animals

Article

Effect of Slow-Release Urea Administration on ProductionPerformance Health Status Diet Digestibility andEnvironmental Sustainability in Lactating Dairy Cows

Silvia Grossi 1 Riccardo Compiani 2 Luciana Rossi 1 Matteo DellrsquoAnno 1 Israel Castillo 3 andCarlo Angelo Sgoifo Rossi 1

Citation Grossi S Compiani R

Rossi L DellrsquoAnno M Castillo I

Sgoifo Rossi CA Effect of

Slow-Release Urea Administration on

Production Performance Health

Status Diet Digestibility and

Environmental Sustainability in

Lactating Dairy Cows Animals 2021

11 2405 httpsdoiorg103390

ani11082405

Academic Editors Adam Cieslak

Amlan Kumar Patra Małgorzata

Szumacher-Strabel and

Zora Vaacuteradyovaacute

Received 1 July 2021

Accepted 12 August 2021

Published 14 August 2021

Publisherrsquos Note MDPI stays neutral

with regard to jurisdictional claims in

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iations

Copyright copy 2021 by the authors

Licensee MDPI Basel Switzerland

This article is an open access article

distributed under the terms and

conditions of the Creative Commons

Attribution (CC BY) license (https

creativecommonsorglicensesby

40)

1 Department of Health Animal Science and Food Safety ldquoCarlo Cantonirdquo (VESPA)Universitagrave degli Studi di Milano 26900 Lodi Italy lucianarossiunimiit (LR)matteodellannounimiit (MD) carlosgoifounimiit (CASR)

2 Animal Science and Food Safety University of Milan 7 20122 Milan Italy riccardocompianigmailcom3 Phytotherapic Solutions SL-Caldes de Montbui 08140 Barcelona Spain israelphytosolutionses Correspondence silviagrossiunimiit

Simple Summary The dairy system is facing many environmental issues such as greenhouse gasemissions land use and consumption of human-edible raw materials as well as increased demandfor milk by the growing world population Dairy cow farming must evolve toward more efficient andsustainable methods of production Strategies to reduce the carbon footprint of the animal feed usedand enhance overall productivity should be considered Feed production especially soybean mealrepresents the second source of total dairy greenhouse gas emissions Moreover there is a positivecorrelation between production efficiency and environmental footprint Using slow-releasing ureasources as an alternative to soybean meal can enhance rumen efficiency functionality and reduceemissions related to the feed used due to a lower carbon footprint

Abstract The effects of partially replacing soybean meal (SBM) with a slow-release urea source(SRU) on production performance feed efficiency digestibility and environmental sustainabilityof dairy cows were evaluated A total of 140 lactating Holstein Frisian cows were allocated intotwo study groups (i) control (diet entirely based on SBM) and (ii) treatment (diet of 022 on drymatter basis (dm)) of SRU Milk yield dry matter intake (DMI) feed conversion rate (FCR) bodycondition score (BCS) reproductive parameters and milk quality were evaluated The chemicalcomposition of the feeds and feces were analyzed to calculate the in vivo digestibility of the two dietsThe carbon footprint (CFP) and predicted methane (CH4) emissions were evaluated The inclusion ofSRU significantly increases milk yield DMI and FCR (p lt 00001) whereas milk quality BCS andreproductive indicators were not affected (p gt 005) In the treatment group the digestibility of crudeprotein (CP) (p = 0012) NDF (p = 0039) and cellulose (p = 0033) was significantly higher while theother nutritional parameters werenrsquot affected All the environmental parameters were significantlyimproved in the treatment group (p lt 00001) Replacing SBM with SRU can be a strategy to enhancedairy cowsrsquo sustainability due to improved production efficiency reduced feed CFP and predictedCH4 production

Keywords dairy slow-release urea efficiency feed digestibility sustainability carbon footprint

1 Introduction

The global population is expected to rise by 2 billion in the next three decades in-creasing in parallel the demand for animal-derived products with higher pressure on thefood market to meet consumer requests [12] In the past 60 years this growth in the needfor animal-derived foods has been met primarily by a steady increase in the number of

Animals 2021 11 2405 httpsdoiorg103390ani11082405 httpswwwmdpicomjournalanimals

Animals 2021 11 2405 2 of 15

animals reared and the nutritional value of the feeds using higher levels of human-ediblecereals and protein sources [3]

Those solutions are no longer feasible In fact zootechnical systems especially dairyand beef cattle farming confront many sustainability challenges such as human-inducedgreenhouse gas (GHG) emissions in which both animal rearing and feed production areinvolved [34]

In response to these concerns more efficient and sustainable dairy production systemsneed to be developed Strategies to reduce the ruminal CH4 production directly enhancethe overall production efficiency and reduce the carbon footprint (CFP) of the feed usedmay be considered In fact there is a positive relationship between production efficiencyand environmental footprint suggesting that strategies improving the productivity of dairycows can lead to a simultaneous improvement in environmental impacts and profitabil-ity [4] Moreover a lower CFP of the diets is related to a lower emission intensity withreduced emissions per unit of milk [5]

The use of alternative protein sources such as nonprotein nitrogen (NPN) to replacesoybean meal (SBM) may be an effective strategy to address those challenges mainly dueto both the high environmental impact of SBM [6ndash8] and the positive effect of NPN atthe ruminal level [9] Traditionally using alternative protein sources such as nonproteinnitrogen (NPN) to replace SBM was conducted primarily to reduce feed costs due to thehigh market prices of SBM and improve dietary protein utilization in dairy cows to enhanceproduction efficiency [9] In recent years this strategy is gaining interest in mitigating theenvironmental impacts of dairy products and improving dairy cowsrsquo productivity andefficiency [6ndash8]

Between the possible sources of NPN feed grade urea was initially the most commonin ruminants due to its low cost [10] However feed grade urea is characterized by rapidhydrolysis in the rumen with a consequent fast release of ammonia exceeding the rateof carbohydrate fermentation Consequently this condition reduces the production flowand availability of microbial protein for milk production and reduces the nitrogen (N)utilization efficiency [11] Moreover the rapid ruminal hydrolysis of urea increases Nexcretion through the urine and elevates blood NH3 levels with a potentially negativeeffect on cattle fertility [1213]

Coating technologies are used to develop slow-release urea (SRU) products for con-trolling the urea degradation rate and release of NH3 into the rumen improving theefficiency of N utilization The effects of SRU instead of SBM and feed grade urea havebeen reviewed in the literature reporting positive effects on both beef [1415] and dairycattle [8ndash17] Cherdthong et al (2010) provided a narrative review of scientific literaturethat highlighted the potential efficacy of SRU in enhancing the efficiency of rumen Ncapture microbial protein synthesis and fiber digestion with a consequent improvementin animalsrsquo productivity and efficiency (cattle buffalo sheep and goat) [18] Specificallyin dairy cows the inclusion of SRU instead of SBM or other traditional protein sourcesresulted in improved production performances namely higher milk yield increased feedefficiency and improved feed conversion rate as a result of a healthier more stable andefficient rumen [8ndash17]

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 SL08140 Caldes de Montbui Barcelona Spain) More information about the product can befound in Supplementary Table S1

We hypothesize that the partial substitution of soybean meal by the new sources ofslow-release urea can be effectively used in dairy cattle due to its effect on rumen function-ality feed digestibility production efficiency and potentially lower environmental impact

The present study aimed to evaluate the effects of the partial substitution of soybeanmeal (SBM) with a coated slow-release urea (SRU) sourcemdashProtigenmdashon the productionperformance digestibility and environmental impact of high pedigree Holstein Frisiandairy cows

Animals 2021 11 2405 3 of 15

2 Materials and Methods21 Animal Groups and Animal Care

The survey was conducted at the Del Santo farm located in Castelgerundo (LodiItaly) which well reflects the typical intensive dairy farm of the Po Valley area due tomanagement and structural characteristics

A total of 140 lactating Holstein Frisian cows were selected between the 200 lactatingHolstein Frisian cows present on the farm at the beginning of the test and later enrolledin the trial The animals were blocked by lactation number and days of lactation to createtwo balanced study groups with 70 cows each (i) control (average lactation number of230 plusmn 069 average days of lactation 5386 plusmn 2536) (ii) treatment (average lactationnumber of 231 plusmn 067 average days of lactation 5186 plusmn 2437)

The animals were reared in two separate groups in the same free housing barn on aconcrete floor with straw-bedded cubicles All the cows were milked twice a day in themorning at 0700 and in the evening at 1700 in a herringbone milking parlor that allowsthe simultaneous milking of 16 cows (8 + 8)

The study lasted for 140 days

22 Diets and Feeding Management

The two groups received two isoenergetic and isonitrogenous diets that differed forthe protein sources used (Table 1) The control diet was based on soybean meal (SBM) asthe main protein source and did not include any sources of slow-release urea (SRU) In thetreatment diet part of SBM (133 as fed from 654 to 521) was replaced with 022 asfed (100 gheadday) of SRU (Protigen) The SRU product used (Protigen) had a contentof 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 71262capacity of 26 cubic meters mixing system with 2 vertical augers Sgariboldi Codogno2685 (LO) Italy) equipped with a balance and designed to weigh both the inclusion of theindividual ingredients and the unloaded TMR Water was available ad libitum

23 Parameters Recorded231 Production Performances Milk Yield Energy Corrected Milk (ECM) Milk QualityFeed Intake Feed Conversion Rate Body Condition Score Reproductive Performances

The daily milk yield (Lheadday) was recorded for each cow in the two groups Themilk yield was stored using a program similar to the DairyComp programs available de-veloped 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 weighingthe feed administered and then the residue in the manger 24 h later The weekly averagefeed intake was calculated for both groups The FCR was calculated comparing the dailyaverage feed intake with the daily average milk yield per group Then the weekly FCRaverage was calculated for both groups

Milk quality analyses were performed monthly Milk samples were analyzed forfat protein lactose urea and somatic cell counts Milk analyses were performed bythe Lombardy Regional Breeders Association (ARAL) laboratory with the Milkoscan TMFT 6500 Plus instrument (Foss Hilleroslashd Denmark) that employs the Fourier TransformInfrared Spectroscopy (FTIR) measuring principle The milk urea content was evaluatedusing a specific kit (Urea Assay Kit Rapid K-URAMR Megazyme Astori Tecnica sncPoncarale (BS) 25020)

Animals 2021 11 2405 4 of 15

Table 1 Composition and nutritional values of the two diets tested as predicted by the rationingsoftware (Plurimix Fabermatica Ostiano (CR) Italy)

Feed as Fed Control Treatment

Corn Silage 5056 5233Corn meal 1139 1012

Grass Silage 843 824High-moisture Corn 674 835

Soybean meal 44 CP 1 654 521RyeGrass Hay 584 572

Alfalfa Hay 420 411Linseed meal 291 257

Rapeseed meal 182 160Min Mix 157 154Protigen 000 022

Analysis of dm 2 in the TMR

dm 5481 5377Energy Mcalkg dm 162 162

CP 1 1502 1500RDP 3 on CP 6269 6533RUP 4 on CP 3731 3467

Sol CP 5 on CP 2883 3440Sol CP on RDP 4598 5266

Sugars 311 297Starch 2793 2825NDF 3471 3533ADF 2022 2048ADL 393 398Fat 290 289

Ca total 084 085p total 036 035

1 CP = crude protein 2 dm = dry matter 3 RDP = rumen degradable protein 4 RUP = rumen undegradableprotein 5 Sol CP = soluble crude protein

Monthly the energy corrected milk (ECM) was evaluated by comparing the values offat and protein obtained from the analyses and average milk production of the same weekThe ECM was calculated following the equation proposed by Tyrrel and Reid (1965) [20]

ECM = 0327 lowast Milk yield (L) + 1295 lowast Fat yield(Kg) + 72 lowast Protein Yield (kg) (1)

The BCS was assessed monthly by the farm veterinary staff on all cows involved in thetrial as proposed by Edmonson et al (1989) [21] and Ferguson et al (1994) [22] througha visual and tactile evaluation of body fat reserves using a 5-point scale with 025-pointincrements (1mdashvery thin cow 5mdashexcessively fat cow) where 3 represents the average bodycondition The evaluation focused on the rump and loin

Reproductive performance was also evaluated in the two groups considering the daysopen 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

232 Characteristics of the Dies Feces and Digestibility of the Feeds

The characteristics of both the diet and feces were monitored twice per month (startand end of each month) using a portable NIR instrument (Polispec IT Photonics FaraVicentino 36030 (VI) Italy) The monthly averages were then calculated The characteristicsof the TMR were analyzed in fresh feed with the portable NIR instrument while consideringthe entire bunk Specifically every time three measurements were gauged with the portableinstrument along the entire length of the feed bunk (beginning middle and end of themanger) Similarly the characteristics of the feces were analyzed for each group in a pool

Animals 2021 11 2405 5 of 15

of fecal material collected the day after each feed analysis The pool of fecal material wascollected directly by a rectal grab in 20 cows per group Samples of feces from the samegroup were then pooled together and mixed to create a single sample for each group Thepooled 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 detergentfiber (NDF) acid detergent lignin (ADL) starch and ash The content of hemicelluloseswas obtained from the difference between NDF and ADF The content of cellulose wasobtained from the difference between ADF and ADL Sugars and pectin were obtained bythe calculation 100 ndash(ash + fats + proteins + NDF + starch)

The digestibility was evaluated through the following formula

Digestibility =

(Xd

ADLd

)minus

(X f

ADL f

)(

XdADLd

) times 100 (2)

where

X = each analytical parameter considered ()ADL = acid detergent lignin ()d = dietf = feces

233 Environmental Impact Diet Carbon Footprint (CFP)

The CFP of the two diets was calculated to evaluate the effect of partial replacementon the traditional SBM with an SRU source on greenhouse gas emissions

The contribution of each feedrsquos raw material to the feedrsquos CFP was estimated bymultiplying the inclusion level of the raw material and the CFP per kilogram of dry matterof raw material (g CO2-eqkg) The CFP of each feedrsquos raw material was obtained fromboth the feed database created by Salami et al (2021) [8] which includes CFP values fromthe 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 productionfeed 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 fromproducts with a similar composition structure and characteristics were used [8]

The average CFP of each TMR was then calculated and expressed as g CO2-eqThe CFP of milk production as related to diet was calculated by dividing the weekly

TMR CFP by the average weekly milk production

234 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 coefficientof determination (R2) value (0880 root mean square error 153) between predicted andobserved values [24] among all the possible equations available [25] The equation isas follows

CH4 (gd) = 254 + 1914 times DMI (3)

where

1 CH4 = enteric methane production2 DMI = dry matter intake (kgheadday)

24 Statistical Analysis

Data analysis was conducted using SAS statistical software (SAS 94 SAS InstituteInc Cary NC USA)

Animals 2021 11 2405 6 of 15

Data distribution and homogeneity of variances were tested using PROC UNIVARI-ATE (SAS 94 SAS Institute Inc Cary NC USA) Data about production performanceand environmental impact were analyzed using a mixed model (PROC MIXED) whichconsidered the fixed effect of treatment and time of detection For digestibility data of thesingle diet component a residual estimate of maximum-likelihood was performed withPROC MIXED (SAS 94 SAS Institute Inc Cary NC USA) on a mixed model consideringthe fixed effects of treatment sampling day their interaction and the random effects of theanimal within the treatment period

A single-subject was used as an experimental unit in all the statistical evaluationsFor all the parameters a p-value le 005 was considered statistically significant

whereas a value le01 was considered a tendency

3 Results and Discussion31 Production Performances Milk Yield Energy Corrected Milk (ECM) Milk Quality FeedIntake Feed Conversion Rate Body Condition Score Reproductive Performances

Data about the production performance are reported in Tables 2 and 3 and Figure 1The partial substitution of SBM with an SRU significantly (p lt 00001) improved the dailymilk production and resulted in an average production increase of 39 during the entiretrial period corresponding to 154 Lheadday Moreover the results of ECM were alsosignificantly higher in the treatment group (p = 00017) As shown in Figure 1 productivitybegan to differ between the two groups in the third week of the study when the differencereached statistical significance The literature also recognized that an integration of the dietaimed at influencing ruminal fermentation requires a period of at least 3 weeks to clearlyshow its effects [26] As visible in Figure 1 milk production decreased from weeks 9 to13 and increased sharply afterward This great variation can be explained by changingenvironmental conditions (T C and humidity) Firstly between weeks 10 and 13 theadverse winter conditions which were very cold with heavy rain and humidity negativelyaffected both the animals and the microenvironment inside the stable These conditionsresulted in reduced feed intake and lower milk production with the declining health of themammary gland Conversely from weeks 14 to 15 the environmental conditions improvedquickly as spring began resulting in better housing conditions (eg drier litter in thecubicles cleaner and drier floors) and a more comfortable microenvironment inside thestable with positive reflexes on mammary health as well as feed intake and milk production

Table 2 Production performance milk yield feed intake feed conversion rate bodycondition scores and reproductive performance in the two groups

GroupSEM p-Value

Control Treatment

Production Performance

Milk yield Lheadday 3934 4089 013 lt00001

ECM 2 kg 4320 4487 037 00017DMI 3 kgheadday 2469 2392 004 lt00001

FCR 4 159 170 0004 lt00001

BCS 4

December week 3 287 2911 003 0351January week 7 302 306 003 0193

February week 12 317 318 003 0852March week 17 316 312 003 0181

Reproductive Performance

Days open 10146 10010 128 0454Services to pregnancy 208 197 009 0402

2 ECM = energy corrected milk 3 DMI = dry matter intake 4 FCR = feed conversion rate 5 BCS = bodycondition score

Animals 2021 11 2405 7 of 15

Table 3 Production performance milk quality analyses

December Week 3 January Week 7 February Week 12 March Week 17

Fat

Control 381 382 384 384Treatment 384 380 385 386

p-value 0720 0786 0901 0864

Proteins

Control 367 369 367 365Treatment 374 366 365 366

p-value 0234 0535 0847 0852

Urea mg100 mL (mmolL)

Control 2145 (3560) 2313 (3839) 2228 (3698) 2229 (3700)Treatment 2101 (3487) 2356 (3910) 2231 (3703) 2296 (3811)

p-value 0471 0479 0486 0274

Lactose

Control 482 483 484 482Treatment 483 482 484 486

p-value 0533 061 0886 0061

Somatic cells x000

Control 42657 48879 49629 47056Treatment 42102 48053 48878 46342

p-value 0957 0936 0942 0945

Fat yield kgday

Control 1517 1563 1497 1510Treatment 1590 1609 1552 1569

p-value 0104 0300 0224 0190

Protein yield kgday

Control 1464 1508 1432 1436Treatment 1550 1553 1476 1491

p-value 0031 0258 0272 01605

The result of the present study agreed with Tikofsky and Harrison (2007) [27] andInostroza et al (2010) [16] who reported a significant increase in milk production of cowsfed diets containing SRU Also Kowalski et al (2010) showed an improvement in milkproduction in high-yielding dairy cows fed with SRU in partial replacement of SBM [17]Supplementation of SRU in ruminant diets fed with high levels of rapidly fermentablecarbohydrates may increase the synchrony between the energy and protein availability atthe rumen level enhancing the microbial protein synthesis thus improving its efficiency ofconverting into milk [28] It should be emphasized that the use of urea (combined withenzyme and cereals as a slower and safer form of ruminally released nitrogen) dairy cowdiets can beneficially modulate ruminal fermentation including microbiota populations(an increase in relative abundances of Megasphaera elsdenii and ammonia-producing bacte-ria) consequently improving production performance as was mentioned by Libera et al2021 [29]

Animals 2021 11 2405 8 of 15

Animals 2021 11 x FOR PEER REVIEW 8 of 15

1 d= day 2ECM = energy corrected milk 3 DMI = dry matter intake 4 FCR = feed conversion rate 5 BCS = body condition score

Figure 1 Average weekly milk production in the two groups (a = p-value le 005 x = p-value le 01)

Table 3 Production performance milk quality analyses

December Week 3 January Week 7 February Week 12 March Week 17 Fat

Control 381 382 384 384 Treatment 384 380 385 386

p-value 0720 0786 0901 0864 Proteins

Control 367 369 367 365 Treatment 374 366 365 366

p-value 0234 0535 0847 0852 Urea mg100 mL (mmolL)

Control 2145 (3560) 2313 (3839) 2228 (3698) 2229 (3700) Treatment 2101 (3487) 2356 (3910) 2231 (3703) 2296 (3811)

p-value 0471 0479 0486 0274 Lactose

Control 482 483 484 482 Treatment 483 482 484 486

p-value 0533 061 0886 0061 Somatic cells x000

Control 42657 48879 49629 47056 Treatment 42102 48053 48878 46342

p-value 0957 0936 0942 0945 Fat yield kgday

Control 1517 1563 1497 1510 Treatment 1590 1609 1552 1569

p-value 0104 0300 0224 0190 Protein yield kgday

Control 1464 1508 1432 1436

Figure 1 Average weekly milk production in the two groups (a = p-value le 005 x = p-value le 01)

Conversely Galo et al (2003) [30] and Giallongo et al (2015) [31] did not show anygain in milk production when SBM was partially replaced by SRU

In the present study DMI was significantly reduced in the treatment group (2392 vs2469 kgheadd in control) (p = 004) positively affecting FCR In fact the FCR significantlyimproved (p lt 00001) during treatment with an overall increase in feed efficiency at 69due to the lower DMI and better milk production

These results agree with the findings of Salami et al (2021) who reported a 3enhancement in feed efficiency due to a significant reduction in feed intake without anyeffects on milk yield when the traditional protein sources were replaced with SRU in dairycowsrsquo diets in Northern Europe [8]

Reproductive performance remained unaffected by the treatment (Table 2) which is inagreement with the findings of Hallajian et al (2021) who reported similar characteristicsof the follicles blood levels of progesterone and milk urea nitrogen (MUN) between dairycows fed exclusively with SBM or with the partial replacement of SBM SRU [32] Theseresults show that feeding with SRU appears to overcome the possible negative effect ofother NPN sources such as feed grade urea on both plasma urea nitrogen and overallreproductive performance [33]

Body condition scores were not influenced by the treatment (Table 2) Similarly Nealet al (2014) [34] and Hallajian et al (2021) [32] did not report significant differences interms of body weight and body condition in dairy Holstein cows fed with diets containingSRU compared with SBM control diets

Also the treatment did not influence milk quality traits as reported in Table 3 Thesefindings align with the main results found in the literature regarding dairy cows fed withan appropriate amount of slow-release urea [16ndash30]

No treatment effects were found in the overall health condition monitored daily bythe farm veterinary staff

The positive results observed after including SRU as a partial substitute for SBMunderlined that ruminal kinetics and fermentation could be optimized in diets with apercentage of soluble protein higher than 30 of the total crude protein and 50 ofdegradable protein if combined with an adequate intake of nonstructural and rapidlyfermentable carbohydrates

Despite the significant increase in the solubility of the protein fraction no changes inmilk quality or reproductive performance were observed Conversely previous studies

Animals 2021 11 2405 9 of 15

showed an increase in the milk urea levels and a reduction in fertility after an increase inprotein solubility [3536]

32 Characteristics of the Diets Feces and Digestibility of the Feeds

Chemical characteristics of the diets are shown in Tables 4 and 5 while chemical char-acteristics of feces are shown in Tables 6 and 7 Results highlighted a good correspondencebetween the projection of the rationing software and the analytical characteristics found

Table 4 Analysis of the composition of the control diet done with the portable NIR instru-ment Polispec

Parameter December January February March April

dm 1 5491 5495 5485 5465 5470Ash dm 920 976 969 923 976

Crude protein dm 1596 1621 1620 1583 1617Fats dm 300 299 315 300 302NDF dm 3635 3665 3620 3643 3668

Cellulose dm 1874 1877 1927 1882 1871Lignin dm 392 389 389 393 394

Hemicellulose dm 1370 1400 1305 1368 1403Starch dm 2800 2815 2807 2815 2819

Sugars and pectins dm 749 625 670 737 6201 dm = dry matter

Table 5 Analysis of the composition of the Treatment diet done with the portable NIR instru-ment Polispec

Parameter December January February March April

dm 1 5379 5389 5378 5463 5455Ash dm 910 980 955 919 972

Crude protein dm 1600 1619 1635 1586 1605Fats dm 288 305 305 300 303NDF dm 3655 3625 3610 3655 3680

Cellulose dm 1880 1846 1844 1882 1873Lignin dm 385 395 390 393 389

Hemicellulose dm 1390 1385 1377 1380 1417Starch dm 2838 2842 2825 2805 2821

Sugars and pectins dm 710 630 670 735 6201 dm = dry matter

Table 6 Analysis of the composition of the Control feces done with the portable NIR instru-ment Polispec

Parameter December January February March April

Moisture 8697 8688 8660 8690 8703Dry matter 1303 1313 1340 1310 1297Ash dm 927 947 897 938 925

Crude protein dm 1700 1705 1715 1727 1700Fats dm 275 255 257 278 264NDF dm 6165 6163 6150 6147 6167

Cellulose dm 3593 3628 3530 3570 3581Lignin dm 1121 1112 1125 1128 1137

Hemicellulose dm 1451 1423 1495 1457 1450Starch dm 590 591 611 568 603

Sugars and pectins dm 244 240 271 242 241dm = dry matter

Animals 2021 11 2405 10 of 15

Table 7 Analysis of the composition of the Treatment feces done with the portable NIR instru-ment Polispec

Parameter December January February March April

Moisture 8684 8725 8600 8690 8703dm 1316 1275 1400 1310 1296

Ash dm 952 1025 925 1003 1003Crude protein dm 1646 1645 1668 1687 1660

Fats dm 270 289 263 285 282NDF dm 6212 6076 6170 6052 6091

Cellulose dm 3575 3485 3426 3422 3501Lignin dm 1135 1158 1193 1128 1137

Hemicellulose dm 1493 1434 1542 1502 1453Starch dm 580 625 600 620 618

Sugars and pectins dm 240 241 275 254 247dm = dry matter

Results for nutrients digestibility during the different months of the survey (Table 8)showed that the partial substitution of SBM with SRU significantly enhanced protein(p = 0012) NDF (p = 0039) and cellulose (p = 0033) digestibility Those results partiallyagreed with Sinclair et al (2008) who reported a significant improvement in ruminaldigestion of fiber in vitro [37] These findings can be partially explained by an increasedabundance and activity of fibrolytic bacteria in the rumen such as Ruminococcaceae whichuses ammonia as its main nitrogen source due to both a higher level of NH3 availabilityand better synchrony between nitrogen and carbohydrates in the rumen when SRU is usedas a partial substitution for SBM [3839] Furthermore Geron et al (2016) found a betterin vivo digestibility of crude protein in sheep fed with SRU as a partial replacement forSBM [40]

Table 8 Digestibility in the two groups

Month December January February March April Average P(g) 1 P(m) 1 P(g m) 1

Group Ash

Control 6481 6611 6792 6456 6714 66100069 0010 0528Treatment 6452 6436 6835 6191 6468 6477

Sem 104 104 104 104 104 046p-value 0848 0264 0775 0103 0127 0069

Crude Protein

Control 6279 6325 6342 6197 6356 63000012 0201 0763Treatment 6506 6537 6666 6290 6459 6492

Sem 099 099 099 099 099 044p-value 0139 0164 0044 0524 0483 0012

Fats

Control 6648 7027 7194 6786 6942 69200621 0018 0324Treatment 6920 6802 7232 6536 6891 6876

Sem 133 134 134 134 134 060p-value 0184 0266 0848 0218 0794 0621

NDF 2

Control 4077 4127 4129 4118 4171 41240039 0821 0952Treatment 4132 4289 4416 4226 4332 4299

Sem 116 116 116 116 116 116p-value 0368 0349 0113 0528 0351 0039

Animals 2021 11 2405 11 of 15

Table 8 Cont

Month December January February March April Average P(g) 1 P(m) 1 P(g m) 1

Cellulose

Control 3302 3247 3659 3388 3366 33930033 0234 0999Treatment 3552 3564 3930 3657 3601 3661

Sem 171 171 171 171 171 171p-value 0329 0221 0292 0295 0357 0033

Hemicellulose

Control 6300 6452 6039 6287 6417 62990470 0343 0783Treatment 6347 6476 6341 6204 6488 6371

Sem 154 154 154 154 154 276p-value 0833 0911 0193 0710 0746 0470

Starch

Control 9264 9266 9247 9297 9258 92660874 0456 0045Treatment 9307 9250 9306 9229 9250 9268

Sem 018 018 018 018 018 008p-value 0132 0550 005 0028 0776 0874

Sugars and Pectins

Control 8865 8658 8588 8858 8654 87250903 0003 0753Treatment 8853 8700 8657 8707 8636 8729

Sem 053 053 053 053 053 023p-value 0883 0583 0382 0441 0817 0903

1 g = group m = month g m = group month 2 NDF = neutral detergent fiber

33 Environmental Impact Carbon Footprint of the Feed (CFP) and Predicted Methane(CH4) Production

The impact of feeding SRU as a partial substitution for SBM on dairy sustainabilitywas evaluated by estimating the CFP of the different diet feeds and predicting the entericCH4 production (Table 9 Figure 2)

Table 9 Environmental impact carbon footprint (CFP) and predicted enteric CH4 production in thetwo groups

GroupSEM p-Value

Control Treatment

Diet CFP 1

CO2 eq 2 gDMI (kgDMI)1248400(1248)

1076400(1076)

2245(022) lt00001

CO2 eq gL milk 32053 26640 079 lt00001

Predicted enteric CH43 Production

CH4 gd 47528 46045 090 lt00001CH4 gL milk 1220 1130 003 lt00001

1 CFP = Carbon footprint of the two feeds 2 CO2 eq = equivalent to the carbon dioxide 3 CH4 = methane

The results revealed that SBM was the dominant contributor to the feed CFP account-ing for 5371 and 4808 of the total CFP of the control and treatment diets respectively(Figure 2) Notably the inclusion of SRU in the treatment diet contributed only 056 of thefeed CFP (Figure 2) These results align with the average values reported in other studiesfocused on intensive dairy cows farming where SBM and other plant-protein sources werereplaced by SRU [8]

