material considers all the emissions derived from the field production, feed processing, and transport, including those derived from land-use changing (LUC). In order to quantify the CFP of the slow-release urea source Protigen, data derived from products with a similar composition, structure, and characteristics were used [
8].
The average CFP of each TMR was then calculated and expressed as g CO2-eq.
The CFP of milk production as related to diet was calculated by dividing the weekly TMR CFP by the average weekly milk production.
2.3.4. Environmental Impact: Predicted Enteric Methane Production
Enteric methane production was estimated according to dry matter intake (DMI) using the equation of Hristov et al. (2013) [
23], characterized by the highest coefficient of determination (R2) value (0.880; root mean square error: 15.3) between predicted and observed values [
24], among all the possible equations available [
25]. The equation is as follows:
where:
2.4. Statistical Analysis
Data analysis was conducted using SAS statistical software (SAS 9.4, SAS Institute Inc., Cary, NC, USA).
Data distribution and homogeneity of variances were tested using PROC UNIVARIATE (SAS 9.4, SAS Institute Inc., Cary, NC, USA). Data about production performance and environmental impact were analyzed using a mixed model (PROC MIXED), which considered the fixed effect of treatment and time of detection. For digestibility data of the single diet component, a residual estimate of maximum-likelihood was performed with PROC MIXED (SAS 9.4 SAS Institute Inc., Cary, NC, USA) on a mixed model considering the fixed effects of treatment, sampling day, their interaction, and the random effects of the animal within the treatment period.
A single-subject was used as an experimental unit in all the statistical evaluations.
For all the parameters, a p-value ≤ 0.05 was considered statistically significant, whereas a value ≤0.1 was considered a tendency.