DAIANE CAROLINE DE MOURA

FARELO DE BRASSICA NA ALIMENTAÇÃO DE VACAS LEITEIRAS

Cuiabá-MT

2018

DAIANE CAROLINE DE MOURA

FARELO DE BRASSICA NA ALIMENTAÇÃO DE VACAS LEITEIRAS

Cuiabá-MT 2018

Tese apresentada ao Programa de Pós-Graduação em Ciência Animal da Universidade Federal de Mato Grosso para a obtenção do título de Doutora em Ciência Animal Área de Concentração: Nutrição de Ruminantes Orientador: Prof. Dr. André Soares de Oliveira Co-Orientadores: Prof. Dr. André Fonseca de Brito e Prof. Dr. Nelcino Francisco de Paula

ii

iii

iv

Aos meus amigos e família. Em especial: A minha mãe Maria Inês de Moura e ao meu esposo

por todo carinho e paciência Flavio Junior Gonçalves Vieira que sempre me apoiaram e

incentivaram em todos os momentos.

Dedico!

v

É saber se sentir infinito

Num universo tão vasto e bonito, é saber sonhar

Então fazer valer a pena

Cada verso daquele poema sobre acreditar

Não é sobre chegar

No topo do mundo e saber que venceu

É sobre escalar e sentir que o caminho te fortaleceu

É sobre ser abrigo

E também ter morada em outros corações

E assim ter amigos contigo em todas as situações

(Ana Vilela)

FORÇA, FOCO E FÉ

Trabalhe para seu EU e não para seu EGO. (Fabio de Melo)

vi

AGRADECIMENTOS A Deus:

“Meu refugio, minha fortaleza,

Meu Deus eu confio em ti”

A minha família:

Á minha amada mãe. EU TE AMO

Ao meu amado esposo Flavio Junior Gonçalves Vieira.

Agradeço a todos, que sempre se preocupam com meus estudos, com meu futuro, sempre

me incentivam, oram por mim que de alguma forma direta ou indiretamente, me ajudam ao longo

desses anos.

Deus abençoe e recompense a todos com suas graças.

Ao meu orientador, pela oportunidade de sua orientada, Professor D.Sc. André Soares de

Oliveira. Obrigada pela compreensão, paciência, pela confiança e amizade, pelos valiosos

ensinamentos.

Agradeço a Capes pela concessão de bolsa de doutorado, o CNPq pela concessão de bolsa

sanduiche e a Fundação de Amparo à Pesquisa do Estado de Mato Grosso (FAPEMAT), projeto

número 483724/2011 PRONEM 006/2011) pelo recurso para execução dos experimentos.

Agradeço a empresa Caramuru Alimentos S.A (Itumbiara, GO, Brasil). pelo fornecimento

do farelo de crambe.

A Universidade Federal de Mato Grosso Campus Sinop e Cuiabá pela minha formação,

pela oportunidade e contribuição científica neste trabalho. Obrigada a todos os funcionários de

uma forma geral.

Aos membros da banca de defesa de tese Dr. André Fonseca de Brito, Dr. Nelcino

Francisco de Paula, Eduardo Henrique Bevitori Kling de Moraes e Erick Darlisson Batista.

A família Peron (Braz, Eloide, Eloir, Josimar, Bia minha amada e Josi), por todo carinho,

atenção e conhecimentos. OBRIGADA POR FAZEREM PARTE DA MINHA FAMÍLIA.

As minhas vaquinhas amadas: Preta, Suja, Miúda, Coração, Mandala, Vermelha, Fumaça,

Tana, Dala, Rapoza, Preta e Capitu por colaborem durante todo o experimento.

A família que conquistei nos Estados Unidos: Adelia, Berquinha e todos de sua família que

me acolherem com muito carinho. Minha querida amada Lia e Rafael por todo carinho cuidados e

passeios. Arlete e família por todo carinho e risadas. A lindíssima Ellen Quirino e suas amigas por

vii

me receber tão bem. Aos amigos de batalhas do frio Ronan, Simone, Vicent e Geraldo, por todo

carinho, conversas e risadas. A Dover Adult Learning Center por todos os ensinamentos de inglês.

As minhas amadas eternas amigas e irmãs que encontrei em momentos muitos difíceis

Mariane Moreno Ferro, Caren Paludo Ghedini, Luiza Ghedini e Juliane Freire. EU AMO VOCÊS

POR DEMAIS.

Aos amigos e irmãos do Núcleo de Pesquisa em Pecuária de leite: Karine Claudia Alessi,

Suziane Rodrigues Soares, Rodrigo Torres, Henrique Melo, Leticia, Mari Lucia, Indiara, Andrea

Donadia, Juliana Carla, Dhulyeli Silveira e Janaine Poli

Aos amigos que ajudaram para execução dos experimentos Robson Miranda, Suziane

Rodrigues Soares, Fabio Woork, Fernanda Norberto, Silvia, Douglas, Daniely da Silva Sousa,

Poliana Oliveira Cordeiro, Luana Molossi, Viviane, Vitor T. Padilha, Maycon Barbosa, Gabriel

Tschope, Fabricio Marquez, Taynara, Hugo Assis, Rafael Moreno Ferro, Cadu Teles, Natan

Cecconello, Laísa Marangoni, Camila Piovezan, Rodrigo Torres, Silvana, Dâmiris.