Animals 2021 11 2405 12 of 15

Animals 2021 11 x FOR PEER REVIEW 12 of 15

The partial substitution of SBM with SRU led to a significant (p lt 00001) reduction in the predicted ruminal CH4 production (Table 9) It is important to underline that the pre-sent study did not quantify the real ruminal CH4 production Instead it was assessed by following the equation of Hristov et al (2013) [23] based on dry matter intake (DMI) The lower CH4 production in the treatment group was a function of both the lower DMI when expressed only as grams of CH4 per day and better feed efficiency and milk yield when expressed as grams of CH4 per liter of milk

Notably there is limited published information on the effect of feeding urea on real enteric CH4 production Alipour et al (2020) [42] and Rebelo et al (2019) [43] showed that feeding two sources of coated and non-coated urea did not affect enteric CH4 yield meas-ured in an in vitro ruminal fermentation system or beef cattle respectively However Libera et al (2021) showed that the CH4 production at the ruminal level reduced after using urea combined with enzymes and cereals in dairy cows This result was due to a reduction in the substrate available for methanogenesis and an influence on methanogens or other rumen microorganisms [29] Although existing information suggests that slow-release urea (SRU) may have little or no effect on enteric CH4 emissions there is a crucial need for future studies to address this lack in the literature

Table 9 Environmental impact carbon footprint (CFP) and predicted enteric CH4 production in the two groups

Group SEM p-Value

Control Treatment

Diet CFP 1

CO2 eq 2 gDMI (kgDMI) 1248400 (1248)

1076400 (1076)

2245 (022)

lt00001

CO2 eq gL milk 32053 26640 079 lt00001 Predicted enteric CH4 3 Production

CH4 gd 47528 46045 090 lt00001 CH4 gL milk 1220 1130 003 lt00001

1 CFP = Carbon footprint of the two feeds 2 CO2 eq = equivalent to the carbon dioxide 3 CH4 = methane

Figure 2 Contribution of different feedsrsquo raw materials to the average carbon footprint of (A) control diet (B) treatment diet

The partial replacement of SBM with SRU decreased the CFP of the treatment dietexpressed per 1 kilogram of diet (minus1098 44991 vs 50541 g CO2-eqkg diet) comparedto the control diet due to the lower global warming potential (GWP) of SRU than SBM [24]The highest GWP of soybean meal is mainly due to large transport distances and emissionsrelated to land-use change (LUC) such as deforestation which can lead to heavier emissionsof greenhouse gases [6ndash41] These results agree with the findings of Salami et al (2021)who showed a 12 reduction in CFP (g CO2-eqkg diet) when SBM and other plant-proteinsources were replaced by SRU [8]

The inclusion of SRU also significantly (p lt 00001) reduced the CFP of the diet on adry matter basis (1076 vs 1248 kg CO2-eqkg DMI) as a result of both the lower DMI andthe lower CFP of the feeds (Table 9) Notably the CFP of the control diet per total DMIaligned with the average values reported by Gislon et al (2020) in a study conducted on171 dairy herds in the same Po Valley area where the present study was conducted [24]Conversely the impact per kilogram of DMI in the treatment diet was lower than the rangeof values reported in that survey [24]

Similarly the CFP of milk related to feed intake was significantly lower (p lt 00001) inthe treatment diet compared with the control diet (minus1689 26640 vs 32053 g CO2-eqLmilk) as a result of both the higher daily milk production and lower CFP of the feeds(Table 9) The reduction of CFP in milk related to the feed intake found in the present studywhich was also higher than the reduction (minus145) found by Salami et al (2021) [8]

The partial substitution of SBM with SRU led to a significant (p lt 00001) reductionin the predicted ruminal CH4 production (Table 9) It is important to underline that thepresent study did not quantify the real ruminal CH4 production Instead it was assessedby following the equation of Hristov et al (2013) [23] based on dry matter intake (DMI)The lower CH4 production in the treatment group was a function of both the lower DMIwhen expressed only as grams of CH4 per day and better feed efficiency and milk yieldwhen expressed as grams of CH4 per liter of milk

Notably there is limited published information on the effect of feeding urea on realenteric CH4 production Alipour et al (2020) [42] and Rebelo et al (2019) [43] showedthat feeding two sources of coated and non-coated urea did not affect enteric CH4 yieldmeasured in an in vitro ruminal fermentation system or beef cattle respectively However

Animals 2021 11 2405 13 of 15

Libera et al (2021) showed that the CH4 production at the ruminal level reduced after usingurea combined with enzymes and cereals in dairy cows This result was due to a reductionin the substrate available for methanogenesis and an influence on methanogens or otherrumen microorganisms [29] Although existing information suggests that slow-releaseurea (SRU) may have little or no effect on enteric CH4 emissions there is a crucial need forfuture studies to address this lack in the literature

4 Conclusions

This work contributes to defining scientific knowledge about the use of SRU in dairycowsrsquo nutrition and filling the present gap in the literature about their effect on environ-mental sustainability

This study showed that the substitution of traditional SBM (133 as fed) with thesource of SRU Protigen (022 as fed 100 gheadd) led to an improvement in dairycowsrsquo efficiency due to an enhanced feed conversion rate a lower dry matter intake ahigher digestibility of the fibrous parts of the diet and to better daily milk productionFurthermore the present study showed that the environmental sustainability of dairycowsrsquo diets could be improved by including SRU as an alternative protein source mainlyreducing the impact of the feed production in terms of greenhouse gas emissions andpredicted ruminal CH4 production Further research is needed to evaluate the effectiverole of SRU on methanogenesis and real ruminal production of methane

Supplementary Materials The following are available online at httpswwwmdpicomarticle103390ani11082405s1 Table S1 Technical information about the product Protigen used in thepresent trial particle size measured in millimetres and in vitro release rate at different time point

Author Contributions Conceptualization data curation writingmdashoriginal draft preparation re-view and editing SG conceptualization data curation RC data curation study validation LRmanuscript review MD data curation manuscript review IC conceptualization project admin-istration and supervision CASR All authors have read and agreed to the published version ofthe manuscript

Funding This research received no external funding

Institutional Review Board Statement The trial was a field and practical study not an experimentalone so it did not require approval For the trial we only used data commonly recorded by the farmer(milk production milk quality judged through monthly analyses reproductive parameters and soon) without any additional or ldquoexperimentalrdquo practices that can or will harm the animals or risktheir welfare The SRU product used in this study is already registered and used in dairy cowsrsquo feed

Data Availability Statement The data presented in this study are available on request from thecorresponding author

Conflicts of Interest The authors declare no conflict of interest

References1 United Nations Department of Economic and Social Affairs Population Division World Population Prospects 2019 Comprehensive

Tables (STESASERA426) United Nations Department of Economic and Social Affairs Population Division New York NYUSA 2019 Volume I

2 Georganas A Giamouri E Pappas AC Papadomichelakis G Galliou F Manios T Tsiplakou E Fegeros K ZervasG Bioactive Compounds in Food Waste A Review on the Transformation of Food Waste to Animal Feed Foods 2020 9 291[CrossRef]

3 Takiya CS Ylioja CM Bennett A Davidson MJ Sudbeck M Wickersham TA Vandehaar MJ Bradford BJ FeedingDairy Cows With ldquoLeftoversrdquo and the Variation in Recovery of Human-Edible Nutrients in Milk Front Sustain Food Syst 2019 3114 [CrossRef]

4 Capper JL Cady RA The effects of improved performance in the US dairy cattle industry on environmental impacts between2007 and 2017 J Anim Sci 2020 1 skz291 [CrossRef]

5 Wilkinson J Garnsworthy P Impact of diet and fertility on greenhouse gas emissions and nitrogen efficiency of milk productionLivestock 2017 22 140ndash144 [CrossRef]

Animals 2021 11 2405 3 of 15

2 Materials and Methods21 Animal Groups and Animal Care

The survey was conducted at the Del Santo farm located in Castelgerundo (LodiItaly) which well reflects the typical intensive dairy farm of the Po Valley area due tomanagement and structural characteristics

A total of 140 lactating Holstein Frisian cows were selected between the 200 lactatingHolstein Frisian cows present on the farm at the beginning of the test and later enrolledin the trial The animals were blocked by lactation number and days of lactation to createtwo balanced study groups with 70 cows each (i) control (average lactation number of230 plusmn 069 average days of lactation 5386 plusmn 2536) (ii) treatment (average lactationnumber of 231 plusmn 067 average days of lactation 5186 plusmn 2437)

The animals were reared in two separate groups in the same free housing barn on aconcrete floor with straw-bedded cubicles All the cows were milked twice a day in themorning at 0700 and in the evening at 1700 in a herringbone milking parlor that allowsthe simultaneous milking of 16 cows (8 + 8)

The study lasted for 140 days

22 Diets and Feeding Management

The two groups received two isoenergetic and isonitrogenous diets that differed forthe protein sources used (Table 1) The control diet was based on soybean meal (SBM) asthe main protein source and did not include any sources of slow-release urea (SRU) In thetreatment diet part of SBM (133 as fed from 654 to 521) was replaced with 022 asfed (100 gheadday) of SRU (Protigen) The SRU product used (Protigen) had a contentof 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 71262capacity of 26 cubic meters mixing system with 2 vertical augers Sgariboldi Codogno2685 (LO) Italy) equipped with a balance and designed to weigh both the inclusion of theindividual ingredients and the unloaded TMR Water was available ad libitum

23 Parameters Recorded231 Production Performances Milk Yield Energy Corrected Milk (ECM) Milk QualityFeed Intake Feed Conversion Rate Body Condition Score Reproductive Performances

The daily milk yield (Lheadday) was recorded for each cow in the two groups Themilk yield was stored using a program similar to the DairyComp programs available de-veloped 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 weighingthe feed administered and then the residue in the manger 24 h later The weekly averagefeed intake was calculated for both groups The FCR was calculated comparing the dailyaverage feed intake with the daily average milk yield per group Then the weekly FCRaverage was calculated for both groups

Milk quality analyses were performed monthly Milk samples were analyzed forfat protein lactose urea and somatic cell counts Milk analyses were performed bythe Lombardy Regional Breeders Association (ARAL) laboratory with the Milkoscan TMFT 6500 Plus instrument (Foss Hilleroslashd Denmark) that employs the Fourier TransformInfrared Spectroscopy (FTIR) measuring principle The milk urea content was evaluatedusing a specific kit (Urea Assay Kit Rapid K-URAMR Megazyme Astori Tecnica sncPoncarale (BS) 25020)

Animals 2021 11 2405 4 of 15

Table 1 Composition and nutritional values of the two diets tested as predicted by the rationingsoftware (Plurimix Fabermatica Ostiano (CR) Italy)

Feed as Fed Control Treatment

Corn Silage 5056 5233Corn meal 1139 1012

Grass Silage 843 824High-moisture Corn 674 835

Soybean meal 44 CP 1 654 521RyeGrass Hay 584 572

Alfalfa Hay 420 411Linseed meal 291 257

Rapeseed meal 182 160Min Mix 157 154Protigen 000 022

Analysis of dm 2 in the TMR

dm 5481 5377Energy Mcalkg dm 162 162

CP 1 1502 1500RDP 3 on CP 6269 6533RUP 4 on CP 3731 3467

Sol CP 5 on CP 2883 3440Sol CP on RDP 4598 5266

Sugars 311 297Starch 2793 2825NDF 3471 3533ADF 2022 2048ADL 393 398Fat 290 289

Ca total 084 085p total 036 035

1 CP = crude protein 2 dm = dry matter 3 RDP = rumen degradable protein 4 RUP = rumen undegradableprotein 5 Sol CP = soluble crude protein

Monthly the energy corrected milk (ECM) was evaluated by comparing the values offat and protein obtained from the analyses and average milk production of the same weekThe ECM was calculated following the equation proposed by Tyrrel and Reid (1965) [20]

ECM = 0327 lowast Milk yield (L) + 1295 lowast Fat yield(Kg) + 72 lowast Protein Yield (kg) (1)

The BCS was assessed monthly by the farm veterinary staff on all cows involved in thetrial as proposed by Edmonson et al (1989) [21] and Ferguson et al (1994) [22] througha visual and tactile evaluation of body fat reserves using a 5-point scale with 025-pointincrements (1mdashvery thin cow 5mdashexcessively fat cow) where 3 represents the average bodycondition The evaluation focused on the rump and loin

Reproductive performance was also evaluated in the two groups considering the daysopen 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

232 Characteristics of the Dies Feces and Digestibility of the Feeds

The characteristics of both the diet and feces were monitored twice per month (startand end of each month) using a portable NIR instrument (Polispec IT Photonics FaraVicentino 36030 (VI) Italy) The monthly averages were then calculated The characteristicsof the TMR were analyzed in fresh feed with the portable NIR instrument while consideringthe entire bunk Specifically every time three measurements were gauged with the portableinstrument along the entire length of the feed bunk (beginning middle and end of themanger) Similarly the characteristics of the feces were analyzed for each group in a pool

Animals 2021 11 2405 5 of 15

of fecal material collected the day after each feed analysis The pool of fecal material wascollected directly by a rectal grab in 20 cows per group Samples of feces from the samegroup were then pooled together and mixed to create a single sample for each group Thepooled 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 detergentfiber (NDF) acid detergent lignin (ADL) starch and ash The content of hemicelluloseswas obtained from the difference between NDF and ADF The content of cellulose wasobtained from the difference between ADF and ADL Sugars and pectin were obtained bythe calculation 100 ndash(ash + fats + proteins + NDF + starch)

The digestibility was evaluated through the following formula

Digestibility =

(Xd

ADLd

)minus

(X f

ADL f

)(

XdADLd

) times 100 (2)

where

X = each analytical parameter considered ()ADL = acid detergent lignin ()d = dietf = feces

233 Environmental Impact Diet Carbon Footprint (CFP)

The CFP of the two diets was calculated to evaluate the effect of partial replacementon the traditional SBM with an SRU source on greenhouse gas emissions

The contribution of each feedrsquos raw material to the feedrsquos CFP was estimated bymultiplying the inclusion level of the raw material and the CFP per kilogram of dry matterof raw material (g CO2-eqkg) The CFP of each feedrsquos raw material was obtained fromboth the feed database created by Salami et al (2021) [8] which includes CFP values fromthe 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 productionfeed 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 fromproducts with a similar composition structure and characteristics were used [8]

The average CFP of each TMR was then calculated and expressed as g CO2-eqThe CFP of milk production as related to diet was calculated by dividing the weekly

TMR CFP by the average weekly milk production

234 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 coefficientof determination (R2) value (0880 root mean square error 153) between predicted andobserved values [24] among all the possible equations available [25] The equation isas follows

CH4 (gd) = 254 + 1914 times DMI (3)

where

1 CH4 = enteric methane production2 DMI = dry matter intake (kgheadday)

24 Statistical Analysis

Data analysis was conducted using SAS statistical software (SAS 94 SAS InstituteInc Cary NC USA)

Animals 2021 11 2405 6 of 15

Data distribution and homogeneity of variances were tested using PROC UNIVARI-ATE (SAS 94 SAS Institute Inc Cary NC USA) Data about production performanceand environmental impact were analyzed using a mixed model (PROC MIXED) whichconsidered the fixed effect of treatment and time of detection For digestibility data of thesingle diet component a residual estimate of maximum-likelihood was performed withPROC MIXED (SAS 94 SAS Institute Inc Cary NC USA) on a mixed model consideringthe fixed effects of treatment sampling day their interaction and the random effects of theanimal within the treatment period

A single-subject was used as an experimental unit in all the statistical evaluationsFor all the parameters a p-value le 005 was considered statistically significant

whereas a value le01 was considered a tendency

3 Results and Discussion31 Production Performances Milk Yield Energy Corrected Milk (ECM) Milk Quality FeedIntake Feed Conversion Rate Body Condition Score Reproductive Performances

Data about the production performance are reported in Tables 2 and 3 and Figure 1The partial substitution of SBM with an SRU significantly (p lt 00001) improved the dailymilk production and resulted in an average production increase of 39 during the entiretrial period corresponding to 154 Lheadday Moreover the results of ECM were alsosignificantly higher in the treatment group (p = 00017) As shown in Figure 1 productivitybegan to differ between the two groups in the third week of the study when the differencereached statistical significance The literature also recognized that an integration of the dietaimed at influencing ruminal fermentation requires a period of at least 3 weeks to clearlyshow its effects [26] As visible in Figure 1 milk production decreased from weeks 9 to13 and increased sharply afterward This great variation can be explained by changingenvironmental conditions (T C and humidity) Firstly between weeks 10 and 13 theadverse winter conditions which were very cold with heavy rain and humidity negativelyaffected both the animals and the microenvironment inside the stable These conditionsresulted in reduced feed intake and lower milk production with the declining health of themammary gland Conversely from weeks 14 to 15 the environmental conditions improvedquickly as spring began resulting in better housing conditions (eg drier litter in thecubicles cleaner and drier floors) and a more comfortable microenvironment inside thestable with positive reflexes on mammary health as well as feed intake and milk production

Table 2 Production performance milk yield feed intake feed conversion rate bodycondition scores and reproductive performance in the two groups

GroupSEM p-Value

Control Treatment

Production Performance

Milk yield Lheadday 3934 4089 013 lt00001

ECM 2 kg 4320 4487 037 00017DMI 3 kgheadday 2469 2392 004 lt00001

FCR 4 159 170 0004 lt00001

BCS 4

December week 3 287 2911 003 0351January week 7 302 306 003 0193

February week 12 317 318 003 0852March week 17 316 312 003 0181

Reproductive Performance

Days open 10146 10010 128 0454Services to pregnancy 208 197 009 0402

2 ECM = energy corrected milk 3 DMI = dry matter intake 4 FCR = feed conversion rate 5 BCS = bodycondition score

Animals 2021 11 2405 7 of 15

Table 3 Production performance milk quality analyses

December Week 3 January Week 7 February Week 12 March Week 17

Fat

Control 381 382 384 384Treatment 384 380 385 386

p-value 0720 0786 0901 0864

Proteins

Control 367 369 367 365Treatment 374 366 365 366

p-value 0234 0535 0847 0852

Urea mg100 mL (mmolL)

Control 2145 (3560) 2313 (3839) 2228 (3698) 2229 (3700)Treatment 2101 (3487) 2356 (3910) 2231 (3703) 2296 (3811)

p-value 0471 0479 0486 0274

Lactose

Control 482 483 484 482Treatment 483 482 484 486

p-value 0533 061 0886 0061

Somatic cells x000

Control 42657 48879 49629 47056Treatment 42102 48053 48878 46342

p-value 0957 0936 0942 0945

Fat yield kgday

Control 1517 1563 1497 1510Treatment 1590 1609 1552 1569

p-value 0104 0300 0224 0190

Protein yield kgday

Control 1464 1508 1432 1436Treatment 1550 1553 1476 1491

p-value 0031 0258 0272 01605

The result of the present study agreed with Tikofsky and Harrison (2007) [27] andInostroza et al (2010) [16] who reported a significant increase in milk production of cowsfed diets containing SRU Also Kowalski et al (2010) showed an improvement in milkproduction in high-yielding dairy cows fed with SRU in partial replacement of SBM [17]Supplementation of SRU in ruminant diets fed with high levels of rapidly fermentablecarbohydrates may increase the synchrony between the energy and protein availability atthe rumen level enhancing the microbial protein synthesis thus improving its efficiency ofconverting into milk [28] It should be emphasized that the use of urea (combined withenzyme and cereals as a slower and safer form of ruminally released nitrogen) dairy cowdiets can beneficially modulate ruminal fermentation including microbiota populations(an increase in relative abundances of Megasphaera elsdenii and ammonia-producing bacte-ria) consequently improving production performance as was mentioned by Libera et al2021 [29]

Animals 2021 11 2405 8 of 15

Animals 2021 11 x FOR PEER REVIEW 8 of 15

1 d= day 2ECM = energy corrected milk 3 DMI = dry matter intake 4 FCR = feed conversion rate 5 BCS = body condition score

Figure 1 Average weekly milk production in the two groups (a = p-value le 005 x = p-value le 01)

Table 3 Production performance milk quality analyses

December Week 3 January Week 7 February Week 12 March Week 17 Fat

Control 381 382 384 384 Treatment 384 380 385 386

p-value 0720 0786 0901 0864 Proteins

Control 367 369 367 365 Treatment 374 366 365 366

p-value 0234 0535 0847 0852 Urea mg100 mL (mmolL)

Control 2145 (3560) 2313 (3839) 2228 (3698) 2229 (3700) Treatment 2101 (3487) 2356 (3910) 2231 (3703) 2296 (3811)

p-value 0471 0479 0486 0274 Lactose

Control 482 483 484 482 Treatment 483 482 484 486

p-value 0533 061 0886 0061 Somatic cells x000

Control 42657 48879 49629 47056 Treatment 42102 48053 48878 46342

p-value 0957 0936 0942 0945 Fat yield kgday

Control 1517 1563 1497 1510 Treatment 1590 1609 1552 1569

p-value 0104 0300 0224 0190 Protein yield kgday

Control 1464 1508 1432 1436

Figure 1 Average weekly milk production in the two groups (a = p-value le 005 x = p-value le 01)

Conversely Galo et al (2003) [30] and Giallongo et al (2015) [31] did not show anygain in milk production when SBM was partially replaced by SRU

In the present study DMI was significantly reduced in the treatment group (2392 vs2469 kgheadd in control) (p = 004) positively affecting FCR In fact the FCR significantlyimproved (p lt 00001) during treatment with an overall increase in feed efficiency at 69due to the lower DMI and better milk production

These results agree with the findings of Salami et al (2021) who reported a 3enhancement in feed efficiency due to a significant reduction in feed intake without anyeffects on milk yield when the traditional protein sources were replaced with SRU in dairycowsrsquo diets in Northern Europe [8]

Reproductive performance remained unaffected by the treatment (Table 2) which is inagreement with the findings of Hallajian et al (2021) who reported similar characteristicsof the follicles blood levels of progesterone and milk urea nitrogen (MUN) between dairycows fed exclusively with SBM or with the partial replacement of SBM SRU [32] Theseresults show that feeding with SRU appears to overcome the possible negative effect ofother NPN sources such as feed grade urea on both plasma urea nitrogen and overallreproductive performance [33]

Body condition scores were not influenced by the treatment (Table 2) Similarly Nealet al (2014) [34] and Hallajian et al (2021) [32] did not report significant differences interms of body weight and body condition in dairy Holstein cows fed with diets containingSRU compared with SBM control diets

Also the treatment did not influence milk quality traits as reported in Table 3 Thesefindings align with the main results found in the literature regarding dairy cows fed withan appropriate amount of slow-release urea [16ndash30]

No treatment effects were found in the overall health condition monitored daily bythe farm veterinary staff

The positive results observed after including SRU as a partial substitute for SBMunderlined that ruminal kinetics and fermentation could be optimized in diets with apercentage of soluble protein higher than 30 of the total crude protein and 50 ofdegradable protein if combined with an adequate intake of nonstructural and rapidlyfermentable carbohydrates

Despite the significant increase in the solubility of the protein fraction no changes inmilk quality or reproductive performance were observed Conversely previous studies

Animals 2021 11 2405 9 of 15

showed an increase in the milk urea levels and a reduction in fertility after an increase inprotein solubility [3536]

32 Characteristics of the Diets Feces and Digestibility of the Feeds

Chemical characteristics of the diets are shown in Tables 4 and 5 while chemical char-acteristics of feces are shown in Tables 6 and 7 Results highlighted a good correspondencebetween the projection of the rationing software and the analytical characteristics found

Table 4 Analysis of the composition of the control diet done with the portable NIR instru-ment Polispec

Parameter December January February March April

dm 1 5491 5495 5485 5465 5470Ash dm 920 976 969 923 976

Crude protein dm 1596 1621 1620 1583 1617Fats dm 300 299 315 300 302NDF dm 3635 3665 3620 3643 3668

Cellulose dm 1874 1877 1927 1882 1871Lignin dm 392 389 389 393 394

Hemicellulose dm 1370 1400 1305 1368 1403Starch dm 2800 2815 2807 2815 2819

Sugars and pectins dm 749 625 670 737 6201 dm = dry matter

Table 5 Analysis of the composition of the Treatment diet done with the portable NIR instru-ment Polispec

Parameter December January February March April

dm 1 5379 5389 5378 5463 5455Ash dm 910 980 955 919 972

Crude protein dm 1600 1619 1635 1586 1605Fats dm 288 305 305 300 303NDF dm 3655 3625 3610 3655 3680

Cellulose dm 1880 1846 1844 1882 1873Lignin dm 385 395 390 393 389

Hemicellulose dm 1390 1385 1377 1380 1417Starch dm 2838 2842 2825 2805 2821

Sugars and pectins dm 710 630 670 735 6201 dm = dry matter

Table 6 Analysis of the composition of the Control feces done with the portable NIR instru-ment Polispec

Parameter December January February March April

Moisture 8697 8688 8660 8690 8703Dry matter 1303 1313 1340 1310 1297Ash dm 927 947 897 938 925

Crude protein dm 1700 1705 1715 1727 1700Fats dm 275 255 257 278 264NDF dm 6165 6163 6150 6147 6167

Cellulose dm 3593 3628 3530 3570 3581Lignin dm 1121 1112 1125 1128 1137

Hemicellulose dm 1451 1423 1495 1457 1450Starch dm 590 591 611 568 603

Sugars and pectins dm 244 240 271 242 241dm = dry matter

Animals 2021 11 2405 10 of 15

Table 7 Analysis of the composition of the Treatment feces done with the portable NIR instru-ment Polispec

Parameter December January February March April

Moisture 8684 8725 8600 8690 8703dm 1316 1275 1400 1310 1296

Ash dm 952 1025 925 1003 1003Crude protein dm 1646 1645 1668 1687 1660

Fats dm 270 289 263 285 282NDF dm 6212 6076 6170 6052 6091

Cellulose dm 3575 3485 3426 3422 3501Lignin dm 1135 1158 1193 1128 1137

Hemicellulose dm 1493 1434 1542 1502 1453Starch dm 580 625 600 620 618

Sugars and pectins dm 240 241 275 254 247dm = dry matter

Results for nutrients digestibility during the different months of the survey (Table 8)showed that the partial substitution of SBM with SRU significantly enhanced protein(p = 0012) NDF (p = 0039) and cellulose (p = 0033) digestibility Those results partiallyagreed with Sinclair et al (2008) who reported a significant improvement in ruminaldigestion of fiber in vitro [37] These findings can be partially explained by an increasedabundance and activity of fibrolytic bacteria in the rumen such as Ruminococcaceae whichuses ammonia as its main nitrogen source due to both a higher level of NH3 availabilityand better synchrony between nitrogen and carbohydrates in the rumen when SRU is usedas a partial substitution for SBM [3839] Furthermore Geron et al (2016) found a betterin vivo digestibility of crude protein in sheep fed with SRU as a partial replacement forSBM [40]

Table 8 Digestibility in the two groups

Month December January February March April Average P(g) 1 P(m) 1 P(g m) 1

Group Ash

Control 6481 6611 6792 6456 6714 66100069 0010 0528Treatment 6452 6436 6835 6191 6468 6477

Sem 104 104 104 104 104 046p-value 0848 0264 0775 0103 0127 0069

Crude Protein

Control 6279 6325 6342 6197 6356 63000012 0201 0763Treatment 6506 6537 6666 6290 6459 6492

Sem 099 099 099 099 099 044p-value 0139 0164 0044 0524 0483 0012

Fats

Control 6648 7027 7194 6786 6942 69200621 0018 0324Treatment 6920 6802 7232 6536 6891 6876

Sem 133 134 134 134 134 060p-value 0184 0266 0848 0218 0794 0621

NDF 2

Control 4077 4127 4129 4118 4171 41240039 0821 0952Treatment 4132 4289 4416 4226 4332 4299

Sem 116 116 116 116 116 116p-value 0368 0349 0113 0528 0351 0039

Animals 2021 11 2405 11 of 15

Table 8 Cont

Month December January February March April Average P(g) 1 P(m) 1 P(g m) 1

Cellulose

Control 3302 3247 3659 3388 3366 33930033 0234 0999Treatment 3552 3564 3930 3657 3601 3661

Sem 171 171 171 171 171 171p-value 0329 0221 0292 0295 0357 0033

Hemicellulose

Control 6300 6452 6039 6287 6417 62990470 0343 0783Treatment 6347 6476 6341 6204 6488 6371

Sem 154 154 154 154 154 276p-value 0833 0911 0193 0710 0746 0470

Starch

Control 9264 9266 9247 9297 9258 92660874 0456 0045Treatment 9307 9250 9306 9229 9250 9268

Sem 018 018 018 018 018 008p-value 0132 0550 005 0028 0776 0874

Sugars and Pectins

Control 8865 8658 8588 8858 8654 87250903 0003 0753Treatment 8853 8700 8657 8707 8636 8729

Sem 053 053 053 053 053 023p-value 0883 0583 0382 0441 0817 0903

1 g = group m = month g m = group month 2 NDF = neutral detergent fiber

33 Environmental Impact Carbon Footprint of the Feed (CFP) and Predicted Methane(CH4) Production

The impact of feeding SRU as a partial substitution for SBM on dairy sustainabilitywas evaluated by estimating the CFP of the different diet feeds and predicting the entericCH4 production (Table 9 Figure 2)

Table 9 Environmental impact carbon footprint (CFP) and predicted enteric CH4 production in thetwo groups

GroupSEM p-Value

Control Treatment

Diet CFP 1

CO2 eq 2 gDMI (kgDMI)1248400(1248)

1076400(1076)

2245(022) lt00001

CO2 eq gL milk 32053 26640 079 lt00001

Predicted enteric CH43 Production

CH4 gd 47528 46045 090 lt00001CH4 gL milk 1220 1130 003 lt00001

1 CFP = Carbon footprint of the two feeds 2 CO2 eq = equivalent to the carbon dioxide 3 CH4 = methane

The results revealed that SBM was the dominant contributor to the feed CFP account-ing for 5371 and 4808 of the total CFP of the control and treatment diets respectively(Figure 2) Notably the inclusion of SRU in the treatment diet contributed only 056 of thefeed CFP (Figure 2) These results align with the average values reported in other studiesfocused on intensive dairy cows farming where SBM and other plant-protein sources werereplaced by SRU [8]

Animals 2021 11 2405 12 of 15

Animals 2021 11 x FOR PEER REVIEW 12 of 15

The partial substitution of SBM with SRU led to a significant (p lt 00001) reduction in the predicted ruminal CH4 production (Table 9) It is important to underline that the pre-sent study did not quantify the real ruminal CH4 production Instead it was assessed by following the equation of Hristov et al (2013) [23] based on dry matter intake (DMI) The lower CH4 production in the treatment group was a function of both the lower DMI when expressed only as grams of CH4 per day and better feed efficiency and milk yield when expressed as grams of CH4 per liter of milk

Notably there is limited published information on the effect of feeding urea on real enteric CH4 production Alipour et al (2020) [42] and Rebelo et al (2019) [43] showed that feeding two sources of coated and non-coated urea did not affect enteric CH4 yield meas-ured in an in vitro ruminal fermentation system or beef cattle respectively However Libera et al (2021) showed that the CH4 production at the ruminal level reduced after using urea combined with enzymes and cereals in dairy cows This result was due to a reduction in the substrate available for methanogenesis and an influence on methanogens or other rumen microorganisms [29] Although existing information suggests that slow-release urea (SRU) may have little or no effect on enteric CH4 emissions there is a crucial need for future studies to address this lack in the literature

Table 9 Environmental impact carbon footprint (CFP) and predicted enteric CH4 production in the two groups

Group SEM p-Value

Control Treatment

Diet CFP 1

CO2 eq 2 gDMI (kgDMI) 1248400 (1248)

1076400 (1076)

2245 (022)

lt00001

CO2 eq gL milk 32053 26640 079 lt00001 Predicted enteric CH4 3 Production

CH4 gd 47528 46045 090 lt00001 CH4 gL milk 1220 1130 003 lt00001

1 CFP = Carbon footprint of the two feeds 2 CO2 eq = equivalent to the carbon dioxide 3 CH4 = methane

Figure 2 Contribution of different feedsrsquo raw materials to the average carbon footprint of (A) control diet (B) treatment diet

The partial replacement of SBM with SRU decreased the CFP of the treatment dietexpressed per 1 kilogram of diet (minus1098 44991 vs 50541 g CO2-eqkg diet) comparedto the control diet due to the lower global warming potential (GWP) of SRU than SBM [24]The highest GWP of soybean meal is mainly due to large transport distances and emissionsrelated to land-use change (LUC) such as deforestation which can lead to heavier emissionsof greenhouse gases [6ndash41] These results agree with the findings of Salami et al (2021)who showed a 12 reduction in CFP (g CO2-eqkg diet) when SBM and other plant-proteinsources were replaced by SRU [8]

The inclusion of SRU also significantly (p lt 00001) reduced the CFP of the diet on adry matter basis (1076 vs 1248 kg CO2-eqkg DMI) as a result of both the lower DMI andthe lower CFP of the feeds (Table 9) Notably the CFP of the control diet per total DMIaligned with the average values reported by Gislon et al (2020) in a study conducted on171 dairy herds in the same Po Valley area where the present study was conducted [24]Conversely the impact per kilogram of DMI in the treatment diet was lower than the rangeof values reported in that survey [24]

Similarly the CFP of milk related to feed intake was significantly lower (p lt 00001) inthe treatment diet compared with the control diet (minus1689 26640 vs 32053 g CO2-eqLmilk) as a result of both the higher daily milk production and lower CFP of the feeds(Table 9) The reduction of CFP in milk related to the feed intake found in the present studywhich was also higher than the reduction (minus145) found by Salami et al (2021) [8]

The partial substitution of SBM with SRU led to a significant (p lt 00001) reductionin the predicted ruminal CH4 production (Table 9) It is important to underline that thepresent study did not quantify the real ruminal CH4 production Instead it was assessedby following the equation of Hristov et al (2013) [23] based on dry matter intake (DMI)The lower CH4 production in the treatment group was a function of both the lower DMIwhen expressed only as grams of CH4 per day and better feed efficiency and milk yieldwhen expressed as grams of CH4 per liter of milk

Notably there is limited published information on the effect of feeding urea on realenteric CH4 production Alipour et al (2020) [42] and Rebelo et al (2019) [43] showedthat feeding two sources of coated and non-coated urea did not affect enteric CH4 yieldmeasured in an in vitro ruminal fermentation system or beef cattle respectively However

Animals 2021 11 2405 13 of 15

Libera et al (2021) showed that the CH4 production at the ruminal level reduced after usingurea combined with enzymes and cereals in dairy cows This result was due to a reductionin the substrate available for methanogenesis and an influence on methanogens or otherrumen microorganisms [29] Although existing information suggests that slow-releaseurea (SRU) may have little or no effect on enteric CH4 emissions there is a crucial need forfuture studies to address this lack in the literature

4 Conclusions

This work contributes to defining scientific knowledge about the use of SRU in dairycowsrsquo nutrition and filling the present gap in the literature about their effect on environ-mental sustainability

This study showed that the substitution of traditional SBM (133 as fed) with thesource of SRU Protigen (022 as fed 100 gheadd) led to an improvement in dairycowsrsquo efficiency due to an enhanced feed conversion rate a lower dry matter intake ahigher digestibility of the fibrous parts of the diet and to better daily milk productionFurthermore the present study showed that the environmental sustainability of dairycowsrsquo diets could be improved by including SRU as an alternative protein source mainlyreducing the impact of the feed production in terms of greenhouse gas emissions andpredicted ruminal CH4 production Further research is needed to evaluate the effectiverole of SRU on methanogenesis and real ruminal production of methane

Supplementary Materials The following are available online at httpswwwmdpicomarticle103390ani11082405s1 Table S1 Technical information about the product Protigen used in thepresent trial particle size measured in millimetres and in vitro release rate at different time point

Author Contributions Conceptualization data curation writingmdashoriginal draft preparation re-view and editing SG conceptualization data curation RC data curation study validation LRmanuscript review MD data curation manuscript review IC conceptualization project admin-istration and supervision CASR All authors have read and agreed to the published version ofthe manuscript

Funding This research received no external funding

Institutional Review Board Statement The trial was a field and practical study not an experimentalone so it did not require approval For the trial we only used data commonly recorded by the farmer(milk production milk quality judged through monthly analyses reproductive parameters and soon) without any additional or ldquoexperimentalrdquo practices that can or will harm the animals or risktheir welfare The SRU product used in this study is already registered and used in dairy cowsrsquo feed

Data Availability Statement The data presented in this study are available on request from thecorresponding author

Conflicts of Interest The authors declare no conflict of interest

References1 United Nations Department of Economic and Social Affairs Population Division World Population Prospects 2019 Comprehensive

Tables (STESASERA426) United Nations Department of Economic and Social Affairs Population Division New York NYUSA 2019 Volume I

2 Georganas A Giamouri E Pappas AC Papadomichelakis G Galliou F Manios T Tsiplakou E Fegeros K ZervasG Bioactive Compounds in Food Waste A Review on the Transformation of Food Waste to Animal Feed Foods 2020 9 291[CrossRef]

3 Takiya CS Ylioja CM Bennett A Davidson MJ Sudbeck M Wickersham TA Vandehaar MJ Bradford BJ FeedingDairy Cows With ldquoLeftoversrdquo and the Variation in Recovery of Human-Edible Nutrients in Milk Front Sustain Food Syst 2019 3114 [CrossRef]

4 Capper JL Cady RA The effects of improved performance in the US dairy cattle industry on environmental impacts between2007 and 2017 J Anim Sci 2020 1 skz291 [CrossRef]

5 Wilkinson J Garnsworthy P Impact of diet and fertility on greenhouse gas emissions and nitrogen efficiency of milk productionLivestock 2017 22 140ndash144 [CrossRef]

Animals 2021 11 2405 4 of 15

Table 1 Composition and nutritional values of the two diets tested as predicted by the rationingsoftware (Plurimix Fabermatica Ostiano (CR) Italy)

Feed as Fed Control Treatment

Corn Silage 5056 5233Corn meal 1139 1012

Grass Silage 843 824High-moisture Corn 674 835

Soybean meal 44 CP 1 654 521RyeGrass Hay 584 572

Alfalfa Hay 420 411Linseed meal 291 257

Rapeseed meal 182 160Min Mix 157 154Protigen 000 022

Analysis of dm 2 in the TMR

dm 5481 5377Energy Mcalkg dm 162 162

CP 1 1502 1500RDP 3 on CP 6269 6533RUP 4 on CP 3731 3467

Sol CP 5 on CP 2883 3440Sol CP on RDP 4598 5266

Sugars 311 297Starch 2793 2825NDF 3471 3533ADF 2022 2048ADL 393 398Fat 290 289

Ca total 084 085p total 036 035

1 CP = crude protein 2 dm = dry matter 3 RDP = rumen degradable protein 4 RUP = rumen undegradableprotein 5 Sol CP = soluble crude protein

Monthly the energy corrected milk (ECM) was evaluated by comparing the values offat and protein obtained from the analyses and average milk production of the same weekThe ECM was calculated following the equation proposed by Tyrrel and Reid (1965) [20]

ECM = 0327 lowast Milk yield (L) + 1295 lowast Fat yield(Kg) + 72 lowast Protein Yield (kg) (1)

The BCS was assessed monthly by the farm veterinary staff on all cows involved in thetrial as proposed by Edmonson et al (1989) [21] and Ferguson et al (1994) [22] througha visual and tactile evaluation of body fat reserves using a 5-point scale with 025-pointincrements (1mdashvery thin cow 5mdashexcessively fat cow) where 3 represents the average bodycondition The evaluation focused on the rump and loin

Reproductive performance was also evaluated in the two groups considering the daysopen 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

232 Characteristics of the Dies Feces and Digestibility of the Feeds

The characteristics of both the diet and feces were monitored twice per month (startand end of each month) using a portable NIR instrument (Polispec IT Photonics FaraVicentino 36030 (VI) Italy) The monthly averages were then calculated The characteristicsof the TMR were analyzed in fresh feed with the portable NIR instrument while consideringthe entire bunk Specifically every time three measurements were gauged with the portableinstrument along the entire length of the feed bunk (beginning middle and end of themanger) Similarly the characteristics of the feces were analyzed for each group in a pool

Animals 2021 11 2405 5 of 15

of fecal material collected the day after each feed analysis The pool of fecal material wascollected directly by a rectal grab in 20 cows per group Samples of feces from the samegroup were then pooled together and mixed to create a single sample for each group Thepooled 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 detergentfiber (NDF) acid detergent lignin (ADL) starch and ash The content of hemicelluloseswas obtained from the difference between NDF and ADF The content of cellulose wasobtained from the difference between ADF and ADL Sugars and pectin were obtained bythe calculation 100 ndash(ash + fats + proteins + NDF + starch)

The digestibility was evaluated through the following formula

Digestibility =

(Xd

ADLd

)minus

(X f

ADL f

)(

XdADLd

) times 100 (2)

where

X = each analytical parameter considered ()ADL = acid detergent lignin ()d = dietf = feces

233 Environmental Impact Diet Carbon Footprint (CFP)

The CFP of the two diets was calculated to evaluate the effect of partial replacementon the traditional SBM with an SRU source on greenhouse gas emissions

The contribution of each feedrsquos raw material to the feedrsquos CFP was estimated bymultiplying the inclusion level of the raw material and the CFP per kilogram of dry matterof raw material (g CO2-eqkg) The CFP of each feedrsquos raw material was obtained fromboth the feed database created by Salami et al (2021) [8] which includes CFP values fromthe 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 productionfeed 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 fromproducts with a similar composition structure and characteristics were used [8]

The average CFP of each TMR was then calculated and expressed as g CO2-eqThe CFP of milk production as related to diet was calculated by dividing the weekly

TMR CFP by the average weekly milk production

234 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 coefficientof determination (R2) value (0880 root mean square error 153) between predicted andobserved values [24] among all the possible equations available [25] The equation isas follows

CH4 (gd) = 254 + 1914 times DMI (3)

where

1 CH4 = enteric methane production2 DMI = dry matter intake (kgheadday)

24 Statistical Analysis

Data analysis was conducted using SAS statistical software (SAS 94 SAS InstituteInc Cary NC USA)

Animals 2021 11 2405 6 of 15

Data distribution and homogeneity of variances were tested using PROC UNIVARI-ATE (SAS 94 SAS Institute Inc Cary NC USA) Data about production performanceand environmental impact were analyzed using a mixed model (PROC MIXED) whichconsidered the fixed effect of treatment and time of detection For digestibility data of thesingle diet component a residual estimate of maximum-likelihood was performed withPROC MIXED (SAS 94 SAS Institute Inc Cary NC USA) on a mixed model consideringthe fixed effects of treatment sampling day their interaction and the random effects of theanimal within the treatment period

A single-subject was used as an experimental unit in all the statistical evaluationsFor all the parameters a p-value le 005 was considered statistically significant

whereas a value le01 was considered a tendency

3 Results and Discussion31 Production Performances Milk Yield Energy Corrected Milk (ECM) Milk Quality FeedIntake Feed Conversion Rate Body Condition Score Reproductive Performances

Data about the production performance are reported in Tables 2 and 3 and Figure 1The partial substitution of SBM with an SRU significantly (p lt 00001) improved the dailymilk production and resulted in an average production increase of 39 during the entiretrial period corresponding to 154 Lheadday Moreover the results of ECM were alsosignificantly higher in the treatment group (p = 00017) As shown in Figure 1 productivitybegan to differ between the two groups in the third week of the study when the differencereached statistical significance The literature also recognized that an integration of the dietaimed at influencing ruminal fermentation requires a period of at least 3 weeks to clearlyshow its effects [26] As visible in Figure 1 milk production decreased from weeks 9 to13 and increased sharply afterward This great variation can be explained by changingenvironmental conditions (T C and humidity) Firstly between weeks 10 and 13 theadverse winter conditions which were very cold with heavy rain and humidity negativelyaffected both the animals and the microenvironment inside the stable These conditionsresulted in reduced feed intake and lower milk production with the declining health of themammary gland Conversely from weeks 14 to 15 the environmental conditions improvedquickly as spring began resulting in better housing conditions (eg drier litter in thecubicles cleaner and drier floors) and a more comfortable microenvironment inside thestable with positive reflexes on mammary health as well as feed intake and milk production

Table 2 Production performance milk yield feed intake feed conversion rate bodycondition scores and reproductive performance in the two groups

GroupSEM p-Value

Control Treatment

Production Performance

Milk yield Lheadday 3934 4089 013 lt00001

ECM 2 kg 4320 4487 037 00017DMI 3 kgheadday 2469 2392 004 lt00001

FCR 4 159 170 0004 lt00001

BCS 4

December week 3 287 2911 003 0351January week 7 302 306 003 0193

February week 12 317 318 003 0852March week 17 316 312 003 0181

Reproductive Performance

Days open 10146 10010 128 0454Services to pregnancy 208 197 009 0402

2 ECM = energy corrected milk 3 DMI = dry matter intake 4 FCR = feed conversion rate 5 BCS = bodycondition score

Animals 2021 11 2405 7 of 15

Table 3 Production performance milk quality analyses

December Week 3 January Week 7 February Week 12 March Week 17

Fat

Control 381 382 384 384Treatment 384 380 385 386

p-value 0720 0786 0901 0864

Proteins

Control 367 369 367 365Treatment 374 366 365 366

p-value 0234 0535 0847 0852

Urea mg100 mL (mmolL)

Control 2145 (3560) 2313 (3839) 2228 (3698) 2229 (3700)Treatment 2101 (3487) 2356 (3910) 2231 (3703) 2296 (3811)

p-value 0471 0479 0486 0274

Lactose

Control 482 483 484 482Treatment 483 482 484 486

p-value 0533 061 0886 0061

Somatic cells x000

Control 42657 48879 49629 47056Treatment 42102 48053 48878 46342

p-value 0957 0936 0942 0945

Fat yield kgday

Control 1517 1563 1497 1510Treatment 1590 1609 1552 1569

p-value 0104 0300 0224 0190

Protein yield kgday

Control 1464 1508 1432 1436Treatment 1550 1553 1476 1491

p-value 0031 0258 0272 01605

The result of the present study agreed with Tikofsky and Harrison (2007) [27] andInostroza et al (2010) [16] who reported a significant increase in milk production of cowsfed diets containing SRU Also Kowalski et al (2010) showed an improvement in milkproduction in high-yielding dairy cows fed with SRU in partial replacement of SBM [17]Supplementation of SRU in ruminant diets fed with high levels of rapidly fermentablecarbohydrates may increase the synchrony between the energy and protein availability atthe rumen level enhancing the microbial protein synthesis thus improving its efficiency ofconverting into milk [28] It should be emphasized that the use of urea (combined withenzyme and cereals as a slower and safer form of ruminally released nitrogen) dairy cowdiets can beneficially modulate ruminal fermentation including microbiota populations(an increase in relative abundances of Megasphaera elsdenii and ammonia-producing bacte-ria) consequently improving production performance as was mentioned by Libera et al2021 [29]

Animals 2021 11 2405 8 of 15

Animals 2021 11 x FOR PEER REVIEW 8 of 15

1 d= day 2ECM = energy corrected milk 3 DMI = dry matter intake 4 FCR = feed conversion rate 5 BCS = body condition score

Figure 1 Average weekly milk production in the two groups (a = p-value le 005 x = p-value le 01)

Table 3 Production performance milk quality analyses

December Week 3 January Week 7 February Week 12 March Week 17 Fat

Control 381 382 384 384 Treatment 384 380 385 386

p-value 0720 0786 0901 0864 Proteins

Control 367 369 367 365 Treatment 374 366 365 366

p-value 0234 0535 0847 0852 Urea mg100 mL (mmolL)

Control 2145 (3560) 2313 (3839) 2228 (3698) 2229 (3700) Treatment 2101 (3487) 2356 (3910) 2231 (3703) 2296 (3811)

p-value 0471 0479 0486 0274 Lactose

Control 482 483 484 482 Treatment 483 482 484 486

p-value 0533 061 0886 0061 Somatic cells x000

Control 42657 48879 49629 47056 Treatment 42102 48053 48878 46342

p-value 0957 0936 0942 0945 Fat yield kgday

Control 1517 1563 1497 1510 Treatment 1590 1609 1552 1569

p-value 0104 0300 0224 0190 Protein yield kgday

Control 1464 1508 1432 1436

Figure 1 Average weekly milk production in the two groups (a = p-value le 005 x = p-value le 01)

Conversely Galo et al (2003) [30] and Giallongo et al (2015) [31] did not show anygain in milk production when SBM was partially replaced by SRU

In the present study DMI was significantly reduced in the treatment group (2392 vs2469 kgheadd in control) (p = 004) positively affecting FCR In fact the FCR significantlyimproved (p lt 00001) during treatment with an overall increase in feed efficiency at 69due to the lower DMI and better milk production

These results agree with the findings of Salami et al (2021) who reported a 3enhancement in feed efficiency due to a significant reduction in feed intake without anyeffects on milk yield when the traditional protein sources were replaced with SRU in dairycowsrsquo diets in Northern Europe [8]

Reproductive performance remained unaffected by the treatment (Table 2) which is inagreement with the findings of Hallajian et al (2021) who reported similar characteristicsof the follicles blood levels of progesterone and milk urea nitrogen (MUN) between dairycows fed exclusively with SBM or with the partial replacement of SBM SRU [32] Theseresults show that feeding with SRU appears to overcome the possible negative effect ofother NPN sources such as feed grade urea on both plasma urea nitrogen and overallreproductive performance [33]

Body condition scores were not influenced by the treatment (Table 2) Similarly Nealet al (2014) [34] and Hallajian et al (2021) [32] did not report significant differences interms of body weight and body condition in dairy Holstein cows fed with diets containingSRU compared with SBM control diets

Also the treatment did not influence milk quality traits as reported in Table 3 Thesefindings align with the main results found in the literature regarding dairy cows fed withan appropriate amount of slow-release urea [16ndash30]

No treatment effects were found in the overall health condition monitored daily bythe farm veterinary staff

The positive results observed after including SRU as a partial substitute for SBMunderlined that ruminal kinetics and fermentation could be optimized in diets with apercentage of soluble protein higher than 30 of the total crude protein and 50 ofdegradable protein if combined with an adequate intake of nonstructural and rapidlyfermentable carbohydrates

Despite the significant increase in the solubility of the protein fraction no changes inmilk quality or reproductive performance were observed Conversely previous studies

Animals 2021 11 2405 9 of 15

showed an increase in the milk urea levels and a reduction in fertility after an increase inprotein solubility [3536]

32 Characteristics of the Diets Feces and Digestibility of the Feeds

Chemical characteristics of the diets are shown in Tables 4 and 5 while chemical char-acteristics of feces are shown in Tables 6 and 7 Results highlighted a good correspondencebetween the projection of the rationing software and the analytical characteristics found

Table 4 Analysis of the composition of the control diet done with the portable NIR instru-ment Polispec

Parameter December January February March April

dm 1 5491 5495 5485 5465 5470Ash dm 920 976 969 923 976

Crude protein dm 1596 1621 1620 1583 1617Fats dm 300 299 315 300 302NDF dm 3635 3665 3620 3643 3668

Cellulose dm 1874 1877 1927 1882 1871Lignin dm 392 389 389 393 394

Hemicellulose dm 1370 1400 1305 1368 1403Starch dm 2800 2815 2807 2815 2819

Sugars and pectins dm 749 625 670 737 6201 dm = dry matter

Table 5 Analysis of the composition of the Treatment diet done with the portable NIR instru-ment Polispec

Parameter December January February March April

dm 1 5379 5389 5378 5463 5455Ash dm 910 980 955 919 972

Crude protein dm 1600 1619 1635 1586 1605Fats dm 288 305 305 300 303NDF dm 3655 3625 3610 3655 3680

Cellulose dm 1880 1846 1844 1882 1873Lignin dm 385 395 390 393 389

Hemicellulose dm 1390 1385 1377 1380 1417Starch dm 2838 2842 2825 2805 2821

Sugars and pectins dm 710 630 670 735 6201 dm = dry matter

Table 6 Analysis of the composition of the Control feces done with the portable NIR instru-ment Polispec

Parameter December January February March April

Moisture 8697 8688 8660 8690 8703Dry matter 1303 1313 1340 1310 1297Ash dm 927 947 897 938 925

Crude protein dm 1700 1705 1715 1727 1700Fats dm 275 255 257 278 264NDF dm 6165 6163 6150 6147 6167

Cellulose dm 3593 3628 3530 3570 3581Lignin dm 1121 1112 1125 1128 1137

Hemicellulose dm 1451 1423 1495 1457 1450Starch dm 590 591 611 568 603

Sugars and pectins dm 244 240 271 242 241dm = dry matter

Animals 2021 11 2405 10 of 15

Table 7 Analysis of the composition of the Treatment feces done with the portable NIR instru-ment Polispec

Parameter December January February March April

Moisture 8684 8725 8600 8690 8703dm 1316 1275 1400 1310 1296

Ash dm 952 1025 925 1003 1003Crude protein dm 1646 1645 1668 1687 1660

Fats dm 270 289 263 285 282NDF dm 6212 6076 6170 6052 6091

Cellulose dm 3575 3485 3426 3422 3501Lignin dm 1135 1158 1193 1128 1137

Hemicellulose dm 1493 1434 1542 1502 1453Starch dm 580 625 600 620 618

Sugars and pectins dm 240 241 275 254 247dm = dry matter

Results for nutrients digestibility during the different months of the survey (Table 8)showed that the partial substitution of SBM with SRU significantly enhanced protein(p = 0012) NDF (p = 0039) and cellulose (p = 0033) digestibility Those results partiallyagreed with Sinclair et al (2008) who reported a significant improvement in ruminaldigestion of fiber in vitro [37] These findings can be partially explained by an increasedabundance and activity of fibrolytic bacteria in the rumen such as Ruminococcaceae whichuses ammonia as its main nitrogen source due to both a higher level of NH3 availabilityand better synchrony between nitrogen and carbohydrates in the rumen when SRU is usedas a partial substitution for SBM [3839] Furthermore Geron et al (2016) found a betterin vivo digestibility of crude protein in sheep fed with SRU as a partial replacement forSBM [40]

Table 8 Digestibility in the two groups

Month December January February March April Average P(g) 1 P(m) 1 P(g m) 1

Group Ash

Control 6481 6611 6792 6456 6714 66100069 0010 0528Treatment 6452 6436 6835 6191 6468 6477

Sem 104 104 104 104 104 046p-value 0848 0264 0775 0103 0127 0069

Crude Protein

Control 6279 6325 6342 6197 6356 63000012 0201 0763Treatment 6506 6537 6666 6290 6459 6492

Sem 099 099 099 099 099 044p-value 0139 0164 0044 0524 0483 0012

Fats

Control 6648 7027 7194 6786 6942 69200621 0018 0324Treatment 6920 6802 7232 6536 6891 6876

Sem 133 134 134 134 134 060p-value 0184 0266 0848 0218 0794 0621

NDF 2

Control 4077 4127 4129 4118 4171 41240039 0821 0952Treatment 4132 4289 4416 4226 4332 4299

Sem 116 116 116 116 116 116p-value 0368 0349 0113 0528 0351 0039

Animals 2021 11 2405 11 of 15

Table 8 Cont

Month December January February March April Average P(g) 1 P(m) 1 P(g m) 1

Cellulose

Control 3302 3247 3659 3388 3366 33930033 0234 0999Treatment 3552 3564 3930 3657 3601 3661

Sem 171 171 171 171 171 171p-value 0329 0221 0292 0295 0357 0033

Hemicellulose

Control 6300 6452 6039 6287 6417 62990470 0343 0783Treatment 6347 6476 6341 6204 6488 6371

Sem 154 154 154 154 154 276p-value 0833 0911 0193 0710 0746 0470

Starch

Control 9264 9266 9247 9297 9258 92660874 0456 0045Treatment 9307 9250 9306 9229 9250 9268

Sem 018 018 018 018 018 008p-value 0132 0550 005 0028 0776 0874

Sugars and Pectins

Control 8865 8658 8588 8858 8654 87250903 0003 0753Treatment 8853 8700 8657 8707 8636 8729

Sem 053 053 053 053 053 023p-value 0883 0583 0382 0441 0817 0903

1 g = group m = month g m = group month 2 NDF = neutral detergent fiber

33 Environmental Impact Carbon Footprint of the Feed (CFP) and Predicted Methane(CH4) Production

The impact of feeding SRU as a partial substitution for SBM on dairy sustainabilitywas evaluated by estimating the CFP of the different diet feeds and predicting the entericCH4 production (Table 9 Figure 2)

Table 9 Environmental impact carbon footprint (CFP) and predicted enteric CH4 production in thetwo groups

GroupSEM p-Value

Control Treatment

Diet CFP 1

CO2 eq 2 gDMI (kgDMI)1248400(1248)

1076400(1076)

2245(022) lt00001

CO2 eq gL milk 32053 26640 079 lt00001

Predicted enteric CH43 Production

CH4 gd 47528 46045 090 lt00001CH4 gL milk 1220 1130 003 lt00001

1 CFP = Carbon footprint of the two feeds 2 CO2 eq = equivalent to the carbon dioxide 3 CH4 = methane

The results revealed that SBM was the dominant contributor to the feed CFP account-ing for 5371 and 4808 of the total CFP of the control and treatment diets respectively(Figure 2) Notably the inclusion of SRU in the treatment diet contributed only 056 of thefeed CFP (Figure 2) These results align with the average values reported in other studiesfocused on intensive dairy cows farming where SBM and other plant-protein sources werereplaced by SRU [8]

Animals 2021 11 2405 12 of 15

Animals 2021 11 x FOR PEER REVIEW 12 of 15

The partial substitution of SBM with SRU led to a significant (p lt 00001) reduction in the predicted ruminal CH4 production (Table 9) It is important to underline that the pre-sent study did not quantify the real ruminal CH4 production Instead it was assessed by following the equation of Hristov et al (2013) [23] based on dry matter intake (DMI) The lower CH4 production in the treatment group was a function of both the lower DMI when expressed only as grams of CH4 per day and better feed efficiency and milk yield when expressed as grams of CH4 per liter of milk

Notably there is limited published information on the effect of feeding urea on real enteric CH4 production Alipour et al (2020) [42] and Rebelo et al (2019) [43] showed that feeding two sources of coated and non-coated urea did not affect enteric CH4 yield meas-ured in an in vitro ruminal fermentation system or beef cattle respectively However Libera et al (2021) showed that the CH4 production at the ruminal level reduced after using urea combined with enzymes and cereals in dairy cows This result was due to a reduction in the substrate available for methanogenesis and an influence on methanogens or other rumen microorganisms [29] Although existing information suggests that slow-release urea (SRU) may have little or no effect on enteric CH4 emissions there is a crucial need for future studies to address this lack in the literature

Table 9 Environmental impact carbon footprint (CFP) and predicted enteric CH4 production in the two groups

Group SEM p-Value

Control Treatment

Diet CFP 1

CO2 eq 2 gDMI (kgDMI) 1248400 (1248)

1076400 (1076)

2245 (022)

lt00001

CO2 eq gL milk 32053 26640 079 lt00001 Predicted enteric CH4 3 Production

CH4 gd 47528 46045 090 lt00001 CH4 gL milk 1220 1130 003 lt00001

1 CFP = Carbon footprint of the two feeds 2 CO2 eq = equivalent to the carbon dioxide 3 CH4 = methane

Figure 2 Contribution of different feedsrsquo raw materials to the average carbon footprint of (A) control diet (B) treatment diet

The partial replacement of SBM with SRU decreased the CFP of the treatment dietexpressed per 1 kilogram of diet (minus1098 44991 vs 50541 g CO2-eqkg diet) comparedto the control diet due to the lower global warming potential (GWP) of SRU than SBM [24]The highest GWP of soybean meal is mainly due to large transport distances and emissionsrelated to land-use change (LUC) such as deforestation which can lead to heavier emissionsof greenhouse gases [6ndash41] These results agree with the findings of Salami et al (2021)who showed a 12 reduction in CFP (g CO2-eqkg diet) when SBM and other plant-proteinsources were replaced by SRU [8]

The inclusion of SRU also significantly (p lt 00001) reduced the CFP of the diet on adry matter basis (1076 vs 1248 kg CO2-eqkg DMI) as a result of both the lower DMI andthe lower CFP of the feeds (Table 9) Notably the CFP of the control diet per total DMIaligned with the average values reported by Gislon et al (2020) in a study conducted on171 dairy herds in the same Po Valley area where the present study was conducted [24]Conversely the impact per kilogram of DMI in the treatment diet was lower than the rangeof values reported in that survey [24]

Similarly the CFP of milk related to feed intake was significantly lower (p lt 00001) inthe treatment diet compared with the control diet (minus1689 26640 vs 32053 g CO2-eqLmilk) as a result of both the higher daily milk production and lower CFP of the feeds(Table 9) The reduction of CFP in milk related to the feed intake found in the present studywhich was also higher than the reduction (minus145) found by Salami et al (2021) [8]

The partial substitution of SBM with SRU led to a significant (p lt 00001) reductionin the predicted ruminal CH4 production (Table 9) It is important to underline that thepresent study did not quantify the real ruminal CH4 production Instead it was assessedby following the equation of Hristov et al (2013) [23] based on dry matter intake (DMI)The lower CH4 production in the treatment group was a function of both the lower DMIwhen expressed only as grams of CH4 per day and better feed efficiency and milk yieldwhen expressed as grams of CH4 per liter of milk

Notably there is limited published information on the effect of feeding urea on realenteric CH4 production Alipour et al (2020) [42] and Rebelo et al (2019) [43] showedthat feeding two sources of coated and non-coated urea did not affect enteric CH4 yieldmeasured in an in vitro ruminal fermentation system or beef cattle respectively However

Animals 2021 11 2405 13 of 15

Libera et al (2021) showed that the CH4 production at the ruminal level reduced after usingurea combined with enzymes and cereals in dairy cows This result was due to a reductionin the substrate available for methanogenesis and an influence on methanogens or otherrumen microorganisms [29] Although existing information suggests that slow-releaseurea (SRU) may have little or no effect on enteric CH4 emissions there is a crucial need forfuture studies to address this lack in the literature

4 Conclusions

This work contributes to defining scientific knowledge about the use of SRU in dairycowsrsquo nutrition and filling the present gap in the literature about their effect on environ-mental sustainability

This study showed that the substitution of traditional SBM (133 as fed) with thesource of SRU Protigen (022 as fed 100 gheadd) led to an improvement in dairycowsrsquo efficiency due to an enhanced feed conversion rate a lower dry matter intake ahigher digestibility of the fibrous parts of the diet and to better daily milk productionFurthermore the present study showed that the environmental sustainability of dairycowsrsquo diets could be improved by including SRU as an alternative protein source mainlyreducing the impact of the feed production in terms of greenhouse gas emissions andpredicted ruminal CH4 production Further research is needed to evaluate the effectiverole of SRU on methanogenesis and real ruminal production of methane

Supplementary Materials The following are available online at httpswwwmdpicomarticle103390ani11082405s1 Table S1 Technical information about the product Protigen used in thepresent trial particle size measured in millimetres and in vitro release rate at different time point

Author Contributions Conceptualization data curation writingmdashoriginal draft preparation re-view and editing SG conceptualization data curation RC data curation study validation LRmanuscript review MD data curation manuscript review IC conceptualization project admin-istration and supervision CASR All authors have read and agreed to the published version ofthe manuscript

Funding This research received no external funding

Institutional Review Board Statement The trial was a field and practical study not an experimentalone so it did not require approval For the trial we only used data commonly recorded by the farmer(milk production milk quality judged through monthly analyses reproductive parameters and soon) without any additional or ldquoexperimentalrdquo practices that can or will harm the animals or risktheir welfare The SRU product used in this study is already registered and used in dairy cowsrsquo feed

Data Availability Statement The data presented in this study are available on request from thecorresponding author

Conflicts of Interest The authors declare no conflict of interest

References1 United Nations Department of Economic and Social Affairs Population Division World Population Prospects 2019 Comprehensive

Tables (STESASERA426) United Nations Department of Economic and Social Affairs Population Division New York NYUSA 2019 Volume I

2 Georganas A Giamouri E Pappas AC Papadomichelakis G Galliou F Manios T Tsiplakou E Fegeros K ZervasG Bioactive Compounds in Food Waste A Review on the Transformation of Food Waste to Animal Feed Foods 2020 9 291[CrossRef]

3 Takiya CS Ylioja CM Bennett A Davidson MJ Sudbeck M Wickersham TA Vandehaar MJ Bradford BJ FeedingDairy Cows With ldquoLeftoversrdquo and the Variation in Recovery of Human-Edible Nutrients in Milk Front Sustain Food Syst 2019 3114 [CrossRef]

4 Capper JL Cady RA The effects of improved performance in the US dairy cattle industry on environmental impacts between2007 and 2017 J Anim Sci 2020 1 skz291 [CrossRef]

5 Wilkinson J Garnsworthy P Impact of diet and fertility on greenhouse gas emissions and nitrogen efficiency of milk productionLivestock 2017 22 140ndash144 [CrossRef]

Animals 2021 11 2405 5 of 15

of fecal material collected the day after each feed analysis The pool of fecal material wascollected directly by a rectal grab in 20 cows per group Samples of feces from the samegroup were then pooled together and mixed to create a single sample for each group Thepooled 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 detergentfiber (NDF) acid detergent lignin (ADL) starch and ash The content of hemicelluloseswas obtained from the difference between NDF and ADF The content of cellulose wasobtained from the difference between ADF and ADL Sugars and pectin were obtained bythe calculation 100 ndash(ash + fats + proteins + NDF + starch)

The digestibility was evaluated through the following formula

Digestibility =

(Xd

ADLd

)minus

(X f

ADL f

)(

XdADLd

) times 100 (2)

where

X = each analytical parameter considered ()ADL = acid detergent lignin ()d = dietf = feces

233 Environmental Impact Diet Carbon Footprint (CFP)

The CFP of the two diets was calculated to evaluate the effect of partial replacementon the traditional SBM with an SRU source on greenhouse gas emissions

The contribution of each feedrsquos raw material to the feedrsquos CFP was estimated bymultiplying the inclusion level of the raw material and the CFP per kilogram of dry matterof raw material (g CO2-eqkg) The CFP of each feedrsquos raw material was obtained fromboth the feed database created by Salami et al (2021) [8] which includes CFP values fromthe 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 productionfeed 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 fromproducts with a similar composition structure and characteristics were used [8]

The average CFP of each TMR was then calculated and expressed as g CO2-eqThe CFP of milk production as related to diet was calculated by dividing the weekly

TMR CFP by the average weekly milk production

234 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 coefficientof determination (R2) value (0880 root mean square error 153) between predicted andobserved values [24] among all the possible equations available [25] The equation isas follows

CH4 (gd) = 254 + 1914 times DMI (3)

where

1 CH4 = enteric methane production2 DMI = dry matter intake (kgheadday)

24 Statistical Analysis

Data analysis was conducted using SAS statistical software (SAS 94 SAS InstituteInc Cary NC USA)

Animals 2021 11 2405 6 of 15

Data distribution and homogeneity of variances were tested using PROC UNIVARI-ATE (SAS 94 SAS Institute Inc Cary NC USA) Data about production performanceand environmental impact were analyzed using a mixed model (PROC MIXED) whichconsidered the fixed effect of treatment and time of detection For digestibility data of thesingle diet component a residual estimate of maximum-likelihood was performed withPROC MIXED (SAS 94 SAS Institute Inc Cary NC USA) on a mixed model consideringthe fixed effects of treatment sampling day their interaction and the random effects of theanimal within the treatment period

A single-subject was used as an experimental unit in all the statistical evaluationsFor all the parameters a p-value le 005 was considered statistically significant

whereas a value le01 was considered a tendency

3 Results and Discussion31 Production Performances Milk Yield Energy Corrected Milk (ECM) Milk Quality FeedIntake Feed Conversion Rate Body Condition Score Reproductive Performances

Data about the production performance are reported in Tables 2 and 3 and Figure 1The partial substitution of SBM with an SRU significantly (p lt 00001) improved the dailymilk production and resulted in an average production increase of 39 during the entiretrial period corresponding to 154 Lheadday Moreover the results of ECM were alsosignificantly higher in the treatment group (p = 00017) As shown in Figure 1 productivitybegan to differ between the two groups in the third week of the study when the differencereached statistical significance The literature also recognized that an integration of the dietaimed at influencing ruminal fermentation requires a period of at least 3 weeks to clearlyshow its effects [26] As visible in Figure 1 milk production decreased from weeks 9 to13 and increased sharply afterward This great variation can be explained by changingenvironmental conditions (T C and humidity) Firstly between weeks 10 and 13 theadverse winter conditions which were very cold with heavy rain and humidity negativelyaffected both the animals and the microenvironment inside the stable These conditionsresulted in reduced feed intake and lower milk production with the declining health of themammary gland Conversely from weeks 14 to 15 the environmental conditions improvedquickly as spring began resulting in better housing conditions (eg drier litter in thecubicles cleaner and drier floors) and a more comfortable microenvironment inside thestable with positive reflexes on mammary health as well as feed intake and milk production

Table 2 Production performance milk yield feed intake feed conversion rate bodycondition scores and reproductive performance in the two groups

GroupSEM p-Value

Control Treatment

Production Performance

Milk yield Lheadday 3934 4089 013 lt00001

ECM 2 kg 4320 4487 037 00017DMI 3 kgheadday 2469 2392 004 lt00001

FCR 4 159 170 0004 lt00001

BCS 4

December week 3 287 2911 003 0351January week 7 302 306 003 0193

February week 12 317 318 003 0852March week 17 316 312 003 0181

Reproductive Performance

Days open 10146 10010 128 0454Services to pregnancy 208 197 009 0402

2 ECM = energy corrected milk 3 DMI = dry matter intake 4 FCR = feed conversion rate 5 BCS = bodycondition score

Animals 2021 11 2405 7 of 15

Table 3 Production performance milk quality analyses

December Week 3 January Week 7 February Week 12 March Week 17

Fat

Control 381 382 384 384Treatment 384 380 385 386

p-value 0720 0786 0901 0864

Proteins

Control 367 369 367 365Treatment 374 366 365 366

p-value 0234 0535 0847 0852

Urea mg100 mL (mmolL)

Control 2145 (3560) 2313 (3839) 2228 (3698) 2229 (3700)Treatment 2101 (3487) 2356 (3910) 2231 (3703) 2296 (3811)

p-value 0471 0479 0486 0274

Lactose

Control 482 483 484 482Treatment 483 482 484 486

p-value 0533 061 0886 0061

Somatic cells x000

Control 42657 48879 49629 47056Treatment 42102 48053 48878 46342

p-value 0957 0936 0942 0945

Fat yield kgday

Control 1517 1563 1497 1510Treatment 1590 1609 1552 1569

p-value 0104 0300 0224 0190

Protein yield kgday

Control 1464 1508 1432 1436Treatment 1550 1553 1476 1491

p-value 0031 0258 0272 01605

The result of the present study agreed with Tikofsky and Harrison (2007) [27] andInostroza et al (2010) [16] who reported a significant increase in milk production of cowsfed diets containing SRU Also Kowalski et al (2010) showed an improvement in milkproduction in high-yielding dairy cows fed with SRU in partial replacement of SBM [17]Supplementation of SRU in ruminant diets fed with high levels of rapidly fermentablecarbohydrates may increase the synchrony between the energy and protein availability atthe rumen level enhancing the microbial protein synthesis thus improving its efficiency ofconverting into milk [28] It should be emphasized that the use of urea (combined withenzyme and cereals as a slower and safer form of ruminally released nitrogen) dairy cowdiets can beneficially modulate ruminal fermentation including microbiota populations(an increase in relative abundances of Megasphaera elsdenii and ammonia-producing bacte-ria) consequently improving production performance as was mentioned by Libera et al2021 [29]

Animals 2021 11 2405 8 of 15

Animals 2021 11 x FOR PEER REVIEW 8 of 15

1 d= day 2ECM = energy corrected milk 3 DMI = dry matter intake 4 FCR = feed conversion rate 5 BCS = body condition score

Figure 1 Average weekly milk production in the two groups (a = p-value le 005 x = p-value le 01)

Table 3 Production performance milk quality analyses

December Week 3 January Week 7 February Week 12 March Week 17 Fat

Control 381 382 384 384 Treatment 384 380 385 386

p-value 0720 0786 0901 0864 Proteins

Control 367 369 367 365 Treatment 374 366 365 366

p-value 0234 0535 0847 0852 Urea mg100 mL (mmolL)

Control 2145 (3560) 2313 (3839) 2228 (3698) 2229 (3700) Treatment 2101 (3487) 2356 (3910) 2231 (3703) 2296 (3811)

p-value 0471 0479 0486 0274 Lactose

Control 482 483 484 482 Treatment 483 482 484 486

p-value 0533 061 0886 0061 Somatic cells x000

Control 42657 48879 49629 47056 Treatment 42102 48053 48878 46342

p-value 0957 0936 0942 0945 Fat yield kgday

Control 1517 1563 1497 1510 Treatment 1590 1609 1552 1569

p-value 0104 0300 0224 0190 Protein yield kgday

Control 1464 1508 1432 1436

Figure 1 Average weekly milk production in the two groups (a = p-value le 005 x = p-value le 01)

Conversely Galo et al (2003) [30] and Giallongo et al (2015) [31] did not show anygain in milk production when SBM was partially replaced by SRU

In the present study DMI was significantly reduced in the treatment group (2392 vs2469 kgheadd in control) (p = 004) positively affecting FCR In fact the FCR significantlyimproved (p lt 00001) during treatment with an overall increase in feed efficiency at 69due to the lower DMI and better milk production

These results agree with the findings of Salami et al (2021) who reported a 3enhancement in feed efficiency due to a significant reduction in feed intake without anyeffects on milk yield when the traditional protein sources were replaced with SRU in dairycowsrsquo diets in Northern Europe [8]

Reproductive performance remained unaffected by the treatment (Table 2) which is inagreement with the findings of Hallajian et al (2021) who reported similar characteristicsof the follicles blood levels of progesterone and milk urea nitrogen (MUN) between dairycows fed exclusively with SBM or with the partial replacement of SBM SRU [32] Theseresults show that feeding with SRU appears to overcome the possible negative effect ofother NPN sources such as feed grade urea on both plasma urea nitrogen and overallreproductive performance [33]

Body condition scores were not influenced by the treatment (Table 2) Similarly Nealet al (2014) [34] and Hallajian et al (2021) [32] did not report significant differences interms of body weight and body condition in dairy Holstein cows fed with diets containingSRU compared with SBM control diets

Also the treatment did not influence milk quality traits as reported in Table 3 Thesefindings align with the main results found in the literature regarding dairy cows fed withan appropriate amount of slow-release urea [16ndash30]

No treatment effects were found in the overall health condition monitored daily bythe farm veterinary staff

The positive results observed after including SRU as a partial substitute for SBMunderlined that ruminal kinetics and fermentation could be optimized in diets with apercentage of soluble protein higher than 30 of the total crude protein and 50 ofdegradable protein if combined with an adequate intake of nonstructural and rapidlyfermentable carbohydrates

Despite the significant increase in the solubility of the protein fraction no changes inmilk quality or reproductive performance were observed Conversely previous studies

Animals 2021 11 2405 9 of 15

showed an increase in the milk urea levels and a reduction in fertility after an increase inprotein solubility [3536]

32 Characteristics of the Diets Feces and Digestibility of the Feeds

Chemical characteristics of the diets are shown in Tables 4 and 5 while chemical char-acteristics of feces are shown in Tables 6 and 7 Results highlighted a good correspondencebetween the projection of the rationing software and the analytical characteristics found

Table 4 Analysis of the composition of the control diet done with the portable NIR instru-ment Polispec

Parameter December January February March April

dm 1 5491 5495 5485 5465 5470Ash dm 920 976 969 923 976

Crude protein dm 1596 1621 1620 1583 1617Fats dm 300 299 315 300 302NDF dm 3635 3665 3620 3643 3668

Cellulose dm 1874 1877 1927 1882 1871Lignin dm 392 389 389 393 394

Hemicellulose dm 1370 1400 1305 1368 1403Starch dm 2800 2815 2807 2815 2819

Sugars and pectins dm 749 625 670 737 6201 dm = dry matter

Table 5 Analysis of the composition of the Treatment diet done with the portable NIR instru-ment Polispec

Parameter December January February March April

dm 1 5379 5389 5378 5463 5455Ash dm 910 980 955 919 972

Crude protein dm 1600 1619 1635 1586 1605Fats dm 288 305 305 300 303NDF dm 3655 3625 3610 3655 3680

Cellulose dm 1880 1846 1844 1882 1873Lignin dm 385 395 390 393 389

Hemicellulose dm 1390 1385 1377 1380 1417Starch dm 2838 2842 2825 2805 2821

Sugars and pectins dm 710 630 670 735 6201 dm = dry matter

Table 6 Analysis of the composition of the Control feces done with the portable NIR instru-ment Polispec

Parameter December January February March April

Moisture 8697 8688 8660 8690 8703Dry matter 1303 1313 1340 1310 1297Ash dm 927 947 897 938 925

Crude protein dm 1700 1705 1715 1727 1700Fats dm 275 255 257 278 264NDF dm 6165 6163 6150 6147 6167

Cellulose dm 3593 3628 3530 3570 3581Lignin dm 1121 1112 1125 1128 1137

Hemicellulose dm 1451 1423 1495 1457 1450Starch dm 590 591 611 568 603

Sugars and pectins dm 244 240 271 242 241dm = dry matter

Animals 2021 11 2405 10 of 15

Table 7 Analysis of the composition of the Treatment feces done with the portable NIR instru-ment Polispec

Parameter December January February March April

Moisture 8684 8725 8600 8690 8703dm 1316 1275 1400 1310 1296

Ash dm 952 1025 925 1003 1003Crude protein dm 1646 1645 1668 1687 1660

Fats dm 270 289 263 285 282NDF dm 6212 6076 6170 6052 6091

Cellulose dm 3575 3485 3426 3422 3501Lignin dm 1135 1158 1193 1128 1137

Hemicellulose dm 1493 1434 1542 1502 1453Starch dm 580 625 600 620 618

Sugars and pectins dm 240 241 275 254 247dm = dry matter

Results for nutrients digestibility during the different months of the survey (Table 8)showed that the partial substitution of SBM with SRU significantly enhanced protein(p = 0012) NDF (p = 0039) and cellulose (p = 0033) digestibility Those results partiallyagreed with Sinclair et al (2008) who reported a significant improvement in ruminaldigestion of fiber in vitro [37] These findings can be partially explained by an increasedabundance and activity of fibrolytic bacteria in the rumen such as Ruminococcaceae whichuses ammonia as its main nitrogen source due to both a higher level of NH3 availabilityand better synchrony between nitrogen and carbohydrates in the rumen when SRU is usedas a partial substitution for SBM [3839] Furthermore Geron et al (2016) found a betterin vivo digestibility of crude protein in sheep fed with SRU as a partial replacement forSBM [40]

Table 8 Digestibility in the two groups

Month December January February March April Average P(g) 1 P(m) 1 P(g m) 1

Group Ash

Control 6481 6611 6792 6456 6714 66100069 0010 0528Treatment 6452 6436 6835 6191 6468 6477

Sem 104 104 104 104 104 046p-value 0848 0264 0775 0103 0127 0069

Crude Protein

Control 6279 6325 6342 6197 6356 63000012 0201 0763Treatment 6506 6537 6666 6290 6459 6492

Sem 099 099 099 099 099 044p-value 0139 0164 0044 0524 0483 0012

Fats

Control 6648 7027 7194 6786 6942 69200621 0018 0324Treatment 6920 6802 7232 6536 6891 6876

Sem 133 134 134 134 134 060p-value 0184 0266 0848 0218 0794 0621

NDF 2

Control 4077 4127 4129 4118 4171 41240039 0821 0952Treatment 4132 4289 4416 4226 4332 4299

Sem 116 116 116 116 116 116p-value 0368 0349 0113 0528 0351 0039

Animals 2021 11 2405 11 of 15

Table 8 Cont

Month December January February March April Average P(g) 1 P(m) 1 P(g m) 1

Cellulose

Control 3302 3247 3659 3388 3366 33930033 0234 0999Treatment 3552 3564 3930 3657 3601 3661

Sem 171 171 171 171 171 171p-value 0329 0221 0292 0295 0357 0033

Hemicellulose

Control 6300 6452 6039 6287 6417 62990470 0343 0783Treatment 6347 6476 6341 6204 6488 6371

Sem 154 154 154 154 154 276p-value 0833 0911 0193 0710 0746 0470

Starch

Control 9264 9266 9247 9297 9258 92660874 0456 0045Treatment 9307 9250 9306 9229 9250 9268

Sem 018 018 018 018 018 008p-value 0132 0550 005 0028 0776 0874

Sugars and Pectins

Control 8865 8658 8588 8858 8654 87250903 0003 0753Treatment 8853 8700 8657 8707 8636 8729

Sem 053 053 053 053 053 023p-value 0883 0583 0382 0441 0817 0903

1 g = group m = month g m = group month 2 NDF = neutral detergent fiber

33 Environmental Impact Carbon Footprint of the Feed (CFP) and Predicted Methane(CH4) Production

The impact of feeding SRU as a partial substitution for SBM on dairy sustainabilitywas evaluated by estimating the CFP of the different diet feeds and predicting the entericCH4 production (Table 9 Figure 2)

Table 9 Environmental impact carbon footprint (CFP) and predicted enteric CH4 production in thetwo groups

GroupSEM p-Value

Control Treatment

Diet CFP 1

CO2 eq 2 gDMI (kgDMI)1248400(1248)

1076400(1076)

2245(022) lt00001

CO2 eq gL milk 32053 26640 079 lt00001

Predicted enteric CH43 Production

CH4 gd 47528 46045 090 lt00001CH4 gL milk 1220 1130 003 lt00001

1 CFP = Carbon footprint of the two feeds 2 CO2 eq = equivalent to the carbon dioxide 3 CH4 = methane

The results revealed that SBM was the dominant contributor to the feed CFP account-ing for 5371 and 4808 of the total CFP of the control and treatment diets respectively(Figure 2) Notably the inclusion of SRU in the treatment diet contributed only 056 of thefeed CFP (Figure 2) These results align with the average values reported in other studiesfocused on intensive dairy cows farming where SBM and other plant-protein sources werereplaced by SRU [8]

Animals 2021 11 2405 12 of 15

Animals 2021 11 x FOR PEER REVIEW 12 of 15

The partial substitution of SBM with SRU led to a significant (p lt 00001) reduction in the predicted ruminal CH4 production (Table 9) It is important to underline that the pre-sent study did not quantify the real ruminal CH4 production Instead it was assessed by following the equation of Hristov et al (2013) [23] based on dry matter intake (DMI) The lower CH4 production in the treatment group was a function of both the lower DMI when expressed only as grams of CH4 per day and better feed efficiency and milk yield when expressed as grams of CH4 per liter of milk

Notably there is limited published information on the effect of feeding urea on real enteric CH4 production Alipour et al (2020) [42] and Rebelo et al (2019) [43] showed that feeding two sources of coated and non-coated urea did not affect enteric CH4 yield meas-ured in an in vitro ruminal fermentation system or beef cattle respectively However Libera et al (2021) showed that the CH4 production at the ruminal level reduced after using urea combined with enzymes and cereals in dairy cows This result was due to a reduction in the substrate available for methanogenesis and an influence on methanogens or other rumen microorganisms [29] Although existing information suggests that slow-release urea (SRU) may have little or no effect on enteric CH4 emissions there is a crucial need for future studies to address this lack in the literature

Table 9 Environmental impact carbon footprint (CFP) and predicted enteric CH4 production in the two groups

Group SEM p-Value

Control Treatment

Diet CFP 1

CO2 eq 2 gDMI (kgDMI) 1248400 (1248)

1076400 (1076)

2245 (022)

lt00001

CO2 eq gL milk 32053 26640 079 lt00001 Predicted enteric CH4 3 Production

CH4 gd 47528 46045 090 lt00001 CH4 gL milk 1220 1130 003 lt00001

1 CFP = Carbon footprint of the two feeds 2 CO2 eq = equivalent to the carbon dioxide 3 CH4 = methane

Figure 2 Contribution of different feedsrsquo raw materials to the average carbon footprint of (A) control diet (B) treatment diet

The partial replacement of SBM with SRU decreased the CFP of the treatment dietexpressed per 1 kilogram of diet (minus1098 44991 vs 50541 g CO2-eqkg diet) comparedto the control diet due to the lower global warming potential (GWP) of SRU than SBM [24]The highest GWP of soybean meal is mainly due to large transport distances and emissionsrelated to land-use change (LUC) such as deforestation which can lead to heavier emissionsof greenhouse gases [6ndash41] These results agree with the findings of Salami et al (2021)who showed a 12 reduction in CFP (g CO2-eqkg diet) when SBM and other plant-proteinsources were replaced by SRU [8]

The inclusion of SRU also significantly (p lt 00001) reduced the CFP of the diet on adry matter basis (1076 vs 1248 kg CO2-eqkg DMI) as a result of both the lower DMI andthe lower CFP of the feeds (Table 9) Notably the CFP of the control diet per total DMIaligned with the average values reported by Gislon et al (2020) in a study conducted on171 dairy herds in the same Po Valley area where the present study was conducted [24]Conversely the impact per kilogram of DMI in the treatment diet was lower than the rangeof values reported in that survey [24]

Similarly the CFP of milk related to feed intake was significantly lower (p lt 00001) inthe treatment diet compared with the control diet (minus1689 26640 vs 32053 g CO2-eqLmilk) as a result of both the higher daily milk production and lower CFP of the feeds(Table 9) The reduction of CFP in milk related to the feed intake found in the present studywhich was also higher than the reduction (minus145) found by Salami et al (2021) [8]

The partial substitution of SBM with SRU led to a significant (p lt 00001) reductionin the predicted ruminal CH4 production (Table 9) It is important to underline that thepresent study did not quantify the real ruminal CH4 production Instead it was assessedby following the equation of Hristov et al (2013) [23] based on dry matter intake (DMI)The lower CH4 production in the treatment group was a function of both the lower DMIwhen expressed only as grams of CH4 per day and better feed efficiency and milk yieldwhen expressed as grams of CH4 per liter of milk

Notably there is limited published information on the effect of feeding urea on realenteric CH4 production Alipour et al (2020) [42] and Rebelo et al (2019) [43] showedthat feeding two sources of coated and non-coated urea did not affect enteric CH4 yieldmeasured in an in vitro ruminal fermentation system or beef cattle respectively However

Animals 2021 11 2405 13 of 15

Libera et al (2021) showed that the CH4 production at the ruminal level reduced after usingurea combined with enzymes and cereals in dairy cows This result was due to a reductionin the substrate available for methanogenesis and an influence on methanogens or otherrumen microorganisms [29] Although existing information suggests that slow-releaseurea (SRU) may have little or no effect on enteric CH4 emissions there is a crucial need forfuture studies to address this lack in the literature

4 Conclusions

This work contributes to defining scientific knowledge about the use of SRU in dairycowsrsquo nutrition and filling the present gap in the literature about their effect on environ-mental sustainability

This study showed that the substitution of traditional SBM (133 as fed) with thesource of SRU Protigen (022 as fed 100 gheadd) led to an improvement in dairycowsrsquo efficiency due to an enhanced feed conversion rate a lower dry matter intake ahigher digestibility of the fibrous parts of the diet and to better daily milk productionFurthermore the present study showed that the environmental sustainability of dairycowsrsquo diets could be improved by including SRU as an alternative protein source mainlyreducing the impact of the feed production in terms of greenhouse gas emissions andpredicted ruminal CH4 production Further research is needed to evaluate the effectiverole of SRU on methanogenesis and real ruminal production of methane

Supplementary Materials The following are available online at httpswwwmdpicomarticle103390ani11082405s1 Table S1 Technical information about the product Protigen used in thepresent trial particle size measured in millimetres and in vitro release rate at different time point

Author Contributions Conceptualization data curation writingmdashoriginal draft preparation re-view and editing SG conceptualization data curation RC data curation study validation LRmanuscript review MD data curation manuscript review IC conceptualization project admin-istration and supervision CASR All authors have read and agreed to the published version ofthe manuscript

Funding This research received no external funding

Institutional Review Board Statement The trial was a field and practical study not an experimentalone so it did not require approval For the trial we only used data commonly recorded by the farmer(milk production milk quality judged through monthly analyses reproductive parameters and soon) without any additional or ldquoexperimentalrdquo practices that can or will harm the animals or risktheir welfare The SRU product used in this study is already registered and used in dairy cowsrsquo feed

Data Availability Statement The data presented in this study are available on request from thecorresponding author

Conflicts of Interest The authors declare no conflict of interest

References1 United Nations Department of Economic and Social Affairs Population Division World Population Prospects 2019 Comprehensive

Tables (STESASERA426) United Nations Department of Economic and Social Affairs Population Division New York NYUSA 2019 Volume I

2 Georganas A Giamouri E Pappas AC Papadomichelakis G Galliou F Manios T Tsiplakou E Fegeros K ZervasG Bioactive Compounds in Food Waste A Review on the Transformation of Food Waste to Animal Feed Foods 2020 9 291[CrossRef]

3 Takiya CS Ylioja CM Bennett A Davidson MJ Sudbeck M Wickersham TA Vandehaar MJ Bradford BJ FeedingDairy Cows With ldquoLeftoversrdquo and the Variation in Recovery of Human-Edible Nutrients in Milk Front Sustain Food Syst 2019 3114 [CrossRef]

4 Capper JL Cady RA The effects of improved performance in the US dairy cattle industry on environmental impacts between2007 and 2017 J Anim Sci 2020 1 skz291 [CrossRef]

5 Wilkinson J Garnsworthy P Impact of diet and fertility on greenhouse gas emissions and nitrogen efficiency of milk productionLivestock 2017 22 140ndash144 [CrossRef]

Animals 2021 11 2405 6 of 15

Data distribution and homogeneity of variances were tested using PROC UNIVARI-ATE (SAS 94 SAS Institute Inc Cary NC USA) Data about production performanceand environmental impact were analyzed using a mixed model (PROC MIXED) whichconsidered the fixed effect of treatment and time of detection For digestibility data of thesingle diet component a residual estimate of maximum-likelihood was performed withPROC MIXED (SAS 94 SAS Institute Inc Cary NC USA) on a mixed model consideringthe fixed effects of treatment sampling day their interaction and the random effects of theanimal within the treatment period

A single-subject was used as an experimental unit in all the statistical evaluationsFor all the parameters a p-value le 005 was considered statistically significant

whereas a value le01 was considered a tendency

3 Results and Discussion31 Production Performances Milk Yield Energy Corrected Milk (ECM) Milk Quality FeedIntake Feed Conversion Rate Body Condition Score Reproductive Performances

Data about the production performance are reported in Tables 2 and 3 and Figure 1The partial substitution of SBM with an SRU significantly (p lt 00001) improved the dailymilk production and resulted in an average production increase of 39 during the entiretrial period corresponding to 154 Lheadday Moreover the results of ECM were alsosignificantly higher in the treatment group (p = 00017) As shown in Figure 1 productivitybegan to differ between the two groups in the third week of the study when the differencereached statistical significance The literature also recognized that an integration of the dietaimed at influencing ruminal fermentation requires a period of at least 3 weeks to clearlyshow its effects [26] As visible in Figure 1 milk production decreased from weeks 9 to13 and increased sharply afterward This great variation can be explained by changingenvironmental conditions (T C and humidity) Firstly between weeks 10 and 13 theadverse winter conditions which were very cold with heavy rain and humidity negativelyaffected both the animals and the microenvironment inside the stable These conditionsresulted in reduced feed intake and lower milk production with the declining health of themammary gland Conversely from weeks 14 to 15 the environmental conditions improvedquickly as spring began resulting in better housing conditions (eg drier litter in thecubicles cleaner and drier floors) and a more comfortable microenvironment inside thestable with positive reflexes on mammary health as well as feed intake and milk production

Table 2 Production performance milk yield feed intake feed conversion rate bodycondition scores and reproductive performance in the two groups

GroupSEM p-Value

Control Treatment

Production Performance

Milk yield Lheadday 3934 4089 013 lt00001

ECM 2 kg 4320 4487 037 00017DMI 3 kgheadday 2469 2392 004 lt00001

FCR 4 159 170 0004 lt00001

BCS 4

December week 3 287 2911 003 0351January week 7 302 306 003 0193

February week 12 317 318 003 0852March week 17 316 312 003 0181

Reproductive Performance

Days open 10146 10010 128 0454Services to pregnancy 208 197 009 0402

2 ECM = energy corrected milk 3 DMI = dry matter intake 4 FCR = feed conversion rate 5 BCS = bodycondition score

Animals 2021 11 2405 7 of 15

Table 3 Production performance milk quality analyses

December Week 3 January Week 7 February Week 12 March Week 17

Fat

Control 381 382 384 384Treatment 384 380 385 386

p-value 0720 0786 0901 0864

Proteins

Control 367 369 367 365Treatment 374 366 365 366

p-value 0234 0535 0847 0852

Urea mg100 mL (mmolL)

Control 2145 (3560) 2313 (3839) 2228 (3698) 2229 (3700)Treatment 2101 (3487) 2356 (3910) 2231 (3703) 2296 (3811)

p-value 0471 0479 0486 0274

Lactose

Control 482 483 484 482Treatment 483 482 484 486

p-value 0533 061 0886 0061

Somatic cells x000

Control 42657 48879 49629 47056Treatment 42102 48053 48878 46342

p-value 0957 0936 0942 0945

Fat yield kgday

Control 1517 1563 1497 1510Treatment 1590 1609 1552 1569

p-value 0104 0300 0224 0190

Protein yield kgday

Control 1464 1508 1432 1436Treatment 1550 1553 1476 1491

p-value 0031 0258 0272 01605

The result of the present study agreed with Tikofsky and Harrison (2007) [27] andInostroza et al (2010) [16] who reported a significant increase in milk production of cowsfed diets containing SRU Also Kowalski et al (2010) showed an improvement in milkproduction in high-yielding dairy cows fed with SRU in partial replacement of SBM [17]Supplementation of SRU in ruminant diets fed with high levels of rapidly fermentablecarbohydrates may increase the synchrony between the energy and protein availability atthe rumen level enhancing the microbial protein synthesis thus improving its efficiency ofconverting into milk [28] It should be emphasized that the use of urea (combined withenzyme and cereals as a slower and safer form of ruminally released nitrogen) dairy cowdiets can beneficially modulate ruminal fermentation including microbiota populations(an increase in relative abundances of Megasphaera elsdenii and ammonia-producing bacte-ria) consequently improving production performance as was mentioned by Libera et al2021 [29]

Animals 2021 11 2405 8 of 15

Animals 2021 11 x FOR PEER REVIEW 8 of 15

1 d= day 2ECM = energy corrected milk 3 DMI = dry matter intake 4 FCR = feed conversion rate 5 BCS = body condition score

Figure 1 Average weekly milk production in the two groups (a = p-value le 005 x = p-value le 01)

Table 3 Production performance milk quality analyses

December Week 3 January Week 7 February Week 12 March Week 17 Fat

Control 381 382 384 384 Treatment 384 380 385 386

p-value 0720 0786 0901 0864 Proteins

Control 367 369 367 365 Treatment 374 366 365 366

p-value 0234 0535 0847 0852 Urea mg100 mL (mmolL)

Control 2145 (3560) 2313 (3839) 2228 (3698) 2229 (3700) Treatment 2101 (3487) 2356 (3910) 2231 (3703) 2296 (3811)

p-value 0471 0479 0486 0274 Lactose

Control 482 483 484 482 Treatment 483 482 484 486

p-value 0533 061 0886 0061 Somatic cells x000

Control 42657 48879 49629 47056 Treatment 42102 48053 48878 46342

p-value 0957 0936 0942 0945 Fat yield kgday

Control 1517 1563 1497 1510 Treatment 1590 1609 1552 1569

p-value 0104 0300 0224 0190 Protein yield kgday

Control 1464 1508 1432 1436

Figure 1 Average weekly milk production in the two groups (a = p-value le 005 x = p-value le 01)

Conversely Galo et al (2003) [30] and Giallongo et al (2015) [31] did not show anygain in milk production when SBM was partially replaced by SRU

In the present study DMI was significantly reduced in the treatment group (2392 vs2469 kgheadd in control) (p = 004) positively affecting FCR In fact the FCR significantlyimproved (p lt 00001) during treatment with an overall increase in feed efficiency at 69due to the lower DMI and better milk production

These results agree with the findings of Salami et al (2021) who reported a 3enhancement in feed efficiency due to a significant reduction in feed intake without anyeffects on milk yield when the traditional protein sources were replaced with SRU in dairycowsrsquo diets in Northern Europe [8]

Reproductive performance remained unaffected by the treatment (Table 2) which is inagreement with the findings of Hallajian et al (2021) who reported similar characteristicsof the follicles blood levels of progesterone and milk urea nitrogen (MUN) between dairycows fed exclusively with SBM or with the partial replacement of SBM SRU [32] Theseresults show that feeding with SRU appears to overcome the possible negative effect ofother NPN sources such as feed grade urea on both plasma urea nitrogen and overallreproductive performance [33]

Body condition scores were not influenced by the treatment (Table 2) Similarly Nealet al (2014) [34] and Hallajian et al (2021) [32] did not report significant differences interms of body weight and body condition in dairy Holstein cows fed with diets containingSRU compared with SBM control diets

Also the treatment did not influence milk quality traits as reported in Table 3 Thesefindings align with the main results found in the literature regarding dairy cows fed withan appropriate amount of slow-release urea [16ndash30]

No treatment effects were found in the overall health condition monitored daily bythe farm veterinary staff

The positive results observed after including SRU as a partial substitute for SBMunderlined that ruminal kinetics and fermentation could be optimized in diets with apercentage of soluble protein higher than 30 of the total crude protein and 50 ofdegradable protein if combined with an adequate intake of nonstructural and rapidlyfermentable carbohydrates

Despite the significant increase in the solubility of the protein fraction no changes inmilk quality or reproductive performance were observed Conversely previous studies

Animals 2021 11 2405 9 of 15

showed an increase in the milk urea levels and a reduction in fertility after an increase inprotein solubility [3536]

32 Characteristics of the Diets Feces and Digestibility of the Feeds

Chemical characteristics of the diets are shown in Tables 4 and 5 while chemical char-acteristics of feces are shown in Tables 6 and 7 Results highlighted a good correspondencebetween the projection of the rationing software and the analytical characteristics found

Table 4 Analysis of the composition of the control diet done with the portable NIR instru-ment Polispec

Parameter December January February March April

dm 1 5491 5495 5485 5465 5470Ash dm 920 976 969 923 976

Crude protein dm 1596 1621 1620 1583 1617Fats dm 300 299 315 300 302NDF dm 3635 3665 3620 3643 3668

Cellulose dm 1874 1877 1927 1882 1871Lignin dm 392 389 389 393 394

Hemicellulose dm 1370 1400 1305 1368 1403Starch dm 2800 2815 2807 2815 2819

Sugars and pectins dm 749 625 670 737 6201 dm = dry matter

Table 5 Analysis of the composition of the Treatment diet done with the portable NIR instru-ment Polispec

Parameter December January February March April

dm 1 5379 5389 5378 5463 5455Ash dm 910 980 955 919 972

Crude protein dm 1600 1619 1635 1586 1605Fats dm 288 305 305 300 303NDF dm 3655 3625 3610 3655 3680

Cellulose dm 1880 1846 1844 1882 1873Lignin dm 385 395 390 393 389

Hemicellulose dm 1390 1385 1377 1380 1417Starch dm 2838 2842 2825 2805 2821

Sugars and pectins dm 710 630 670 735 6201 dm = dry matter

Table 6 Analysis of the composition of the Control feces done with the portable NIR instru-ment Polispec

Parameter December January February March April

Moisture 8697 8688 8660 8690 8703Dry matter 1303 1313 1340 1310 1297Ash dm 927 947 897 938 925

Crude protein dm 1700 1705 1715 1727 1700Fats dm 275 255 257 278 264NDF dm 6165 6163 6150 6147 6167

Cellulose dm 3593 3628 3530 3570 3581Lignin dm 1121 1112 1125 1128 1137

Hemicellulose dm 1451 1423 1495 1457 1450Starch dm 590 591 611 568 603

Sugars and pectins dm 244 240 271 242 241dm = dry matter

Animals 2021 11 2405 10 of 15

Table 7 Analysis of the composition of the Treatment feces done with the portable NIR instru-ment Polispec

Parameter December January February March April

Moisture 8684 8725 8600 8690 8703dm 1316 1275 1400 1310 1296

Ash dm 952 1025 925 1003 1003Crude protein dm 1646 1645 1668 1687 1660

Fats dm 270 289 263 285 282NDF dm 6212 6076 6170 6052 6091

Cellulose dm 3575 3485 3426 3422 3501Lignin dm 1135 1158 1193 1128 1137

Hemicellulose dm 1493 1434 1542 1502 1453Starch dm 580 625 600 620 618

Sugars and pectins dm 240 241 275 254 247dm = dry matter

Results for nutrients digestibility during the different months of the survey (Table 8)showed that the partial substitution of SBM with SRU significantly enhanced protein(p = 0012) NDF (p = 0039) and cellulose (p = 0033) digestibility Those results partiallyagreed with Sinclair et al (2008) who reported a significant improvement in ruminaldigestion of fiber in vitro [37] These findings can be partially explained by an increasedabundance and activity of fibrolytic bacteria in the rumen such as Ruminococcaceae whichuses ammonia as its main nitrogen source due to both a higher level of NH3 availabilityand better synchrony between nitrogen and carbohydrates in the rumen when SRU is usedas a partial substitution for SBM [3839] Furthermore Geron et al (2016) found a betterin vivo digestibility of crude protein in sheep fed with SRU as a partial replacement forSBM [40]

Table 8 Digestibility in the two groups

Month December January February March April Average P(g) 1 P(m) 1 P(g m) 1

Group Ash

Control 6481 6611 6792 6456 6714 66100069 0010 0528Treatment 6452 6436 6835 6191 6468 6477

Sem 104 104 104 104 104 046p-value 0848 0264 0775 0103 0127 0069

Crude Protein

Control 6279 6325 6342 6197 6356 63000012 0201 0763Treatment 6506 6537 6666 6290 6459 6492

Sem 099 099 099 099 099 044p-value 0139 0164 0044 0524 0483 0012

Fats

Control 6648 7027 7194 6786 6942 69200621 0018 0324Treatment 6920 6802 7232 6536 6891 6876

Sem 133 134 134 134 134 060p-value 0184 0266 0848 0218 0794 0621

NDF 2

Control 4077 4127 4129 4118 4171 41240039 0821 0952Treatment 4132 4289 4416 4226 4332 4299

Sem 116 116 116 116 116 116p-value 0368 0349 0113 0528 0351 0039

Animals 2021 11 2405 11 of 15

Table 8 Cont

Month December January February March April Average P(g) 1 P(m) 1 P(g m) 1

Cellulose

Control 3302 3247 3659 3388 3366 33930033 0234 0999Treatment 3552 3564 3930 3657 3601 3661

Sem 171 171 171 171 171 171p-value 0329 0221 0292 0295 0357 0033

Hemicellulose

Control 6300 6452 6039 6287 6417 62990470 0343 0783Treatment 6347 6476 6341 6204 6488 6371

Sem 154 154 154 154 154 276p-value 0833 0911 0193 0710 0746 0470

Starch

Control 9264 9266 9247 9297 9258 92660874 0456 0045Treatment 9307 9250 9306 9229 9250 9268

Sem 018 018 018 018 018 008p-value 0132 0550 005 0028 0776 0874

Sugars and Pectins

Control 8865 8658 8588 8858 8654 87250903 0003 0753Treatment 8853 8700 8657 8707 8636 8729

Sem 053 053 053 053 053 023p-value 0883 0583 0382 0441 0817 0903

1 g = group m = month g m = group month 2 NDF = neutral detergent fiber

33 Environmental Impact Carbon Footprint of the Feed (CFP) and Predicted Methane(CH4) Production

The impact of feeding SRU as a partial substitution for SBM on dairy sustainabilitywas evaluated by estimating the CFP of the different diet feeds and predicting the entericCH4 production (Table 9 Figure 2)

Table 9 Environmental impact carbon footprint (CFP) and predicted enteric CH4 production in thetwo groups

GroupSEM p-Value

Control Treatment

Diet CFP 1

CO2 eq 2 gDMI (kgDMI)1248400(1248)

1076400(1076)

2245(022) lt00001

CO2 eq gL milk 32053 26640 079 lt00001

Predicted enteric CH43 Production

CH4 gd 47528 46045 090 lt00001CH4 gL milk 1220 1130 003 lt00001

1 CFP = Carbon footprint of the two feeds 2 CO2 eq = equivalent to the carbon dioxide 3 CH4 = methane

The results revealed that SBM was the dominant contributor to the feed CFP account-ing for 5371 and 4808 of the total CFP of the control and treatment diets respectively(Figure 2) Notably the inclusion of SRU in the treatment diet contributed only 056 of thefeed CFP (Figure 2) These results align with the average values reported in other studiesfocused on intensive dairy cows farming where SBM and other plant-protein sources werereplaced by SRU [8]

Animals 2021 11 2405 12 of 15

Animals 2021 11 x FOR PEER REVIEW 12 of 15

The partial substitution of SBM with SRU led to a significant (p lt 00001) reduction in the predicted ruminal CH4 production (Table 9) It is important to underline that the pre-sent study did not quantify the real ruminal CH4 production Instead it was assessed by following the equation of Hristov et al (2013) [23] based on dry matter intake (DMI) The lower CH4 production in the treatment group was a function of both the lower DMI when expressed only as grams of CH4 per day and better feed efficiency and milk yield when expressed as grams of CH4 per liter of milk

Notably there is limited published information on the effect of feeding urea on real enteric CH4 production Alipour et al (2020) [42] and Rebelo et al (2019) [43] showed that feeding two sources of coated and non-coated urea did not affect enteric CH4 yield meas-ured in an in vitro ruminal fermentation system or beef cattle respectively However Libera et al (2021) showed that the CH4 production at the ruminal level reduced after using urea combined with enzymes and cereals in dairy cows This result was due to a reduction in the substrate available for methanogenesis and an influence on methanogens or other rumen microorganisms [29] Although existing information suggests that slow-release urea (SRU) may have little or no effect on enteric CH4 emissions there is a crucial need for future studies to address this lack in the literature

Table 9 Environmental impact carbon footprint (CFP) and predicted enteric CH4 production in the two groups

Group SEM p-Value

Control Treatment

Diet CFP 1

CO2 eq 2 gDMI (kgDMI) 1248400 (1248)

1076400 (1076)

2245 (022)

lt00001

CO2 eq gL milk 32053 26640 079 lt00001 Predicted enteric CH4 3 Production

CH4 gd 47528 46045 090 lt00001 CH4 gL milk 1220 1130 003 lt00001

1 CFP = Carbon footprint of the two feeds 2 CO2 eq = equivalent to the carbon dioxide 3 CH4 = methane

Figure 2 Contribution of different feedsrsquo raw materials to the average carbon footprint of (A) control diet (B) treatment diet

The partial replacement of SBM with SRU decreased the CFP of the treatment dietexpressed per 1 kilogram of diet (minus1098 44991 vs 50541 g CO2-eqkg diet) comparedto the control diet due to the lower global warming potential (GWP) of SRU than SBM [24]The highest GWP of soybean meal is mainly due to large transport distances and emissionsrelated to land-use change (LUC) such as deforestation which can lead to heavier emissionsof greenhouse gases [6ndash41] These results agree with the findings of Salami et al (2021)who showed a 12 reduction in CFP (g CO2-eqkg diet) when SBM and other plant-proteinsources were replaced by SRU [8]

The inclusion of SRU also significantly (p lt 00001) reduced the CFP of the diet on adry matter basis (1076 vs 1248 kg CO2-eqkg DMI) as a result of both the lower DMI andthe lower CFP of the feeds (Table 9) Notably the CFP of the control diet per total DMIaligned with the average values reported by Gislon et al (2020) in a study conducted on171 dairy herds in the same Po Valley area where the present study was conducted [24]Conversely the impact per kilogram of DMI in the treatment diet was lower than the rangeof values reported in that survey [24]

Similarly the CFP of milk related to feed intake was significantly lower (p lt 00001) inthe treatment diet compared with the control diet (minus1689 26640 vs 32053 g CO2-eqLmilk) as a result of both the higher daily milk production and lower CFP of the feeds(Table 9) The reduction of CFP in milk related to the feed intake found in the present studywhich was also higher than the reduction (minus145) found by Salami et al (2021) [8]

The partial substitution of SBM with SRU led to a significant (p lt 00001) reductionin the predicted ruminal CH4 production (Table 9) It is important to underline that thepresent study did not quantify the real ruminal CH4 production Instead it was assessedby following the equation of Hristov et al (2013) [23] based on dry matter intake (DMI)The lower CH4 production in the treatment group was a function of both the lower DMIwhen expressed only as grams of CH4 per day and better feed efficiency and milk yieldwhen expressed as grams of CH4 per liter of milk

Notably there is limited published information on the effect of feeding urea on realenteric CH4 production Alipour et al (2020) [42] and Rebelo et al (2019) [43] showedthat feeding two sources of coated and non-coated urea did not affect enteric CH4 yieldmeasured in an in vitro ruminal fermentation system or beef cattle respectively However

Animals 2021 11 2405 13 of 15

Libera et al (2021) showed that the CH4 production at the ruminal level reduced after usingurea combined with enzymes and cereals in dairy cows This result was due to a reductionin the substrate available for methanogenesis and an influence on methanogens or otherrumen microorganisms [29] Although existing information suggests that slow-releaseurea (SRU) may have little or no effect on enteric CH4 emissions there is a crucial need forfuture studies to address this lack in the literature

4 Conclusions

This work contributes to defining scientific knowledge about the use of SRU in dairycowsrsquo nutrition and filling the present gap in the literature about their effect on environ-mental sustainability

This study showed that the substitution of traditional SBM (133 as fed) with thesource of SRU Protigen (022 as fed 100 gheadd) led to an improvement in dairycowsrsquo efficiency due to an enhanced feed conversion rate a lower dry matter intake ahigher digestibility of the fibrous parts of the diet and to better daily milk productionFurthermore the present study showed that the environmental sustainability of dairycowsrsquo diets could be improved by including SRU as an alternative protein source mainlyreducing the impact of the feed production in terms of greenhouse gas emissions andpredicted ruminal CH4 production Further research is needed to evaluate the effectiverole of SRU on methanogenesis and real ruminal production of methane

Supplementary Materials The following are available online at httpswwwmdpicomarticle103390ani11082405s1 Table S1 Technical information about the product Protigen used in thepresent trial particle size measured in millimetres and in vitro release rate at different time point

Author Contributions Conceptualization data curation writingmdashoriginal draft preparation re-view and editing SG conceptualization data curation RC data curation study validation LRmanuscript review MD data curation manuscript review IC conceptualization project admin-istration and supervision CASR All authors have read and agreed to the published version ofthe manuscript

Funding This research received no external funding

Institutional Review Board Statement The trial was a field and practical study not an experimentalone so it did not require approval For the trial we only used data commonly recorded by the farmer(milk production milk quality judged through monthly analyses reproductive parameters and soon) without any additional or ldquoexperimentalrdquo practices that can or will harm the animals or risktheir welfare The SRU product used in this study is already registered and used in dairy cowsrsquo feed

Data Availability Statement The data presented in this study are available on request from thecorresponding author

Conflicts of Interest The authors declare no conflict of interest

References1 United Nations Department of Economic and Social Affairs Population Division World Population Prospects 2019 Comprehensive

Tables (STESASERA426) United Nations Department of Economic and Social Affairs Population Division New York NYUSA 2019 Volume I

2 Georganas A Giamouri E Pappas AC Papadomichelakis G Galliou F Manios T Tsiplakou E Fegeros K ZervasG Bioactive Compounds in Food Waste A Review on the Transformation of Food Waste to Animal Feed Foods 2020 9 291[CrossRef]

3 Takiya CS Ylioja CM Bennett A Davidson MJ Sudbeck M Wickersham TA Vandehaar MJ Bradford BJ FeedingDairy Cows With ldquoLeftoversrdquo and the Variation in Recovery of Human-Edible Nutrients in Milk Front Sustain Food Syst 2019 3114 [CrossRef]

4 Capper JL Cady RA The effects of improved performance in the US dairy cattle industry on environmental impacts between2007 and 2017 J Anim Sci 2020 1 skz291 [CrossRef]

5 Wilkinson J Garnsworthy P Impact of diet and fertility on greenhouse gas emissions and nitrogen efficiency of milk productionLivestock 2017 22 140ndash144 [CrossRef]

Animals 2021 11 2405 7 of 15

Table 3 Production performance milk quality analyses

December Week 3 January Week 7 February Week 12 March Week 17

Fat

Control 381 382 384 384Treatment 384 380 385 386

p-value 0720 0786 0901 0864

Proteins

Control 367 369 367 365Treatment 374 366 365 366

p-value 0234 0535 0847 0852

Urea mg100 mL (mmolL)

Control 2145 (3560) 2313 (3839) 2228 (3698) 2229 (3700)Treatment 2101 (3487) 2356 (3910) 2231 (3703) 2296 (3811)

p-value 0471 0479 0486 0274

Lactose

Control 482 483 484 482Treatment 483 482 484 486

p-value 0533 061 0886 0061

Somatic cells x000

Control 42657 48879 49629 47056Treatment 42102 48053 48878 46342

p-value 0957 0936 0942 0945

Fat yield kgday

Control 1517 1563 1497 1510Treatment 1590 1609 1552 1569

p-value 0104 0300 0224 0190

Protein yield kgday

Control 1464 1508 1432 1436Treatment 1550 1553 1476 1491

p-value 0031 0258 0272 01605

The result of the present study agreed with Tikofsky and Harrison (2007) [27] andInostroza et al (2010) [16] who reported a significant increase in milk production of cowsfed diets containing SRU Also Kowalski et al (2010) showed an improvement in milkproduction in high-yielding dairy cows fed with SRU in partial replacement of SBM [17]Supplementation of SRU in ruminant diets fed with high levels of rapidly fermentablecarbohydrates may increase the synchrony between the energy and protein availability atthe rumen level enhancing the microbial protein synthesis thus improving its efficiency ofconverting into milk [28] It should be emphasized that the use of urea (combined withenzyme and cereals as a slower and safer form of ruminally released nitrogen) dairy cowdiets can beneficially modulate ruminal fermentation including microbiota populations(an increase in relative abundances of Megasphaera elsdenii and ammonia-producing bacte-ria) consequently improving production performance as was mentioned by Libera et al2021 [29]

Animals 2021 11 2405 8 of 15

Animals 2021 11 x FOR PEER REVIEW 8 of 15

1 d= day 2ECM = energy corrected milk 3 DMI = dry matter intake 4 FCR = feed conversion rate 5 BCS = body condition score

Figure 1 Average weekly milk production in the two groups (a = p-value le 005 x = p-value le 01)

Table 3 Production performance milk quality analyses

December Week 3 January Week 7 February Week 12 March Week 17 Fat

Control 381 382 384 384 Treatment 384 380 385 386

p-value 0720 0786 0901 0864 Proteins

Control 367 369 367 365 Treatment 374 366 365 366

p-value 0234 0535 0847 0852 Urea mg100 mL (mmolL)

Control 2145 (3560) 2313 (3839) 2228 (3698) 2229 (3700) Treatment 2101 (3487) 2356 (3910) 2231 (3703) 2296 (3811)

p-value 0471 0479 0486 0274 Lactose

Control 482 483 484 482 Treatment 483 482 484 486

p-value 0533 061 0886 0061 Somatic cells x000

Control 42657 48879 49629 47056 Treatment 42102 48053 48878 46342

p-value 0957 0936 0942 0945 Fat yield kgday

Control 1517 1563 1497 1510 Treatment 1590 1609 1552 1569

p-value 0104 0300 0224 0190 Protein yield kgday

Control 1464 1508 1432 1436

Figure 1 Average weekly milk production in the two groups (a = p-value le 005 x = p-value le 01)

Conversely Galo et al (2003) [30] and Giallongo et al (2015) [31] did not show anygain in milk production when SBM was partially replaced by SRU

In the present study DMI was significantly reduced in the treatment group (2392 vs2469 kgheadd in control) (p = 004) positively affecting FCR In fact the FCR significantlyimproved (p lt 00001) during treatment with an overall increase in feed efficiency at 69due to the lower DMI and better milk production

These results agree with the findings of Salami et al (2021) who reported a 3enhancement in feed efficiency due to a significant reduction in feed intake without anyeffects on milk yield when the traditional protein sources were replaced with SRU in dairycowsrsquo diets in Northern Europe [8]

Reproductive performance remained unaffected by the treatment (Table 2) which is inagreement with the findings of Hallajian et al (2021) who reported similar characteristicsof the follicles blood levels of progesterone and milk urea nitrogen (MUN) between dairycows fed exclusively with SBM or with the partial replacement of SBM SRU [32] Theseresults show that feeding with SRU appears to overcome the possible negative effect ofother NPN sources such as feed grade urea on both plasma urea nitrogen and overallreproductive performance [33]

Body condition scores were not influenced by the treatment (Table 2) Similarly Nealet al (2014) [34] and Hallajian et al (2021) [32] did not report significant differences interms of body weight and body condition in dairy Holstein cows fed with diets containingSRU compared with SBM control diets

Also the treatment did not influence milk quality traits as reported in Table 3 Thesefindings align with the main results found in the literature regarding dairy cows fed withan appropriate amount of slow-release urea [16ndash30]

No treatment effects were found in the overall health condition monitored daily bythe farm veterinary staff

The positive results observed after including SRU as a partial substitute for SBMunderlined that ruminal kinetics and fermentation could be optimized in diets with apercentage of soluble protein higher than 30 of the total crude protein and 50 ofdegradable protein if combined with an adequate intake of nonstructural and rapidlyfermentable carbohydrates

Despite the significant increase in the solubility of the protein fraction no changes inmilk quality or reproductive performance were observed Conversely previous studies

Animals 2021 11 2405 9 of 15

showed an increase in the milk urea levels and a reduction in fertility after an increase inprotein solubility [3536]

32 Characteristics of the Diets Feces and Digestibility of the Feeds

Chemical characteristics of the diets are shown in Tables 4 and 5 while chemical char-acteristics of feces are shown in Tables 6 and 7 Results highlighted a good correspondencebetween the projection of the rationing software and the analytical characteristics found

Table 4 Analysis of the composition of the control diet done with the portable NIR instru-ment Polispec

Parameter December January February March April

dm 1 5491 5495 5485 5465 5470Ash dm 920 976 969 923 976

Crude protein dm 1596 1621 1620 1583 1617Fats dm 300 299 315 300 302NDF dm 3635 3665 3620 3643 3668

Cellulose dm 1874 1877 1927 1882 1871Lignin dm 392 389 389 393 394

Hemicellulose dm 1370 1400 1305 1368 1403Starch dm 2800 2815 2807 2815 2819

Sugars and pectins dm 749 625 670 737 6201 dm = dry matter

Table 5 Analysis of the composition of the Treatment diet done with the portable NIR instru-ment Polispec

Parameter December January February March April

dm 1 5379 5389 5378 5463 5455Ash dm 910 980 955 919 972

Crude protein dm 1600 1619 1635 1586 1605Fats dm 288 305 305 300 303NDF dm 3655 3625 3610 3655 3680

Cellulose dm 1880 1846 1844 1882 1873Lignin dm 385 395 390 393 389

Hemicellulose dm 1390 1385 1377 1380 1417Starch dm 2838 2842 2825 2805 2821

Sugars and pectins dm 710 630 670 735 6201 dm = dry matter

Table 6 Analysis of the composition of the Control feces done with the portable NIR instru-ment Polispec

Parameter December January February March April

Moisture 8697 8688 8660 8690 8703Dry matter 1303 1313 1340 1310 1297Ash dm 927 947 897 938 925

Crude protein dm 1700 1705 1715 1727 1700Fats dm 275 255 257 278 264NDF dm 6165 6163 6150 6147 6167

Cellulose dm 3593 3628 3530 3570 3581Lignin dm 1121 1112 1125 1128 1137

Hemicellulose dm 1451 1423 1495 1457 1450Starch dm 590 591 611 568 603

Sugars and pectins dm 244 240 271 242 241dm = dry matter

Animals 2021 11 2405 10 of 15

Table 7 Analysis of the composition of the Treatment feces done with the portable NIR instru-ment Polispec

Parameter December January February March April

Moisture 8684 8725 8600 8690 8703dm 1316 1275 1400 1310 1296

Ash dm 952 1025 925 1003 1003Crude protein dm 1646 1645 1668 1687 1660

Fats dm 270 289 263 285 282NDF dm 6212 6076 6170 6052 6091

Cellulose dm 3575 3485 3426 3422 3501Lignin dm 1135 1158 1193 1128 1137

Hemicellulose dm 1493 1434 1542 1502 1453Starch dm 580 625 600 620 618

Sugars and pectins dm 240 241 275 254 247dm = dry matter

Results for nutrients digestibility during the different months of the survey (Table 8)showed that the partial substitution of SBM with SRU significantly enhanced protein(p = 0012) NDF (p = 0039) and cellulose (p = 0033) digestibility Those results partiallyagreed with Sinclair et al (2008) who reported a significant improvement in ruminaldigestion of fiber in vitro [37] These findings can be partially explained by an increasedabundance and activity of fibrolytic bacteria in the rumen such as Ruminococcaceae whichuses ammonia as its main nitrogen source due to both a higher level of NH3 availabilityand better synchrony between nitrogen and carbohydrates in the rumen when SRU is usedas a partial substitution for SBM [3839] Furthermore Geron et al (2016) found a betterin vivo digestibility of crude protein in sheep fed with SRU as a partial replacement forSBM [40]

Table 8 Digestibility in the two groups

Month December January February March April Average P(g) 1 P(m) 1 P(g m) 1

Group Ash

Control 6481 6611 6792 6456 6714 66100069 0010 0528Treatment 6452 6436 6835 6191 6468 6477

Sem 104 104 104 104 104 046p-value 0848 0264 0775 0103 0127 0069

Crude Protein

Control 6279 6325 6342 6197 6356 63000012 0201 0763Treatment 6506 6537 6666 6290 6459 6492

Sem 099 099 099 099 099 044p-value 0139 0164 0044 0524 0483 0012

Fats

Control 6648 7027 7194 6786 6942 69200621 0018 0324Treatment 6920 6802 7232 6536 6891 6876

Sem 133 134 134 134 134 060p-value 0184 0266 0848 0218 0794 0621

NDF 2

Control 4077 4127 4129 4118 4171 41240039 0821 0952Treatment 4132 4289 4416 4226 4332 4299

Sem 116 116 116 116 116 116p-value 0368 0349 0113 0528 0351 0039

Animals 2021 11 2405 11 of 15

Table 8 Cont

Month December January February March April Average P(g) 1 P(m) 1 P(g m) 1

Cellulose

Control 3302 3247 3659 3388 3366 33930033 0234 0999Treatment 3552 3564 3930 3657 3601 3661

Sem 171 171 171 171 171 171p-value 0329 0221 0292 0295 0357 0033

Hemicellulose

Control 6300 6452 6039 6287 6417 62990470 0343 0783Treatment 6347 6476 6341 6204 6488 6371

Sem 154 154 154 154 154 276p-value 0833 0911 0193 0710 0746 0470

Starch

Control 9264 9266 9247 9297 9258 92660874 0456 0045Treatment 9307 9250 9306 9229 9250 9268

Sem 018 018 018 018 018 008p-value 0132 0550 005 0028 0776 0874

Sugars and Pectins

Control 8865 8658 8588 8858 8654 87250903 0003 0753Treatment 8853 8700 8657 8707 8636 8729

Sem 053 053 053 053 053 023p-value 0883 0583 0382 0441 0817 0903

1 g = group m = month g m = group month 2 NDF = neutral detergent fiber

33 Environmental Impact Carbon Footprint of the Feed (CFP) and Predicted Methane(CH4) Production

The impact of feeding SRU as a partial substitution for SBM on dairy sustainabilitywas evaluated by estimating the CFP of the different diet feeds and predicting the entericCH4 production (Table 9 Figure 2)

Table 9 Environmental impact carbon footprint (CFP) and predicted enteric CH4 production in thetwo groups

GroupSEM p-Value

Control Treatment

Diet CFP 1

CO2 eq 2 gDMI (kgDMI)1248400(1248)

1076400(1076)

2245(022) lt00001

CO2 eq gL milk 32053 26640 079 lt00001

Predicted enteric CH43 Production

CH4 gd 47528 46045 090 lt00001CH4 gL milk 1220 1130 003 lt00001

1 CFP = Carbon footprint of the two feeds 2 CO2 eq = equivalent to the carbon dioxide 3 CH4 = methane

The results revealed that SBM was the dominant contributor to the feed CFP account-ing for 5371 and 4808 of the total CFP of the control and treatment diets respectively(Figure 2) Notably the inclusion of SRU in the treatment diet contributed only 056 of thefeed CFP (Figure 2) These results align with the average values reported in other studiesfocused on intensive dairy cows farming where SBM and other plant-protein sources werereplaced by SRU [8]

Animals 2021 11 2405 12 of 15

Animals 2021 11 x FOR PEER REVIEW 12 of 15

The partial substitution of SBM with SRU led to a significant (p lt 00001) reduction in the predicted ruminal CH4 production (Table 9) It is important to underline that the pre-sent study did not quantify the real ruminal CH4 production Instead it was assessed by following the equation of Hristov et al (2013) [23] based on dry matter intake (DMI) The lower CH4 production in the treatment group was a function of both the lower DMI when expressed only as grams of CH4 per day and better feed efficiency and milk yield when expressed as grams of CH4 per liter of milk

Notably there is limited published information on the effect of feeding urea on real enteric CH4 production Alipour et al (2020) [42] and Rebelo et al (2019) [43] showed that feeding two sources of coated and non-coated urea did not affect enteric CH4 yield meas-ured in an in vitro ruminal fermentation system or beef cattle respectively However Libera et al (2021) showed that the CH4 production at the ruminal level reduced after using urea combined with enzymes and cereals in dairy cows This result was due to a reduction in the substrate available for methanogenesis and an influence on methanogens or other rumen microorganisms [29] Although existing information suggests that slow-release urea (SRU) may have little or no effect on enteric CH4 emissions there is a crucial need for future studies to address this lack in the literature

Table 9 Environmental impact carbon footprint (CFP) and predicted enteric CH4 production in the two groups

Group SEM p-Value

Control Treatment

Diet CFP 1

CO2 eq 2 gDMI (kgDMI) 1248400 (1248)

1076400 (1076)

2245 (022)

lt00001

CO2 eq gL milk 32053 26640 079 lt00001 Predicted enteric CH4 3 Production

CH4 gd 47528 46045 090 lt00001 CH4 gL milk 1220 1130 003 lt00001

1 CFP = Carbon footprint of the two feeds 2 CO2 eq = equivalent to the carbon dioxide 3 CH4 = methane

Figure 2 Contribution of different feedsrsquo raw materials to the average carbon footprint of (A) control diet (B) treatment diet

The partial replacement of SBM with SRU decreased the CFP of the treatment dietexpressed per 1 kilogram of diet (minus1098 44991 vs 50541 g CO2-eqkg diet) comparedto the control diet due to the lower global warming potential (GWP) of SRU than SBM [24]The highest GWP of soybean meal is mainly due to large transport distances and emissionsrelated to land-use change (LUC) such as deforestation which can lead to heavier emissionsof greenhouse gases [6ndash41] These results agree with the findings of Salami et al (2021)who showed a 12 reduction in CFP (g CO2-eqkg diet) when SBM and other plant-proteinsources were replaced by SRU [8]

The inclusion of SRU also significantly (p lt 00001) reduced the CFP of the diet on adry matter basis (1076 vs 1248 kg CO2-eqkg DMI) as a result of both the lower DMI andthe lower CFP of the feeds (Table 9) Notably the CFP of the control diet per total DMIaligned with the average values reported by Gislon et al (2020) in a study conducted on171 dairy herds in the same Po Valley area where the present study was conducted [24]Conversely the impact per kilogram of DMI in the treatment diet was lower than the rangeof values reported in that survey [24]

Similarly the CFP of milk related to feed intake was significantly lower (p lt 00001) inthe treatment diet compared with the control diet (minus1689 26640 vs 32053 g CO2-eqLmilk) as a result of both the higher daily milk production and lower CFP of the feeds(Table 9) The reduction of CFP in milk related to the feed intake found in the present studywhich was also higher than the reduction (minus145) found by Salami et al (2021) [8]

The partial substitution of SBM with SRU led to a significant (p lt 00001) reductionin the predicted ruminal CH4 production (Table 9) It is important to underline that thepresent study did not quantify the real ruminal CH4 production Instead it was assessedby following the equation of Hristov et al (2013) [23] based on dry matter intake (DMI)The lower CH4 production in the treatment group was a function of both the lower DMIwhen expressed only as grams of CH4 per day and better feed efficiency and milk yieldwhen expressed as grams of CH4 per liter of milk

Notably there is limited published information on the effect of feeding urea on realenteric CH4 production Alipour et al (2020) [42] and Rebelo et al (2019) [43] showedthat feeding two sources of coated and non-coated urea did not affect enteric CH4 yieldmeasured in an in vitro ruminal fermentation system or beef cattle respectively However

Animals 2021 11 2405 13 of 15

Libera et al (2021) showed that the CH4 production at the ruminal level reduced after usingurea combined with enzymes and cereals in dairy cows This result was due to a reductionin the substrate available for methanogenesis and an influence on methanogens or otherrumen microorganisms [29] Although existing information suggests that slow-releaseurea (SRU) may have little or no effect on enteric CH4 emissions there is a crucial need forfuture studies to address this lack in the literature

4 Conclusions

This work contributes to defining scientific knowledge about the use of SRU in dairycowsrsquo nutrition and filling the present gap in the literature about their effect on environ-mental sustainability

This study showed that the substitution of traditional SBM (133 as fed) with thesource of SRU Protigen (022 as fed 100 gheadd) led to an improvement in dairycowsrsquo efficiency due to an enhanced feed conversion rate a lower dry matter intake ahigher digestibility of the fibrous parts of the diet and to better daily milk productionFurthermore the present study showed that the environmental sustainability of dairycowsrsquo diets could be improved by including SRU as an alternative protein source mainlyreducing the impact of the feed production in terms of greenhouse gas emissions andpredicted ruminal CH4 production Further research is needed to evaluate the effectiverole of SRU on methanogenesis and real ruminal production of methane

Supplementary Materials The following are available online at httpswwwmdpicomarticle103390ani11082405s1 Table S1 Technical information about the product Protigen used in thepresent trial particle size measured in millimetres and in vitro release rate at different time point

Author Contributions Conceptualization data curation writingmdashoriginal draft preparation re-view and editing SG conceptualization data curation RC data curation study validation LRmanuscript review MD data curation manuscript review IC conceptualization project admin-istration and supervision CASR All authors have read and agreed to the published version ofthe manuscript

Funding This research received no external funding

Institutional Review Board Statement The trial was a field and practical study not an experimentalone so it did not require approval For the trial we only used data commonly recorded by the farmer(milk production milk quality judged through monthly analyses reproductive parameters and soon) without any additional or ldquoexperimentalrdquo practices that can or will harm the animals or risktheir welfare The SRU product used in this study is already registered and used in dairy cowsrsquo feed

Data Availability Statement The data presented in this study are available on request from thecorresponding author

Conflicts of Interest The authors declare no conflict of interest

References1 United Nations Department of Economic and Social Affairs Population Division World Population Prospects 2019 Comprehensive

Tables (STESASERA426) United Nations Department of Economic and Social Affairs Population Division New York NYUSA 2019 Volume I

2 Georganas A Giamouri E Pappas AC Papadomichelakis G Galliou F Manios T Tsiplakou E Fegeros K ZervasG Bioactive Compounds in Food Waste A Review on the Transformation of Food Waste to Animal Feed Foods 2020 9 291[CrossRef]

3 Takiya CS Ylioja CM Bennett A Davidson MJ Sudbeck M Wickersham TA Vandehaar MJ Bradford BJ FeedingDairy Cows With ldquoLeftoversrdquo and the Variation in Recovery of Human-Edible Nutrients in Milk Front Sustain Food Syst 2019 3114 [CrossRef]

4 Capper JL Cady RA The effects of improved performance in the US dairy cattle industry on environmental impacts between2007 and 2017 J Anim Sci 2020 1 skz291 [CrossRef]

5 Wilkinson J Garnsworthy P Impact of diet and fertility on greenhouse gas emissions and nitrogen efficiency of milk productionLivestock 2017 22 140ndash144 [CrossRef]

Animals 2021 11 2405 8 of 15

Animals 2021 11 x FOR PEER REVIEW 8 of 15

1 d= day 2ECM = energy corrected milk 3 DMI = dry matter intake 4 FCR = feed conversion rate 5 BCS = body condition score

Figure 1 Average weekly milk production in the two groups (a = p-value le 005 x = p-value le 01)

Table 3 Production performance milk quality analyses

December Week 3 January Week 7 February Week 12 March Week 17 Fat

Control 381 382 384 384 Treatment 384 380 385 386

p-value 0720 0786 0901 0864 Proteins

Control 367 369 367 365 Treatment 374 366 365 366

p-value 0234 0535 0847 0852 Urea mg100 mL (mmolL)

Control 2145 (3560) 2313 (3839) 2228 (3698) 2229 (3700) Treatment 2101 (3487) 2356 (3910) 2231 (3703) 2296 (3811)

p-value 0471 0479 0486 0274 Lactose

Control 482 483 484 482 Treatment 483 482 484 486

p-value 0533 061 0886 0061 Somatic cells x000

Control 42657 48879 49629 47056 Treatment 42102 48053 48878 46342

p-value 0957 0936 0942 0945 Fat yield kgday

Control 1517 1563 1497 1510 Treatment 1590 1609 1552 1569

p-value 0104 0300 0224 0190 Protein yield kgday

Control 1464 1508 1432 1436

Figure 1 Average weekly milk production in the two groups (a = p-value le 005 x = p-value le 01)

Conversely Galo et al (2003) [30] and Giallongo et al (2015) [31] did not show anygain in milk production when SBM was partially replaced by SRU

In the present study DMI was significantly reduced in the treatment group (2392 vs2469 kgheadd in control) (p = 004) positively affecting FCR In fact the FCR significantlyimproved (p lt 00001) during treatment with an overall increase in feed efficiency at 69due to the lower DMI and better milk production

These results agree with the findings of Salami et al (2021) who reported a 3enhancement in feed efficiency due to a significant reduction in feed intake without anyeffects on milk yield when the traditional protein sources were replaced with SRU in dairycowsrsquo diets in Northern Europe [8]

Reproductive performance remained unaffected by the treatment (Table 2) which is inagreement with the findings of Hallajian et al (2021) who reported similar characteristicsof the follicles blood levels of progesterone and milk urea nitrogen (MUN) between dairycows fed exclusively with SBM or with the partial replacement of SBM SRU [32] Theseresults show that feeding with SRU appears to overcome the possible negative effect ofother NPN sources such as feed grade urea on both plasma urea nitrogen and overallreproductive performance [33]

Body condition scores were not influenced by the treatment (Table 2) Similarly Nealet al (2014) [34] and Hallajian et al (2021) [32] did not report significant differences interms of body weight and body condition in dairy Holstein cows fed with diets containingSRU compared with SBM control diets

Also the treatment did not influence milk quality traits as reported in Table 3 Thesefindings align with the main results found in the literature regarding dairy cows fed withan appropriate amount of slow-release urea [16ndash30]

No treatment effects were found in the overall health condition monitored daily bythe farm veterinary staff

The positive results observed after including SRU as a partial substitute for SBMunderlined that ruminal kinetics and fermentation could be optimized in diets with apercentage of soluble protein higher than 30 of the total crude protein and 50 ofdegradable protein if combined with an adequate intake of nonstructural and rapidlyfermentable carbohydrates

Despite the significant increase in the solubility of the protein fraction no changes inmilk quality or reproductive performance were observed Conversely previous studies

Animals 2021 11 2405 9 of 15

showed an increase in the milk urea levels and a reduction in fertility after an increase inprotein solubility [3536]

32 Characteristics of the Diets Feces and Digestibility of the Feeds

Chemical characteristics of the diets are shown in Tables 4 and 5 while chemical char-acteristics of feces are shown in Tables 6 and 7 Results highlighted a good correspondencebetween the projection of the rationing software and the analytical characteristics found

Table 4 Analysis of the composition of the control diet done with the portable NIR instru-ment Polispec

Parameter December January February March April

dm 1 5491 5495 5485 5465 5470Ash dm 920 976 969 923 976

Crude protein dm 1596 1621 1620 1583 1617Fats dm 300 299 315 300 302NDF dm 3635 3665 3620 3643 3668

Cellulose dm 1874 1877 1927 1882 1871Lignin dm 392 389 389 393 394

Hemicellulose dm 1370 1400 1305 1368 1403Starch dm 2800 2815 2807 2815 2819

Sugars and pectins dm 749 625 670 737 6201 dm = dry matter

Table 5 Analysis of the composition of the Treatment diet done with the portable NIR instru-ment Polispec

Parameter December January February March April

dm 1 5379 5389 5378 5463 5455Ash dm 910 980 955 919 972

Crude protein dm 1600 1619 1635 1586 1605Fats dm 288 305 305 300 303NDF dm 3655 3625 3610 3655 3680

Cellulose dm 1880 1846 1844 1882 1873Lignin dm 385 395 390 393 389

Hemicellulose dm 1390 1385 1377 1380 1417Starch dm 2838 2842 2825 2805 2821

Sugars and pectins dm 710 630 670 735 6201 dm = dry matter

Table 6 Analysis of the composition of the Control feces done with the portable NIR instru-ment Polispec

Parameter December January February March April

Moisture 8697 8688 8660 8690 8703Dry matter 1303 1313 1340 1310 1297Ash dm 927 947 897 938 925

Crude protein dm 1700 1705 1715 1727 1700Fats dm 275 255 257 278 264NDF dm 6165 6163 6150 6147 6167

Cellulose dm 3593 3628 3530 3570 3581Lignin dm 1121 1112 1125 1128 1137

Hemicellulose dm 1451 1423 1495 1457 1450Starch dm 590 591 611 568 603

Sugars and pectins dm 244 240 271 242 241dm = dry matter

Animals 2021 11 2405 10 of 15

Table 7 Analysis of the composition of the Treatment feces done with the portable NIR instru-ment Polispec

Parameter December January February March April

Moisture 8684 8725 8600 8690 8703dm 1316 1275 1400 1310 1296

Ash dm 952 1025 925 1003 1003Crude protein dm 1646 1645 1668 1687 1660

Fats dm 270 289 263 285 282NDF dm 6212 6076 6170 6052 6091

Cellulose dm 3575 3485 3426 3422 3501Lignin dm 1135 1158 1193 1128 1137

Hemicellulose dm 1493 1434 1542 1502 1453Starch dm 580 625 600 620 618

Sugars and pectins dm 240 241 275 254 247dm = dry matter

Results for nutrients digestibility during the different months of the survey (Table 8)showed that the partial substitution of SBM with SRU significantly enhanced protein(p = 0012) NDF (p = 0039) and cellulose (p = 0033) digestibility Those results partiallyagreed with Sinclair et al (2008) who reported a significant improvement in ruminaldigestion of fiber in vitro [37] These findings can be partially explained by an increasedabundance and activity of fibrolytic bacteria in the rumen such as Ruminococcaceae whichuses ammonia as its main nitrogen source due to both a higher level of NH3 availabilityand better synchrony between nitrogen and carbohydrates in the rumen when SRU is usedas a partial substitution for SBM [3839] Furthermore Geron et al (2016) found a betterin vivo digestibility of crude protein in sheep fed with SRU as a partial replacement forSBM [40]

Table 8 Digestibility in the two groups

Month December January February March April Average P(g) 1 P(m) 1 P(g m) 1

Group Ash

Control 6481 6611 6792 6456 6714 66100069 0010 0528Treatment 6452 6436 6835 6191 6468 6477

Sem 104 104 104 104 104 046p-value 0848 0264 0775 0103 0127 0069

Crude Protein

Control 6279 6325 6342 6197 6356 63000012 0201 0763Treatment 6506 6537 6666 6290 6459 6492

Sem 099 099 099 099 099 044p-value 0139 0164 0044 0524 0483 0012

Fats

Control 6648 7027 7194 6786 6942 69200621 0018 0324Treatment 6920 6802 7232 6536 6891 6876

Sem 133 134 134 134 134 060p-value 0184 0266 0848 0218 0794 0621

NDF 2

Control 4077 4127 4129 4118 4171 41240039 0821 0952Treatment 4132 4289 4416 4226 4332 4299

Sem 116 116 116 116 116 116p-value 0368 0349 0113 0528 0351 0039

Animals 2021 11 2405 11 of 15

Table 8 Cont

Month December January February March April Average P(g) 1 P(m) 1 P(g m) 1

Cellulose

Control 3302 3247 3659 3388 3366 33930033 0234 0999Treatment 3552 3564 3930 3657 3601 3661

Sem 171 171 171 171 171 171p-value 0329 0221 0292 0295 0357 0033

Hemicellulose

Control 6300 6452 6039 6287 6417 62990470 0343 0783Treatment 6347 6476 6341 6204 6488 6371

Sem 154 154 154 154 154 276p-value 0833 0911 0193 0710 0746 0470

Starch

Control 9264 9266 9247 9297 9258 92660874 0456 0045Treatment 9307 9250 9306 9229 9250 9268

Sem 018 018 018 018 018 008p-value 0132 0550 005 0028 0776 0874

Sugars and Pectins

Control 8865 8658 8588 8858 8654 87250903 0003 0753Treatment 8853 8700 8657 8707 8636 8729

Sem 053 053 053 053 053 023p-value 0883 0583 0382 0441 0817 0903

1 g = group m = month g m = group month 2 NDF = neutral detergent fiber

33 Environmental Impact Carbon Footprint of the Feed (CFP) and Predicted Methane(CH4) Production

The impact of feeding SRU as a partial substitution for SBM on dairy sustainabilitywas evaluated by estimating the CFP of the different diet feeds and predicting the entericCH4 production (Table 9 Figure 2)

Table 9 Environmental impact carbon footprint (CFP) and predicted enteric CH4 production in thetwo groups

GroupSEM p-Value

Control Treatment

Diet CFP 1

CO2 eq 2 gDMI (kgDMI)1248400(1248)

1076400(1076)

2245(022) lt00001

CO2 eq gL milk 32053 26640 079 lt00001

Predicted enteric CH43 Production

CH4 gd 47528 46045 090 lt00001CH4 gL milk 1220 1130 003 lt00001

1 CFP = Carbon footprint of the two feeds 2 CO2 eq = equivalent to the carbon dioxide 3 CH4 = methane

The results revealed that SBM was the dominant contributor to the feed CFP account-ing for 5371 and 4808 of the total CFP of the control and treatment diets respectively(Figure 2) Notably the inclusion of SRU in the treatment diet contributed only 056 of thefeed CFP (Figure 2) These results align with the average values reported in other studiesfocused on intensive dairy cows farming where SBM and other plant-protein sources werereplaced by SRU [8]

Animals 2021 11 2405 12 of 15

Animals 2021 11 x FOR PEER REVIEW 12 of 15

The partial substitution of SBM with SRU led to a significant (p lt 00001) reduction in the predicted ruminal CH4 production (Table 9) It is important to underline that the pre-sent study did not quantify the real ruminal CH4 production Instead it was assessed by following the equation of Hristov et al (2013) [23] based on dry matter intake (DMI) The lower CH4 production in the treatment group was a function of both the lower DMI when expressed only as grams of CH4 per day and better feed efficiency and milk yield when expressed as grams of CH4 per liter of milk

Notably there is limited published information on the effect of feeding urea on real enteric CH4 production Alipour et al (2020) [42] and Rebelo et al (2019) [43] showed that feeding two sources of coated and non-coated urea did not affect enteric CH4 yield meas-ured in an in vitro ruminal fermentation system or beef cattle respectively However Libera et al (2021) showed that the CH4 production at the ruminal level reduced after using urea combined with enzymes and cereals in dairy cows This result was due to a reduction in the substrate available for methanogenesis and an influence on methanogens or other rumen microorganisms [29] Although existing information suggests that slow-release urea (SRU) may have little or no effect on enteric CH4 emissions there is a crucial need for future studies to address this lack in the literature

Table 9 Environmental impact carbon footprint (CFP) and predicted enteric CH4 production in the two groups

Group SEM p-Value

Control Treatment

Diet CFP 1

CO2 eq 2 gDMI (kgDMI) 1248400 (1248)

1076400 (1076)

2245 (022)

lt00001

CO2 eq gL milk 32053 26640 079 lt00001 Predicted enteric CH4 3 Production

CH4 gd 47528 46045 090 lt00001 CH4 gL milk 1220 1130 003 lt00001

1 CFP = Carbon footprint of the two feeds 2 CO2 eq = equivalent to the carbon dioxide 3 CH4 = methane

Figure 2 Contribution of different feedsrsquo raw materials to the average carbon footprint of (A) control diet (B) treatment diet

The partial replacement of SBM with SRU decreased the CFP of the treatment dietexpressed per 1 kilogram of diet (minus1098 44991 vs 50541 g CO2-eqkg diet) comparedto the control diet due to the lower global warming potential (GWP) of SRU than SBM [24]The highest GWP of soybean meal is mainly due to large transport distances and emissionsrelated to land-use change (LUC) such as deforestation which can lead to heavier emissionsof greenhouse gases [6ndash41] These results agree with the findings of Salami et al (2021)who showed a 12 reduction in CFP (g CO2-eqkg diet) when SBM and other plant-proteinsources were replaced by SRU [8]

The inclusion of SRU also significantly (p lt 00001) reduced the CFP of the diet on adry matter basis (1076 vs 1248 kg CO2-eqkg DMI) as a result of both the lower DMI andthe lower CFP of the feeds (Table 9) Notably the CFP of the control diet per total DMIaligned with the average values reported by Gislon et al (2020) in a study conducted on171 dairy herds in the same Po Valley area where the present study was conducted [24]Conversely the impact per kilogram of DMI in the treatment diet was lower than the rangeof values reported in that survey [24]

Similarly the CFP of milk related to feed intake was significantly lower (p lt 00001) inthe treatment diet compared with the control diet (minus1689 26640 vs 32053 g CO2-eqLmilk) as a result of both the higher daily milk production and lower CFP of the feeds(Table 9) The reduction of CFP in milk related to the feed intake found in the present studywhich was also higher than the reduction (minus145) found by Salami et al (2021) [8]

The partial substitution of SBM with SRU led to a significant (p lt 00001) reductionin the predicted ruminal CH4 production (Table 9) It is important to underline that thepresent study did not quantify the real ruminal CH4 production Instead it was assessedby following the equation of Hristov et al (2013) [23] based on dry matter intake (DMI)The lower CH4 production in the treatment group was a function of both the lower DMIwhen expressed only as grams of CH4 per day and better feed efficiency and milk yieldwhen expressed as grams of CH4 per liter of milk

Notably there is limited published information on the effect of feeding urea on realenteric CH4 production Alipour et al (2020) [42] and Rebelo et al (2019) [43] showedthat feeding two sources of coated and non-coated urea did not affect enteric CH4 yieldmeasured in an in vitro ruminal fermentation system or beef cattle respectively However

Animals 2021 11 2405 13 of 15

Libera et al (2021) showed that the CH4 production at the ruminal level reduced after usingurea combined with enzymes and cereals in dairy cows This result was due to a reductionin the substrate available for methanogenesis and an influence on methanogens or otherrumen microorganisms [29] Although existing information suggests that slow-releaseurea (SRU) may have little or no effect on enteric CH4 emissions there is a crucial need forfuture studies to address this lack in the literature

4 Conclusions

This work contributes to defining scientific knowledge about the use of SRU in dairycowsrsquo nutrition and filling the present gap in the literature about their effect on environ-mental sustainability

This study showed that the substitution of traditional SBM (133 as fed) with thesource of SRU Protigen (022 as fed 100 gheadd) led to an improvement in dairycowsrsquo efficiency due to an enhanced feed conversion rate a lower dry matter intake ahigher digestibility of the fibrous parts of the diet and to better daily milk productionFurthermore the present study showed that the environmental sustainability of dairycowsrsquo diets could be improved by including SRU as an alternative protein source mainlyreducing the impact of the feed production in terms of greenhouse gas emissions andpredicted ruminal CH4 production Further research is needed to evaluate the effectiverole of SRU on methanogenesis and real ruminal production of methane

Supplementary Materials The following are available online at httpswwwmdpicomarticle103390ani11082405s1 Table S1 Technical information about the product Protigen used in thepresent trial particle size measured in millimetres and in vitro release rate at different time point

Author Contributions Conceptualization data curation writingmdashoriginal draft preparation re-view and editing SG conceptualization data curation RC data curation study validation LRmanuscript review MD data curation manuscript review IC conceptualization project admin-istration and supervision CASR All authors have read and agreed to the published version ofthe manuscript

Funding This research received no external funding

Institutional Review Board Statement The trial was a field and practical study not an experimentalone so it did not require approval For the trial we only used data commonly recorded by the farmer(milk production milk quality judged through monthly analyses reproductive parameters and soon) without any additional or ldquoexperimentalrdquo practices that can or will harm the animals or risktheir welfare The SRU product used in this study is already registered and used in dairy cowsrsquo feed

Data Availability Statement The data presented in this study are available on request from thecorresponding author

Conflicts of Interest The authors declare no conflict of interest

References1 United Nations Department of Economic and Social Affairs Population Division World Population Prospects 2019 Comprehensive

Tables (STESASERA426) United Nations Department of Economic and Social Affairs Population Division New York NYUSA 2019 Volume I

2 Georganas A Giamouri E Pappas AC Papadomichelakis G Galliou F Manios T Tsiplakou E Fegeros K ZervasG Bioactive Compounds in Food Waste A Review on the Transformation of Food Waste to Animal Feed Foods 2020 9 291[CrossRef]

3 Takiya CS Ylioja CM Bennett A Davidson MJ Sudbeck M Wickersham TA Vandehaar MJ Bradford BJ FeedingDairy Cows With ldquoLeftoversrdquo and the Variation in Recovery of Human-Edible Nutrients in Milk Front Sustain Food Syst 2019 3114 [CrossRef]

4 Capper JL Cady RA The effects of improved performance in the US dairy cattle industry on environmental impacts between2007 and 2017 J Anim Sci 2020 1 skz291 [CrossRef]

5 Wilkinson J Garnsworthy P Impact of diet and fertility on greenhouse gas emissions and nitrogen efficiency of milk productionLivestock 2017 22 140ndash144 [CrossRef]

Animals 2021 11 2405 9 of 15

showed an increase in the milk urea levels and a reduction in fertility after an increase inprotein solubility [3536]

32 Characteristics of the Diets Feces and Digestibility of the Feeds

Chemical characteristics of the diets are shown in Tables 4 and 5 while chemical char-acteristics of feces are shown in Tables 6 and 7 Results highlighted a good correspondencebetween the projection of the rationing software and the analytical characteristics found

Table 4 Analysis of the composition of the control diet done with the portable NIR instru-ment Polispec

Parameter December January February March April

dm 1 5491 5495 5485 5465 5470Ash dm 920 976 969 923 976

Crude protein dm 1596 1621 1620 1583 1617Fats dm 300 299 315 300 302NDF dm 3635 3665 3620 3643 3668

Cellulose dm 1874 1877 1927 1882 1871Lignin dm 392 389 389 393 394

Hemicellulose dm 1370 1400 1305 1368 1403Starch dm 2800 2815 2807 2815 2819

Sugars and pectins dm 749 625 670 737 6201 dm = dry matter

Table 5 Analysis of the composition of the Treatment diet done with the portable NIR instru-ment Polispec

Parameter December January February March April

dm 1 5379 5389 5378 5463 5455Ash dm 910 980 955 919 972

Crude protein dm 1600 1619 1635 1586 1605Fats dm 288 305 305 300 303NDF dm 3655 3625 3610 3655 3680

Cellulose dm 1880 1846 1844 1882 1873Lignin dm 385 395 390 393 389

Hemicellulose dm 1390 1385 1377 1380 1417Starch dm 2838 2842 2825 2805 2821

Sugars and pectins dm 710 630 670 735 6201 dm = dry matter

Table 6 Analysis of the composition of the Control feces done with the portable NIR instru-ment Polispec

Parameter December January February March April

Moisture 8697 8688 8660 8690 8703Dry matter 1303 1313 1340 1310 1297Ash dm 927 947 897 938 925

Crude protein dm 1700 1705 1715 1727 1700Fats dm 275 255 257 278 264NDF dm 6165 6163 6150 6147 6167

Cellulose dm 3593 3628 3530 3570 3581Lignin dm 1121 1112 1125 1128 1137

Hemicellulose dm 1451 1423 1495 1457 1450Starch dm 590 591 611 568 603

Sugars and pectins dm 244 240 271 242 241dm = dry matter

Animals 2021 11 2405 10 of 15

Table 7 Analysis of the composition of the Treatment feces done with the portable NIR instru-ment Polispec

Parameter December January February March April

Moisture 8684 8725 8600 8690 8703dm 1316 1275 1400 1310 1296

Ash dm 952 1025 925 1003 1003Crude protein dm 1646 1645 1668 1687 1660

Fats dm 270 289 263 285 282NDF dm 6212 6076 6170 6052 6091

Cellulose dm 3575 3485 3426 3422 3501Lignin dm 1135 1158 1193 1128 1137

Hemicellulose dm 1493 1434 1542 1502 1453Starch dm 580 625 600 620 618

Sugars and pectins dm 240 241 275 254 247dm = dry matter

Results for nutrients digestibility during the different months of the survey (Table 8)showed that the partial substitution of SBM with SRU significantly enhanced protein(p = 0012) NDF (p = 0039) and cellulose (p = 0033) digestibility Those results partiallyagreed with Sinclair et al (2008) who reported a significant improvement in ruminaldigestion of fiber in vitro [37] These findings can be partially explained by an increasedabundance and activity of fibrolytic bacteria in the rumen such as Ruminococcaceae whichuses ammonia as its main nitrogen source due to both a higher level of NH3 availabilityand better synchrony between nitrogen and carbohydrates in the rumen when SRU is usedas a partial substitution for SBM [3839] Furthermore Geron et al (2016) found a betterin vivo digestibility of crude protein in sheep fed with SRU as a partial replacement forSBM [40]

Table 8 Digestibility in the two groups

Month December January February March April Average P(g) 1 P(m) 1 P(g m) 1

Group Ash

Control 6481 6611 6792 6456 6714 66100069 0010 0528Treatment 6452 6436 6835 6191 6468 6477

Sem 104 104 104 104 104 046p-value 0848 0264 0775 0103 0127 0069

Crude Protein

Control 6279 6325 6342 6197 6356 63000012 0201 0763Treatment 6506 6537 6666 6290 6459 6492

Sem 099 099 099 099 099 044p-value 0139 0164 0044 0524 0483 0012

Fats

Control 6648 7027 7194 6786 6942 69200621 0018 0324Treatment 6920 6802 7232 6536 6891 6876

Sem 133 134 134 134 134 060p-value 0184 0266 0848 0218 0794 0621

NDF 2

Control 4077 4127 4129 4118 4171 41240039 0821 0952Treatment 4132 4289 4416 4226 4332 4299

Sem 116 116 116 116 116 116p-value 0368 0349 0113 0528 0351 0039

Animals 2021 11 2405 11 of 15

Table 8 Cont

Month December January February March April Average P(g) 1 P(m) 1 P(g m) 1

Cellulose

Control 3302 3247 3659 3388 3366 33930033 0234 0999Treatment 3552 3564 3930 3657 3601 3661

Sem 171 171 171 171 171 171p-value 0329 0221 0292 0295 0357 0033

Hemicellulose

Control 6300 6452 6039 6287 6417 62990470 0343 0783Treatment 6347 6476 6341 6204 6488 6371

Sem 154 154 154 154 154 276p-value 0833 0911 0193 0710 0746 0470

Starch

Control 9264 9266 9247 9297 9258 92660874 0456 0045Treatment 9307 9250 9306 9229 9250 9268

Sem 018 018 018 018 018 008p-value 0132 0550 005 0028 0776 0874

Sugars and Pectins

Control 8865 8658 8588 8858 8654 87250903 0003 0753Treatment 8853 8700 8657 8707 8636 8729

Sem 053 053 053 053 053 023p-value 0883 0583 0382 0441 0817 0903

1 g = group m = month g m = group month 2 NDF = neutral detergent fiber

33 Environmental Impact Carbon Footprint of the Feed (CFP) and Predicted Methane(CH4) Production

The impact of feeding SRU as a partial substitution for SBM on dairy sustainabilitywas evaluated by estimating the CFP of the different diet feeds and predicting the entericCH4 production (Table 9 Figure 2)

Table 9 Environmental impact carbon footprint (CFP) and predicted enteric CH4 production in thetwo groups

GroupSEM p-Value

Control Treatment

Diet CFP 1

CO2 eq 2 gDMI (kgDMI)1248400(1248)

1076400(1076)

2245(022) lt00001

CO2 eq gL milk 32053 26640 079 lt00001

Predicted enteric CH43 Production

CH4 gd 47528 46045 090 lt00001CH4 gL milk 1220 1130 003 lt00001

1 CFP = Carbon footprint of the two feeds 2 CO2 eq = equivalent to the carbon dioxide 3 CH4 = methane

The results revealed that SBM was the dominant contributor to the feed CFP account-ing for 5371 and 4808 of the total CFP of the control and treatment diets respectively(Figure 2) Notably the inclusion of SRU in the treatment diet contributed only 056 of thefeed CFP (Figure 2) These results align with the average values reported in other studiesfocused on intensive dairy cows farming where SBM and other plant-protein sources werereplaced by SRU [8]

Animals 2021 11 2405 12 of 15

Animals 2021 11 x FOR PEER REVIEW 12 of 15

The partial substitution of SBM with SRU led to a significant (p lt 00001) reduction in the predicted ruminal CH4 production (Table 9) It is important to underline that the pre-sent study did not quantify the real ruminal CH4 production Instead it was assessed by following the equation of Hristov et al (2013) [23] based on dry matter intake (DMI) The lower CH4 production in the treatment group was a function of both the lower DMI when expressed only as grams of CH4 per day and better feed efficiency and milk yield when expressed as grams of CH4 per liter of milk

Notably there is limited published information on the effect of feeding urea on real enteric CH4 production Alipour et al (2020) [42] and Rebelo et al (2019) [43] showed that feeding two sources of coated and non-coated urea did not affect enteric CH4 yield meas-ured in an in vitro ruminal fermentation system or beef cattle respectively However Libera et al (2021) showed that the CH4 production at the ruminal level reduced after using urea combined with enzymes and cereals in dairy cows This result was due to a reduction in the substrate available for methanogenesis and an influence on methanogens or other rumen microorganisms [29] Although existing information suggests that slow-release urea (SRU) may have little or no effect on enteric CH4 emissions there is a crucial need for future studies to address this lack in the literature

Table 9 Environmental impact carbon footprint (CFP) and predicted enteric CH4 production in the two groups

Group SEM p-Value

Control Treatment

Diet CFP 1

CO2 eq 2 gDMI (kgDMI) 1248400 (1248)

1076400 (1076)

2245 (022)

lt00001

CO2 eq gL milk 32053 26640 079 lt00001 Predicted enteric CH4 3 Production

CH4 gd 47528 46045 090 lt00001 CH4 gL milk 1220 1130 003 lt00001

1 CFP = Carbon footprint of the two feeds 2 CO2 eq = equivalent to the carbon dioxide 3 CH4 = methane

Figure 2 Contribution of different feedsrsquo raw materials to the average carbon footprint of (A) control diet (B) treatment diet

The partial replacement of SBM with SRU decreased the CFP of the treatment dietexpressed per 1 kilogram of diet (minus1098 44991 vs 50541 g CO2-eqkg diet) comparedto the control diet due to the lower global warming potential (GWP) of SRU than SBM [24]The highest GWP of soybean meal is mainly due to large transport distances and emissionsrelated to land-use change (LUC) such as deforestation which can lead to heavier emissionsof greenhouse gases [6ndash41] These results agree with the findings of Salami et al (2021)who showed a 12 reduction in CFP (g CO2-eqkg diet) when SBM and other plant-proteinsources were replaced by SRU [8]

The inclusion of SRU also significantly (p lt 00001) reduced the CFP of the diet on adry matter basis (1076 vs 1248 kg CO2-eqkg DMI) as a result of both the lower DMI andthe lower CFP of the feeds (Table 9) Notably the CFP of the control diet per total DMIaligned with the average values reported by Gislon et al (2020) in a study conducted on171 dairy herds in the same Po Valley area where the present study was conducted [24]Conversely the impact per kilogram of DMI in the treatment diet was lower than the rangeof values reported in that survey [24]

Similarly the CFP of milk related to feed intake was significantly lower (p lt 00001) inthe treatment diet compared with the control diet (minus1689 26640 vs 32053 g CO2-eqLmilk) as a result of both the higher daily milk production and lower CFP of the feeds(Table 9) The reduction of CFP in milk related to the feed intake found in the present studywhich was also higher than the reduction (minus145) found by Salami et al (2021) [8]

The partial substitution of SBM with SRU led to a significant (p lt 00001) reductionin the predicted ruminal CH4 production (Table 9) It is important to underline that thepresent study did not quantify the real ruminal CH4 production Instead it was assessedby following the equation of Hristov et al (2013) [23] based on dry matter intake (DMI)The lower CH4 production in the treatment group was a function of both the lower DMIwhen expressed only as grams of CH4 per day and better feed efficiency and milk yieldwhen expressed as grams of CH4 per liter of milk

Notably there is limited published information on the effect of feeding urea on realenteric CH4 production Alipour et al (2020) [42] and Rebelo et al (2019) [43] showedthat feeding two sources of coated and non-coated urea did not affect enteric CH4 yieldmeasured in an in vitro ruminal fermentation system or beef cattle respectively However

Animals 2021 11 2405 13 of 15

Libera et al (2021) showed that the CH4 production at the ruminal level reduced after usingurea combined with enzymes and cereals in dairy cows This result was due to a reductionin the substrate available for methanogenesis and an influence on methanogens or otherrumen microorganisms [29] Although existing information suggests that slow-releaseurea (SRU) may have little or no effect on enteric CH4 emissions there is a crucial need forfuture studies to address this lack in the literature

4 Conclusions

This work contributes to defining scientific knowledge about the use of SRU in dairycowsrsquo nutrition and filling the present gap in the literature about their effect on environ-mental sustainability

This study showed that the substitution of traditional SBM (133 as fed) with thesource of SRU Protigen (022 as fed 100 gheadd) led to an improvement in dairycowsrsquo efficiency due to an enhanced feed conversion rate a lower dry matter intake ahigher digestibility of the fibrous parts of the diet and to better daily milk productionFurthermore the present study showed that the environmental sustainability of dairycowsrsquo diets could be improved by including SRU as an alternative protein source mainlyreducing the impact of the feed production in terms of greenhouse gas emissions andpredicted ruminal CH4 production Further research is needed to evaluate the effectiverole of SRU on methanogenesis and real ruminal production of methane

Supplementary Materials The following are available online at httpswwwmdpicomarticle103390ani11082405s1 Table S1 Technical information about the product Protigen used in thepresent trial particle size measured in millimetres and in vitro release rate at different time point

Author Contributions Conceptualization data curation writingmdashoriginal draft preparation re-view and editing SG conceptualization data curation RC data curation study validation LRmanuscript review MD data curation manuscript review IC conceptualization project admin-istration and supervision CASR All authors have read and agreed to the published version ofthe manuscript

Funding This research received no external funding

Institutional Review Board Statement The trial was a field and practical study not an experimentalone so it did not require approval For the trial we only used data commonly recorded by the farmer(milk production milk quality judged through monthly analyses reproductive parameters and soon) without any additional or ldquoexperimentalrdquo practices that can or will harm the animals or risktheir welfare The SRU product used in this study is already registered and used in dairy cowsrsquo feed

Data Availability Statement The data presented in this study are available on request from thecorresponding author

Conflicts of Interest The authors declare no conflict of interest

References1 United Nations Department of Economic and Social Affairs Population Division World Population Prospects 2019 Comprehensive

Tables (STESASERA426) United Nations Department of Economic and Social Affairs Population Division New York NYUSA 2019 Volume I

2 Georganas A Giamouri E Pappas AC Papadomichelakis G Galliou F Manios T Tsiplakou E Fegeros K ZervasG Bioactive Compounds in Food Waste A Review on the Transformation of Food Waste to Animal Feed Foods 2020 9 291[CrossRef]

3 Takiya CS Ylioja CM Bennett A Davidson MJ Sudbeck M Wickersham TA Vandehaar MJ Bradford BJ FeedingDairy Cows With ldquoLeftoversrdquo and the Variation in Recovery of Human-Edible Nutrients in Milk Front Sustain Food Syst 2019 3114 [CrossRef]

4 Capper JL Cady RA The effects of improved performance in the US dairy cattle industry on environmental impacts between2007 and 2017 J Anim Sci 2020 1 skz291 [CrossRef]

5 Wilkinson J Garnsworthy P Impact of diet and fertility on greenhouse gas emissions and nitrogen efficiency of milk productionLivestock 2017 22 140ndash144 [CrossRef]

Animals 2021 11 2405 10 of 15

Table 7 Analysis of the composition of the Treatment feces done with the portable NIR instru-ment Polispec

Parameter December January February March April

Moisture 8684 8725 8600 8690 8703dm 1316 1275 1400 1310 1296

Ash dm 952 1025 925 1003 1003Crude protein dm 1646 1645 1668 1687 1660

Fats dm 270 289 263 285 282NDF dm 6212 6076 6170 6052 6091

Cellulose dm 3575 3485 3426 3422 3501Lignin dm 1135 1158 1193 1128 1137

Hemicellulose dm 1493 1434 1542 1502 1453Starch dm 580 625 600 620 618

Sugars and pectins dm 240 241 275 254 247dm = dry matter

Results for nutrients digestibility during the different months of the survey (Table 8)showed that the partial substitution of SBM with SRU significantly enhanced protein(p = 0012) NDF (p = 0039) and cellulose (p = 0033) digestibility Those results partiallyagreed with Sinclair et al (2008) who reported a significant improvement in ruminaldigestion of fiber in vitro [37] These findings can be partially explained by an increasedabundance and activity of fibrolytic bacteria in the rumen such as Ruminococcaceae whichuses ammonia as its main nitrogen source due to both a higher level of NH3 availabilityand better synchrony between nitrogen and carbohydrates in the rumen when SRU is usedas a partial substitution for SBM [3839] Furthermore Geron et al (2016) found a betterin vivo digestibility of crude protein in sheep fed with SRU as a partial replacement forSBM [40]

Table 8 Digestibility in the two groups

Month December January February March April Average P(g) 1 P(m) 1 P(g m) 1

Group Ash

Control 6481 6611 6792 6456 6714 66100069 0010 0528Treatment 6452 6436 6835 6191 6468 6477

Sem 104 104 104 104 104 046p-value 0848 0264 0775 0103 0127 0069

Crude Protein

Control 6279 6325 6342 6197 6356 63000012 0201 0763Treatment 6506 6537 6666 6290 6459 6492

Sem 099 099 099 099 099 044p-value 0139 0164 0044 0524 0483 0012

Fats

Control 6648 7027 7194 6786 6942 69200621 0018 0324Treatment 6920 6802 7232 6536 6891 6876

Sem 133 134 134 134 134 060p-value 0184 0266 0848 0218 0794 0621

NDF 2

Control 4077 4127 4129 4118 4171 41240039 0821 0952Treatment 4132 4289 4416 4226 4332 4299

Sem 116 116 116 116 116 116p-value 0368 0349 0113 0528 0351 0039

Animals 2021 11 2405 11 of 15

Table 8 Cont

Month December January February March April Average P(g) 1 P(m) 1 P(g m) 1

Cellulose

Control 3302 3247 3659 3388 3366 33930033 0234 0999Treatment 3552 3564 3930 3657 3601 3661

Sem 171 171 171 171 171 171p-value 0329 0221 0292 0295 0357 0033

Hemicellulose

Control 6300 6452 6039 6287 6417 62990470 0343 0783Treatment 6347 6476 6341 6204 6488 6371

Sem 154 154 154 154 154 276p-value 0833 0911 0193 0710 0746 0470

Starch

Control 9264 9266 9247 9297 9258 92660874 0456 0045Treatment 9307 9250 9306 9229 9250 9268

Sem 018 018 018 018 018 008p-value 0132 0550 005 0028 0776 0874

Sugars and Pectins

Control 8865 8658 8588 8858 8654 87250903 0003 0753Treatment 8853 8700 8657 8707 8636 8729

Sem 053 053 053 053 053 023p-value 0883 0583 0382 0441 0817 0903

1 g = group m = month g m = group month 2 NDF = neutral detergent fiber

33 Environmental Impact Carbon Footprint of the Feed (CFP) and Predicted Methane(CH4) Production

The impact of feeding SRU as a partial substitution for SBM on dairy sustainabilitywas evaluated by estimating the CFP of the different diet feeds and predicting the entericCH4 production (Table 9 Figure 2)

Table 9 Environmental impact carbon footprint (CFP) and predicted enteric CH4 production in thetwo groups

GroupSEM p-Value

Control Treatment

Diet CFP 1

CO2 eq 2 gDMI (kgDMI)1248400(1248)

1076400(1076)

2245(022) lt00001

CO2 eq gL milk 32053 26640 079 lt00001

Predicted enteric CH43 Production

CH4 gd 47528 46045 090 lt00001CH4 gL milk 1220 1130 003 lt00001

1 CFP = Carbon footprint of the two feeds 2 CO2 eq = equivalent to the carbon dioxide 3 CH4 = methane

The results revealed that SBM was the dominant contributor to the feed CFP account-ing for 5371 and 4808 of the total CFP of the control and treatment diets respectively(Figure 2) Notably the inclusion of SRU in the treatment diet contributed only 056 of thefeed CFP (Figure 2) These results align with the average values reported in other studiesfocused on intensive dairy cows farming where SBM and other plant-protein sources werereplaced by SRU [8]

Animals 2021 11 2405 12 of 15

Animals 2021 11 x FOR PEER REVIEW 12 of 15

The partial substitution of SBM with SRU led to a significant (p lt 00001) reduction in the predicted ruminal CH4 production (Table 9) It is important to underline that the pre-sent study did not quantify the real ruminal CH4 production Instead it was assessed by following the equation of Hristov et al (2013) [23] based on dry matter intake (DMI) The lower CH4 production in the treatment group was a function of both the lower DMI when expressed only as grams of CH4 per day and better feed efficiency and milk yield when expressed as grams of CH4 per liter of milk

Notably there is limited published information on the effect of feeding urea on real enteric CH4 production Alipour et al (2020) [42] and Rebelo et al (2019) [43] showed that feeding two sources of coated and non-coated urea did not affect enteric CH4 yield meas-ured in an in vitro ruminal fermentation system or beef cattle respectively However Libera et al (2021) showed that the CH4 production at the ruminal level reduced after using urea combined with enzymes and cereals in dairy cows This result was due to a reduction in the substrate available for methanogenesis and an influence on methanogens or other rumen microorganisms [29] Although existing information suggests that slow-release urea (SRU) may have little or no effect on enteric CH4 emissions there is a crucial need for future studies to address this lack in the literature

Table 9 Environmental impact carbon footprint (CFP) and predicted enteric CH4 production in the two groups

Group SEM p-Value

Control Treatment

Diet CFP 1

CO2 eq 2 gDMI (kgDMI) 1248400 (1248)

1076400 (1076)

2245 (022)

lt00001

CO2 eq gL milk 32053 26640 079 lt00001 Predicted enteric CH4 3 Production

CH4 gd 47528 46045 090 lt00001 CH4 gL milk 1220 1130 003 lt00001

1 CFP = Carbon footprint of the two feeds 2 CO2 eq = equivalent to the carbon dioxide 3 CH4 = methane

Figure 2 Contribution of different feedsrsquo raw materials to the average carbon footprint of (A) control diet (B) treatment diet

The partial replacement of SBM with SRU decreased the CFP of the treatment dietexpressed per 1 kilogram of diet (minus1098 44991 vs 50541 g CO2-eqkg diet) comparedto the control diet due to the lower global warming potential (GWP) of SRU than SBM [24]The highest GWP of soybean meal is mainly due to large transport distances and emissionsrelated to land-use change (LUC) such as deforestation which can lead to heavier emissionsof greenhouse gases [6ndash41] These results agree with the findings of Salami et al (2021)who showed a 12 reduction in CFP (g CO2-eqkg diet) when SBM and other plant-proteinsources were replaced by SRU [8]

The inclusion of SRU also significantly (p lt 00001) reduced the CFP of the diet on adry matter basis (1076 vs 1248 kg CO2-eqkg DMI) as a result of both the lower DMI andthe lower CFP of the feeds (Table 9) Notably the CFP of the control diet per total DMIaligned with the average values reported by Gislon et al (2020) in a study conducted on171 dairy herds in the same Po Valley area where the present study was conducted [24]Conversely the impact per kilogram of DMI in the treatment diet was lower than the rangeof values reported in that survey [24]

Similarly the CFP of milk related to feed intake was significantly lower (p lt 00001) inthe treatment diet compared with the control diet (minus1689 26640 vs 32053 g CO2-eqLmilk) as a result of both the higher daily milk production and lower CFP of the feeds(Table 9) The reduction of CFP in milk related to the feed intake found in the present studywhich was also higher than the reduction (minus145) found by Salami et al (2021) [8]

The partial substitution of SBM with SRU led to a significant (p lt 00001) reductionin the predicted ruminal CH4 production (Table 9) It is important to underline that thepresent study did not quantify the real ruminal CH4 production Instead it was assessedby following the equation of Hristov et al (2013) [23] based on dry matter intake (DMI)The lower CH4 production in the treatment group was a function of both the lower DMIwhen expressed only as grams of CH4 per day and better feed efficiency and milk yieldwhen expressed as grams of CH4 per liter of milk

Notably there is limited published information on the effect of feeding urea on realenteric CH4 production Alipour et al (2020) [42] and Rebelo et al (2019) [43] showedthat feeding two sources of coated and non-coated urea did not affect enteric CH4 yieldmeasured in an in vitro ruminal fermentation system or beef cattle respectively However

Animals 2021 11 2405 13 of 15

Libera et al (2021) showed that the CH4 production at the ruminal level reduced after usingurea combined with enzymes and cereals in dairy cows This result was due to a reductionin the substrate available for methanogenesis and an influence on methanogens or otherrumen microorganisms [29] Although existing information suggests that slow-releaseurea (SRU) may have little or no effect on enteric CH4 emissions there is a crucial need forfuture studies to address this lack in the literature

4 Conclusions

This work contributes to defining scientific knowledge about the use of SRU in dairycowsrsquo nutrition and filling the present gap in the literature about their effect on environ-mental sustainability

This study showed that the substitution of traditional SBM (133 as fed) with thesource of SRU Protigen (022 as fed 100 gheadd) led to an improvement in dairycowsrsquo efficiency due to an enhanced feed conversion rate a lower dry matter intake ahigher digestibility of the fibrous parts of the diet and to better daily milk productionFurthermore the present study showed that the environmental sustainability of dairycowsrsquo diets could be improved by including SRU as an alternative protein source mainlyreducing the impact of the feed production in terms of greenhouse gas emissions andpredicted ruminal CH4 production Further research is needed to evaluate the effectiverole of SRU on methanogenesis and real ruminal production of methane

Supplementary Materials The following are available online at httpswwwmdpicomarticle103390ani11082405s1 Table S1 Technical information about the product Protigen used in thepresent trial particle size measured in millimetres and in vitro release rate at different time point

Author Contributions Conceptualization data curation writingmdashoriginal draft preparation re-view and editing SG conceptualization data curation RC data curation study validation LRmanuscript review MD data curation manuscript review IC conceptualization project admin-istration and supervision CASR All authors have read and agreed to the published version ofthe manuscript

Funding This research received no external funding

Institutional Review Board Statement The trial was a field and practical study not an experimentalone so it did not require approval For the trial we only used data commonly recorded by the farmer(milk production milk quality judged through monthly analyses reproductive parameters and soon) without any additional or ldquoexperimentalrdquo practices that can or will harm the animals or risktheir welfare The SRU product used in this study is already registered and used in dairy cowsrsquo feed

Data Availability Statement The data presented in this study are available on request from thecorresponding author

Conflicts of Interest The authors declare no conflict of interest

References1 United Nations Department of Economic and Social Affairs Population Division World Population Prospects 2019 Comprehensive

Tables (STESASERA426) United Nations Department of Economic and Social Affairs Population Division New York NYUSA 2019 Volume I

2 Georganas A Giamouri E Pappas AC Papadomichelakis G Galliou F Manios T Tsiplakou E Fegeros K ZervasG Bioactive Compounds in Food Waste A Review on the Transformation of Food Waste to Animal Feed Foods 2020 9 291[CrossRef]

3 Takiya CS Ylioja CM Bennett A Davidson MJ Sudbeck M Wickersham TA Vandehaar MJ Bradford BJ FeedingDairy Cows With ldquoLeftoversrdquo and the Variation in Recovery of Human-Edible Nutrients in Milk Front Sustain Food Syst 2019 3114 [CrossRef]

4 Capper JL Cady RA The effects of improved performance in the US dairy cattle industry on environmental impacts between2007 and 2017 J Anim Sci 2020 1 skz291 [CrossRef]

5 Wilkinson J Garnsworthy P Impact of diet and fertility on greenhouse gas emissions and nitrogen efficiency of milk productionLivestock 2017 22 140ndash144 [CrossRef]

Animals 2021 11 2405 11 of 15

Table 8 Cont

Month December January February March April Average P(g) 1 P(m) 1 P(g m) 1

Cellulose

Control 3302 3247 3659 3388 3366 33930033 0234 0999Treatment 3552 3564 3930 3657 3601 3661

Sem 171 171 171 171 171 171p-value 0329 0221 0292 0295 0357 0033

Hemicellulose

Control 6300 6452 6039 6287 6417 62990470 0343 0783Treatment 6347 6476 6341 6204 6488 6371

Sem 154 154 154 154 154 276p-value 0833 0911 0193 0710 0746 0470

Starch

Control 9264 9266 9247 9297 9258 92660874 0456 0045Treatment 9307 9250 9306 9229 9250 9268

Sem 018 018 018 018 018 008p-value 0132 0550 005 0028 0776 0874

Sugars and Pectins

Control 8865 8658 8588 8858 8654 87250903 0003 0753Treatment 8853 8700 8657 8707 8636 8729

Sem 053 053 053 053 053 023p-value 0883 0583 0382 0441 0817 0903

1 g = group m = month g m = group month 2 NDF = neutral detergent fiber

33 Environmental Impact Carbon Footprint of the Feed (CFP) and Predicted Methane(CH4) Production

The impact of feeding SRU as a partial substitution for SBM on dairy sustainabilitywas evaluated by estimating the CFP of the different diet feeds and predicting the entericCH4 production (Table 9 Figure 2)

Table 9 Environmental impact carbon footprint (CFP) and predicted enteric CH4 production in thetwo groups

GroupSEM p-Value

Control Treatment

Diet CFP 1

CO2 eq 2 gDMI (kgDMI)1248400(1248)

1076400(1076)

2245(022) lt00001

CO2 eq gL milk 32053 26640 079 lt00001

Predicted enteric CH43 Production

CH4 gd 47528 46045 090 lt00001CH4 gL milk 1220 1130 003 lt00001

1 CFP = Carbon footprint of the two feeds 2 CO2 eq = equivalent to the carbon dioxide 3 CH4 = methane

The results revealed that SBM was the dominant contributor to the feed CFP account-ing for 5371 and 4808 of the total CFP of the control and treatment diets respectively(Figure 2) Notably the inclusion of SRU in the treatment diet contributed only 056 of thefeed CFP (Figure 2) These results align with the average values reported in other studiesfocused on intensive dairy cows farming where SBM and other plant-protein sources werereplaced by SRU [8]

Animals 2021 11 2405 12 of 15

Animals 2021 11 x FOR PEER REVIEW 12 of 15

The partial substitution of SBM with SRU led to a significant (p lt 00001) reduction in the predicted ruminal CH4 production (Table 9) It is important to underline that the pre-sent study did not quantify the real ruminal CH4 production Instead it was assessed by following the equation of Hristov et al (2013) [23] based on dry matter intake (DMI) The lower CH4 production in the treatment group was a function of both the lower DMI when expressed only as grams of CH4 per day and better feed efficiency and milk yield when expressed as grams of CH4 per liter of milk

Notably there is limited published information on the effect of feeding urea on real enteric CH4 production Alipour et al (2020) [42] and Rebelo et al (2019) [43] showed that feeding two sources of coated and non-coated urea did not affect enteric CH4 yield meas-ured in an in vitro ruminal fermentation system or beef cattle respectively However Libera et al (2021) showed that the CH4 production at the ruminal level reduced after using urea combined with enzymes and cereals in dairy cows This result was due to a reduction in the substrate available for methanogenesis and an influence on methanogens or other rumen microorganisms [29] Although existing information suggests that slow-release urea (SRU) may have little or no effect on enteric CH4 emissions there is a crucial need for future studies to address this lack in the literature

Table 9 Environmental impact carbon footprint (CFP) and predicted enteric CH4 production in the two groups

Group SEM p-Value

Control Treatment

Diet CFP 1

CO2 eq 2 gDMI (kgDMI) 1248400 (1248)

1076400 (1076)

2245 (022)

lt00001

CO2 eq gL milk 32053 26640 079 lt00001 Predicted enteric CH4 3 Production

CH4 gd 47528 46045 090 lt00001 CH4 gL milk 1220 1130 003 lt00001

1 CFP = Carbon footprint of the two feeds 2 CO2 eq = equivalent to the carbon dioxide 3 CH4 = methane

Figure 2 Contribution of different feedsrsquo raw materials to the average carbon footprint of (A) control diet (B) treatment diet

The partial replacement of SBM with SRU decreased the CFP of the treatment dietexpressed per 1 kilogram of diet (minus1098 44991 vs 50541 g CO2-eqkg diet) comparedto the control diet due to the lower global warming potential (GWP) of SRU than SBM [24]The highest GWP of soybean meal is mainly due to large transport distances and emissionsrelated to land-use change (LUC) such as deforestation which can lead to heavier emissionsof greenhouse gases [6ndash41] These results agree with the findings of Salami et al (2021)who showed a 12 reduction in CFP (g CO2-eqkg diet) when SBM and other plant-proteinsources were replaced by SRU [8]

The inclusion of SRU also significantly (p lt 00001) reduced the CFP of the diet on adry matter basis (1076 vs 1248 kg CO2-eqkg DMI) as a result of both the lower DMI andthe lower CFP of the feeds (Table 9) Notably the CFP of the control diet per total DMIaligned with the average values reported by Gislon et al (2020) in a study conducted on171 dairy herds in the same Po Valley area where the present study was conducted [24]Conversely the impact per kilogram of DMI in the treatment diet was lower than the rangeof values reported in that survey [24]

Similarly the CFP of milk related to feed intake was significantly lower (p lt 00001) inthe treatment diet compared with the control diet (minus1689 26640 vs 32053 g CO2-eqLmilk) as a result of both the higher daily milk production and lower CFP of the feeds(Table 9) The reduction of CFP in milk related to the feed intake found in the present studywhich was also higher than the reduction (minus145) found by Salami et al (2021) [8]

The partial substitution of SBM with SRU led to a significant (p lt 00001) reductionin the predicted ruminal CH4 production (Table 9) It is important to underline that thepresent study did not quantify the real ruminal CH4 production Instead it was assessedby following the equation of Hristov et al (2013) [23] based on dry matter intake (DMI)The lower CH4 production in the treatment group was a function of both the lower DMIwhen expressed only as grams of CH4 per day and better feed efficiency and milk yieldwhen expressed as grams of CH4 per liter of milk

Notably there is limited published information on the effect of feeding urea on realenteric CH4 production Alipour et al (2020) [42] and Rebelo et al (2019) [43] showedthat feeding two sources of coated and non-coated urea did not affect enteric CH4 yieldmeasured in an in vitro ruminal fermentation system or beef cattle respectively However

Animals 2021 11 2405 13 of 15

Libera et al (2021) showed that the CH4 production at the ruminal level reduced after usingurea combined with enzymes and cereals in dairy cows This result was due to a reductionin the substrate available for methanogenesis and an influence on methanogens or otherrumen microorganisms [29] Although existing information suggests that slow-releaseurea (SRU) may have little or no effect on enteric CH4 emissions there is a crucial need forfuture studies to address this lack in the literature

4 Conclusions

This work contributes to defining scientific knowledge about the use of SRU in dairycowsrsquo nutrition and filling the present gap in the literature about their effect on environ-mental sustainability

This study showed that the substitution of traditional SBM (133 as fed) with thesource of SRU Protigen (022 as fed 100 gheadd) led to an improvement in dairycowsrsquo efficiency due to an enhanced feed conversion rate a lower dry matter intake ahigher digestibility of the fibrous parts of the diet and to better daily milk productionFurthermore the present study showed that the environmental sustainability of dairycowsrsquo diets could be improved by including SRU as an alternative protein source mainlyreducing the impact of the feed production in terms of greenhouse gas emissions andpredicted ruminal CH4 production Further research is needed to evaluate the effectiverole of SRU on methanogenesis and real ruminal production of methane

Supplementary Materials The following are available online at httpswwwmdpicomarticle103390ani11082405s1 Table S1 Technical information about the product Protigen used in thepresent trial particle size measured in millimetres and in vitro release rate at different time point

Author Contributions Conceptualization data curation writingmdashoriginal draft preparation re-view and editing SG conceptualization data curation RC data curation study validation LRmanuscript review MD data curation manuscript review IC conceptualization project admin-istration and supervision CASR All authors have read and agreed to the published version ofthe manuscript

Funding This research received no external funding

Institutional Review Board Statement The trial was a field and practical study not an experimentalone so it did not require approval For the trial we only used data commonly recorded by the farmer(milk production milk quality judged through monthly analyses reproductive parameters and soon) without any additional or ldquoexperimentalrdquo practices that can or will harm the animals or risktheir welfare The SRU product used in this study is already registered and used in dairy cowsrsquo feed

Data Availability Statement The data presented in this study are available on request from thecorresponding author

Conflicts of Interest The authors declare no conflict of interest

References1 United Nations Department of Economic and Social Affairs Population Division World Population Prospects 2019 Comprehensive

Tables (STESASERA426) United Nations Department of Economic and Social Affairs Population Division New York NYUSA 2019 Volume I

2 Georganas A Giamouri E Pappas AC Papadomichelakis G Galliou F Manios T Tsiplakou E Fegeros K ZervasG Bioactive Compounds in Food Waste A Review on the Transformation of Food Waste to Animal Feed Foods 2020 9 291[CrossRef]

3 Takiya CS Ylioja CM Bennett A Davidson MJ Sudbeck M Wickersham TA Vandehaar MJ Bradford BJ FeedingDairy Cows With ldquoLeftoversrdquo and the Variation in Recovery of Human-Edible Nutrients in Milk Front Sustain Food Syst 2019 3114 [CrossRef]

4 Capper JL Cady RA The effects of improved performance in the US dairy cattle industry on environmental impacts between2007 and 2017 J Anim Sci 2020 1 skz291 [CrossRef]

5 Wilkinson J Garnsworthy P Impact of diet and fertility on greenhouse gas emissions and nitrogen efficiency of milk productionLivestock 2017 22 140ndash144 [CrossRef]

Animals 2021 11 2405 12 of 15

Animals 2021 11 x FOR PEER REVIEW 12 of 15

The partial substitution of SBM with SRU led to a significant (p lt 00001) reduction in the predicted ruminal CH4 production (Table 9) It is important to underline that the pre-sent study did not quantify the real ruminal CH4 production Instead it was assessed by following the equation of Hristov et al (2013) [23] based on dry matter intake (DMI) The lower CH4 production in the treatment group was a function of both the lower DMI when expressed only as grams of CH4 per day and better feed efficiency and milk yield when expressed as grams of CH4 per liter of milk

Notably there is limited published information on the effect of feeding urea on real enteric CH4 production Alipour et al (2020) [42] and Rebelo et al (2019) [43] showed that feeding two sources of coated and non-coated urea did not affect enteric CH4 yield meas-ured in an in vitro ruminal fermentation system or beef cattle respectively However Libera et al (2021) showed that the CH4 production at the ruminal level reduced after using urea combined with enzymes and cereals in dairy cows This result was due to a reduction in the substrate available for methanogenesis and an influence on methanogens or other rumen microorganisms [29] Although existing information suggests that slow-release urea (SRU) may have little or no effect on enteric CH4 emissions there is a crucial need for future studies to address this lack in the literature

Table 9 Environmental impact carbon footprint (CFP) and predicted enteric CH4 production in the two groups

Group SEM p-Value

Control Treatment

Diet CFP 1

CO2 eq 2 gDMI (kgDMI) 1248400 (1248)

1076400 (1076)

2245 (022)

lt00001

CO2 eq gL milk 32053 26640 079 lt00001 Predicted enteric CH4 3 Production

CH4 gd 47528 46045 090 lt00001 CH4 gL milk 1220 1130 003 lt00001

1 CFP = Carbon footprint of the two feeds 2 CO2 eq = equivalent to the carbon dioxide 3 CH4 = methane

Figure 2 Contribution of different feedsrsquo raw materials to the average carbon footprint of (A) control diet (B) treatment diet

The partial replacement of SBM with SRU decreased the CFP of the treatment dietexpressed per 1 kilogram of diet (minus1098 44991 vs 50541 g CO2-eqkg diet) comparedto the control diet due to the lower global warming potential (GWP) of SRU than SBM [24]The highest GWP of soybean meal is mainly due to large transport distances and emissionsrelated to land-use change (LUC) such as deforestation which can lead to heavier emissionsof greenhouse gases [6ndash41] These results agree with the findings of Salami et al (2021)who showed a 12 reduction in CFP (g CO2-eqkg diet) when SBM and other plant-proteinsources were replaced by SRU [8]

The inclusion of SRU also significantly (p lt 00001) reduced the CFP of the diet on adry matter basis (1076 vs 1248 kg CO2-eqkg DMI) as a result of both the lower DMI andthe lower CFP of the feeds (Table 9) Notably the CFP of the control diet per total DMIaligned with the average values reported by Gislon et al (2020) in a study conducted on171 dairy herds in the same Po Valley area where the present study was conducted [24]Conversely the impact per kilogram of DMI in the treatment diet was lower than the rangeof values reported in that survey [24]

Similarly the CFP of milk related to feed intake was significantly lower (p lt 00001) inthe treatment diet compared with the control diet (minus1689 26640 vs 32053 g CO2-eqLmilk) as a result of both the higher daily milk production and lower CFP of the feeds(Table 9) The reduction of CFP in milk related to the feed intake found in the present studywhich was also higher than the reduction (minus145) found by Salami et al (2021) [8]

The partial substitution of SBM with SRU led to a significant (p lt 00001) reductionin the predicted ruminal CH4 production (Table 9) It is important to underline that thepresent study did not quantify the real ruminal CH4 production Instead it was assessedby following the equation of Hristov et al (2013) [23] based on dry matter intake (DMI)The lower CH4 production in the treatment group was a function of both the lower DMIwhen expressed only as grams of CH4 per day and better feed efficiency and milk yieldwhen expressed as grams of CH4 per liter of milk

Notably there is limited published information on the effect of feeding urea on realenteric CH4 production Alipour et al (2020) [42] and Rebelo et al (2019) [43] showedthat feeding two sources of coated and non-coated urea did not affect enteric CH4 yieldmeasured in an in vitro ruminal fermentation system or beef cattle respectively However

Animals 2021 11 2405 13 of 15

Libera et al (2021) showed that the CH4 production at the ruminal level reduced after usingurea combined with enzymes and cereals in dairy cows This result was due to a reductionin the substrate available for methanogenesis and an influence on methanogens or otherrumen microorganisms [29] Although existing information suggests that slow-releaseurea (SRU) may have little or no effect on enteric CH4 emissions there is a crucial need forfuture studies to address this lack in the literature

4 Conclusions

This work contributes to defining scientific knowledge about the use of SRU in dairycowsrsquo nutrition and filling the present gap in the literature about their effect on environ-mental sustainability

This study showed that the substitution of traditional SBM (133 as fed) with thesource of SRU Protigen (022 as fed 100 gheadd) led to an improvement in dairycowsrsquo efficiency due to an enhanced feed conversion rate a lower dry matter intake ahigher digestibility of the fibrous parts of the diet and to better daily milk productionFurthermore the present study showed that the environmental sustainability of dairycowsrsquo diets could be improved by including SRU as an alternative protein source mainlyreducing the impact of the feed production in terms of greenhouse gas emissions andpredicted ruminal CH4 production Further research is needed to evaluate the effectiverole of SRU on methanogenesis and real ruminal production of methane

Supplementary Materials The following are available online at httpswwwmdpicomarticle103390ani11082405s1 Table S1 Technical information about the product Protigen used in thepresent trial particle size measured in millimetres and in vitro release rate at different time point

Author Contributions Conceptualization data curation writingmdashoriginal draft preparation re-view and editing SG conceptualization data curation RC data curation study validation LRmanuscript review MD data curation manuscript review IC conceptualization project admin-istration and supervision CASR All authors have read and agreed to the published version ofthe manuscript

Funding This research received no external funding

Institutional Review Board Statement The trial was a field and practical study not an experimentalone so it did not require approval For the trial we only used data commonly recorded by the farmer(milk production milk quality judged through monthly analyses reproductive parameters and soon) without any additional or ldquoexperimentalrdquo practices that can or will harm the animals or risktheir welfare The SRU product used in this study is already registered and used in dairy cowsrsquo feed

Data Availability Statement The data presented in this study are available on request from thecorresponding author

Conflicts of Interest The authors declare no conflict of interest

References1 United Nations Department of Economic and Social Affairs Population Division World Population Prospects 2019 Comprehensive

Tables (STESASERA426) United Nations Department of Economic and Social Affairs Population Division New York NYUSA 2019 Volume I

2 Georganas A Giamouri E Pappas AC Papadomichelakis G Galliou F Manios T Tsiplakou E Fegeros K ZervasG Bioactive Compounds in Food Waste A Review on the Transformation of Food Waste to Animal Feed Foods 2020 9 291[CrossRef]

3 Takiya CS Ylioja CM Bennett A Davidson MJ Sudbeck M Wickersham TA Vandehaar MJ Bradford BJ FeedingDairy Cows With ldquoLeftoversrdquo and the Variation in Recovery of Human-Edible Nutrients in Milk Front Sustain Food Syst 2019 3114 [CrossRef]

4 Capper JL Cady RA The effects of improved performance in the US dairy cattle industry on environmental impacts between2007 and 2017 J Anim Sci 2020 1 skz291 [CrossRef]

5 Wilkinson J Garnsworthy P Impact of diet and fertility on greenhouse gas emissions and nitrogen efficiency of milk productionLivestock 2017 22 140ndash144 [CrossRef]

Animals 2021 11 2405 13 of 15

Libera et al (2021) showed that the CH4 production at the ruminal level reduced after usingurea combined with enzymes and cereals in dairy cows This result was due to a reductionin the substrate available for methanogenesis and an influence on methanogens or otherrumen microorganisms [29] Although existing information suggests that slow-releaseurea (SRU) may have little or no effect on enteric CH4 emissions there is a crucial need forfuture studies to address this lack in the literature

4 Conclusions

This work contributes to defining scientific knowledge about the use of SRU in dairycowsrsquo nutrition and filling the present gap in the literature about their effect on environ-mental sustainability

This study showed that the substitution of traditional SBM (133 as fed) with thesource of SRU Protigen (022 as fed 100 gheadd) led to an improvement in dairycowsrsquo efficiency due to an enhanced feed conversion rate a lower dry matter intake ahigher digestibility of the fibrous parts of the diet and to better daily milk productionFurthermore the present study showed that the environmental sustainability of dairycowsrsquo diets could be improved by including SRU as an alternative protein source mainlyreducing the impact of the feed production in terms of greenhouse gas emissions andpredicted ruminal CH4 production Further research is needed to evaluate the effectiverole of SRU on methanogenesis and real ruminal production of methane

Supplementary Materials The following are available online at httpswwwmdpicomarticle103390ani11082405s1 Table S1 Technical information about the product Protigen used in thepresent trial particle size measured in millimetres and in vitro release rate at different time point

Author Contributions Conceptualization data curation writingmdashoriginal draft preparation re-view and editing SG conceptualization data curation RC data curation study validation LRmanuscript review MD data curation manuscript review IC conceptualization project admin-istration and supervision CASR All authors have read and agreed to the published version ofthe manuscript

Funding This research received no external funding

Institutional Review Board Statement The trial was a field and practical study not an experimentalone so it did not require approval For the trial we only used data commonly recorded by the farmer(milk production milk quality judged through monthly analyses reproductive parameters and soon) without any additional or ldquoexperimentalrdquo practices that can or will harm the animals or risktheir welfare The SRU product used in this study is already registered and used in dairy cowsrsquo feed

Data Availability Statement The data presented in this study are available on request from thecorresponding author

Conflicts of Interest The authors declare no conflict of interest

References1 United Nations Department of Economic and Social Affairs Population Division World Population Prospects 2019 Comprehensive

Tables (STESASERA426) United Nations Department of Economic and Social Affairs Population Division New York NYUSA 2019 Volume I

2 Georganas A Giamouri E Pappas AC Papadomichelakis G Galliou F Manios T Tsiplakou E Fegeros K ZervasG Bioactive Compounds in Food Waste A Review on the Transformation of Food Waste to Animal Feed Foods 2020 9 291[CrossRef]

3 Takiya CS Ylioja CM Bennett A Davidson MJ Sudbeck M Wickersham TA Vandehaar MJ Bradford BJ FeedingDairy Cows With ldquoLeftoversrdquo and the Variation in Recovery of Human-Edible Nutrients in Milk Front Sustain Food Syst 2019 3114 [CrossRef]

4 Capper JL Cady RA The effects of improved performance in the US dairy cattle industry on environmental impacts between2007 and 2017 J Anim Sci 2020 1 skz291 [CrossRef]

5 Wilkinson J Garnsworthy P Impact of diet and fertility on greenhouse gas emissions and nitrogen efficiency of milk productionLivestock 2017 22 140ndash144 [CrossRef]

Animals 2021 11 2405 14 of 15

6 Foley JA Asner GP Costa MH Coe MT DeFries R Gibbs HK Howard EA Olson S Patz J Ramankutty N et alAmazonia revealed Forest degradation and loss of ecosystem goods and services in the Amazon Basin Front Ecol Environ 20075 25ndash32 [CrossRef]

7 da Silva VP Van der Werf HMG Spies A Soares SR Variability in environmental impacts of Brazilian soybean according tocrop production and transport scenarios J Environ Manag 2010 91 1831ndash1839 [CrossRef]

8 Salami SA Moran CA Warren HE Taylor-Pickard J Meta-analysis and sustainability of feeding slow-release urea in dairyproduction PLoS ONE 2021 16 e0246922 [CrossRef]

9 Calomeni GD Gardinal R Venturelli BC de Freitas JE Jr Vendramini THA Takiya C de Souza HN Rennoacute FPEffects of polymer-coated slow-release urea on performance ruminal fermentation and blood metabolites in dairy cows RevBras Zootec 2015 44 327ndash334 [CrossRef]

10 Santiago BT Villela SDJ Leonel FdP Zervoudakis JT Araujo RP Machado HVN Slow-release urea in diets for lactatingcrossbred cows Rev Bras Zootec 2015 44 193ndash199 [CrossRef]

11 Owens F Qi S Sapienza D Invited Review Applied protein nutrition of ruminants Current status and future directions ProfAnim Sci 2014 30 150ndash179 [CrossRef]

12 Kertz A Urea feeding to dairy cattle A historical perspective and review Prof Anim Sci 2010 26 257ndash272 [CrossRef]13 Sinclair L Blake C Griffin P Jones G The partial replacement of soyabean meal and rapeseed meal with feed grade urea

or a slow-release urea and its effect on the performance metabolism and digestibility in dairy cows Animal 2012 6 920ndash927[CrossRef] [PubMed]

14 Sgoifo Rossi CA Compiani R Baldi G Vandoni S Agovino M Effect of a source of sustained-release non-protein nitrogenon beef cattle In Proceedings of the European Association of Animal Production (EAAP) 3rd Annual Meeting BratislavaSlovakia 2012

15 Salami SA Moran CA Warren HE Taylor-Pickard J A Meta-Analysis of the Effects of Slow-Release Urea Supplementationon the Performance of Beef Cattle Animals 2020 10 657 [CrossRef]

16 Inostroza JF Shaver RD Cabrera VE Tricaacuterico JM Effect of Diets Containing a Controlled-Release Urea Product on MilkYield Milk Composition and Milk Component Yields in Commercial Wisconsin Dairy Herds and Economic Implications ProfAnim Sci 2010 26 175ndash180 [CrossRef]

17 Kowalski ZM Andrieu S Micek P On farm impact Optigenregin diets fed high yielding dairy cows In Proceedings of theAlltechrsquos 23rd Annual Symposium Lexington KY USA 20ndash23 May 2007 Lyons TP Jacques KA Eds Alltech NicholasvilleKY USA 2010

18 Cherdthong A Wanapat M Development of urea products as rumen slow-release feed for ruminant production A reviewAust J Basic Appl Sci 2010 4 2232ndash2241

19 National Research Council Nutrient Requirements of Dairy Cattle Seventh Revised Edition 2001 The National AcademiesWashington DC USA 2001

20 Tyrrell HF Reid JT Prediction of the energy value of cowrsquos milk J Dairy Sci 1965 48 1215ndash1223 [CrossRef]21 Edmonson AJ Lean IJ Weaver LD Farver T Webster G A body condition scoring chart for Holstein Dairy Cows J Dairy

Sci 1989 72 68ndash78 [CrossRef]22 Ferguson JD Galligan DT Thomsen N Principal descriptors of body condition score in Holstein cows J Dairy Sci 1994 7

2695ndash2703 [CrossRef]23 Hristov AN Oh J Firkins JL Dijkstra J Kebreab E Waghorn G Makkar HPS Adesogan AT Yang W Lee C et al

Special topicsmdashMitigation of methane and nitrous oxide emissions from animal operations I A review of enteric methanemitigation options J Anim Sci 2013 91 5045ndash5069 [CrossRef]

24 Gislon G Bava L Colombini S Zucali M Crovetto GM Sandrucci A Looking for high-production and sustainable dietsfor lactating cows A survey in Italy J Dairy Sci 2020 103 4863ndash4873 [CrossRef]

25 Appuhamy JA France J Kebreab E Models for predicting enteric methane emissions from dairy cows in North AmericaEurope and Australia and New Zealand Glob Chang Biol 2016 22 3039ndash3056 [CrossRef]

26 Hart KJ Jones HG Waddams KE Worgan HJ Zweifel B Newbold CJ An Essential Oil Blend Decreases MethaneEmissions and Increases Milk Yield in Dairy Cows Open J Anim Sci 2019 9 259ndash267 [CrossRef]

27 Tikofsky J Harrison GA Optigen II Improving the efficiency of nitrogen utilization in the dairy cow p 373 in NutritionalBiotechnology in the Feed and Food Industrie In Proceedings of the Alltechrsquos 23rd Annual Symposium Lexington KY USA20ndash23 May 2007 Lyons TP Jacques KA Eds Allte Nicholasville KY USA

28 Broderick GA Reynal SM Effect of source of rumen-degraded protein on production and ruminal metabolism in lactatingdairy cows J Dairy Sci 2009 92 2822 [CrossRef]

29 Libera K Szumacher-Strabel M Vazirigohar M Zielinski W Lukow R Wysocka K Kołodziejski P Lechniak DVaradyova Z Patra A et al Effects of feeding urea-treated triticale and oat grain mixtures on ruminal fermentation microbialpopulation and milk production performance of midlactation dairy cows Ann Anim Sci 2021 21 1007ndash1025 [CrossRef]

30 Galo E Emanuele SM Sniffen CJ White JH Knapp JR Effects of a polymer-coated urea product on nitrogen metabolismin lactating Holstein dairy cattle J Dairy Sci 2003 86 2154 [CrossRef]

Animals 2021 11 2405 15 of 15

31 Giallongo F Hristov AN Oh J Frederick T Weeks H Werner J Lapierre H Patton RA Gehman A Parys C Effects ofslow-release urea and rumen-protected methionine and histidine on performance of dairy cows J Dairy Sci 2015 98 3292ndash3308[CrossRef] [PubMed]

32 Hallajian S Fakhraei J Yarahamdi HM Khorshidi KJ Effects of replacing soybean meal with slow-release urea on milkproduction of holstein dairy cows S Afr J Anim Sci 2021 51 53ndash64 [CrossRef]

33 Westwood CT Lean IJ Garvin JK Factors influencing fertility of Holstein dairy cows A multivariate description J Dairy Sci2000 85 3225ndash3237 [CrossRef]

34 Neal K Eun JS Young AJ Mjoun K Hall JO Feeding protein supplements in alfalfa hay-based lactation diets improvesnutrient utilization lactational performance and feed efficiency of dairy cows J Dairy Sci 2014 97 7716ndash7728 [CrossRef]

35 Lean I Celi P Raadsma H Mcnamara J Rabiee A Effects of dietary crude protein on fertility Meta-analysis and meta-regression Anim Feed Sci Tech 2012 171 31ndash42 [CrossRef]

36 Butler WR Calaman JJ Beam SW Plasma and milk urea nitrogen in relation to pregnancy rate in lactating dairy cattle JAnim Sci 1996 74 858ndash865 [CrossRef]

37 Sinclair LA Rate of nitrogen and energy release in the rumen and effects on feed utilisation and animal performance In GutEfficiency The Key Ingredient in Ruminant Production Andrieu S Wilde D Eds Wageningen Academic Publishers WageningenThe Netherlands 2008 pp 61ndash78

38 Russell JB OrsquoConnor JD Fox DG Van Soest PJ Sniffen CJ A net carbohydrate and protein system for evaluating cattlediets 1 Ruminal fermentation J Anim Sci 1992 70 3551ndash3561 [CrossRef] [PubMed]

39 Hackmann TJ Firkins JL Maximizing efficiency of rumen microbial protein production Front Microbiol 2015 15 465[CrossRef] [PubMed]

40 Geron LJV Garcia J de Aguiar SC da Costa FG da Silva AP Neto ELS de Carvalho JTH Roberto LS CoelhoKSM Santos IS Effect of slow-release urea in sheep feed on nitrogen balance Semin Ciecircncias Agraacuterias 2016 39 683 [CrossRef]

41 Zanten van HH Bikker P Mollenhorst H Meerburg BG de Boer IJ Environmental impact of replacing soybean meal withrapeseed meal in diets of finishing pigs Animal 2015 9 1866ndash1874 [CrossRef] [PubMed]

42 Alipour D Saleem AM Sanderson H Brand T Santos LV Mahmoudi-Abyane M Effect of combinations of feed-gradeurea and slow-release urea in a finishing beef diet on fermentation in an artificial rumen system Transl Anim Sci 2020 4839ndash847 [CrossRef]

43 Rebelo LR Luna IC Messana JD Araujo RC Simioni TA Granja-Salcedo YT Effect of replacing soybean meal with ureaor encapsulated nitrate with or without elemental sulfur on nitrogen digestion and methane emissions in feedlot cattle AnimFeed Sci Technol 2019 257 1142 [CrossRef]