Quem tem um amigo tem um grande tesouro

viii

BIOGRAFIA

Daiane Caroline de Moura, filha de Dirceu Figueiredo e Maria Inês de Moura, nasceu em

Vilhena, Rondônia, no dia 20 de janeiro de 1988.

Em agosto de 2006 ingressou no Curso de Graduação em Zootecnia pela Universidade do

Estado de Mato Grosso, no Campus Universitário de Pontes e Lacerda tendo concluído o curso

em fevereiro de 2012.

Em março de 2012, iniciou o curso de Mestrado em Ciência Animal pela Universidade Federal

de Mato Grosso, desenvolvendo estudos na área de Nutrição e Produção de Ruminantes,

submetendo-se à defesa de tese em 26 de fevereiro de 2014.

Em fevereiro de 2014, iniciou o curso de Doutorado em Ciência Animal pela Universidade

Federal de Mato Grosso e em novembro de 2016 realizou doutorado sanduiche na University of

New Hampshire nos Estados Unidos, em ambas Universidade desenvolvendo estudos na área de

Nutrição e Produção de Ruminantes, submetendo-se à defesa de tese em 05 de Março de 2018.

ix

RESUMO GERAL

MOURA, D. C. Farelo de brassica na alimentação de vacas de leiteiras. 2018. 65f. Tese

(Doutorado em Ciência Animal), Faculdade de Agronomia e Medicina Veterinária, Universidade

Federal de Mato Grosso, Cuiabá, 2018.

Este trabalho está dividido em 2 capítulos. Primeiro capítulo: objetivou-se investigar os efeitos de

inclusão de farelo de crambe (FC, 382,4 g de proteína bruta (PB) / kg de matéria seca (MS), 450

mg de glicosinolatos / kg de MS) na dieta total parcial (DTP; 0, 45, 90 e 135 g / kg DM), sobre o

desempenho produtivo, eficiência de utilização de nutrientes em vacas leiteiras e preferência de

queijo para consumidores não treinados. Foram utilizadas doze vacas leiteiras sendo oito vacas

mestiças de Holandês × Zebu (456 ± 91 kg de peso corporal) e quatros vacas

Jersey (384 ± 32.29 kg de peso corporal) distribuídas em três quadrado latino 4 × 4 simultâneos

com quatro períodos experimentais de 21 dias cada. As DTP foram isonitrogenadas (130 g PB/kg

MS) e oferecido ad libitum entre a ordenha das 7:00 da manhã e 18:30 tarde. As vacas entre o

horário 19:00 da tarde até 6:30 da manhã permaneceram em pastagens Panicum Maximum cv.

Mombaça (564 g de fibra detergente neutro/kg MS e 90,4 g PB/kg MS). A produção de leite e o

consumo de DTP foram registrados do dia 15º ao dia 21º de cada período experimental. As

amostras de leite foram coletadas nos dias 17º e 18º de cada período experimental. As amostras de

fezes de cada animal foram coletadas entre 17º a 21º para estimativa de excreção fecal (usando

dióxido de titânio como indicador externo) e para o consumo de pastagem (utilizando fibra

insolúvel detergente neutro indigestível após 288 incubação ruminal in situ). A inclusão de FC não

afetou o consumo de DTP (P = 0,173; 11,47 ± 0,20 kg de MS/dia), consumo de forragem (P =

0,185; 0,90 ± 0,07 kg MS/dia), consumo de PB (P = 0,481; 1,49 ± 0,01 kg de PB/dia),

digestibilidade da matéria orgânica (P = 0,254; 0,749 ± 0,01 g/g), digestibilidade da PB(P = 0,545;

0,747 ± 0,02 g/g), síntese de proteína microbiana (P = 0,348; 0,83 ± 0,08 kg/d), produção de leite

(P = 0,462; 13,29 ± 0,24 kg/dia), nitrogênio (N) no leite (P = 0,566; 64,2 ± 1,3 g/dia), ureia no

leite (P = 0,178; 10,6 ± 0,94 mg/dL), excreção urinária de nitrogênio (P = 0,717; 90,9 ± 1,9 g/dia),

eficiência de nitrogênio no leite (P = 0,622; 0,268 ± 0,01 g consumo nitrogênio/nitrogênio do

leite), função hepática IU/mL (GGT 32,05 ± 2,94; ALT 15,98 ± 0,44 e AST 48,02 ± 5,71),

rendimento de queijos (0,21 ± 0,01 kg/kg de leite) e análise sensorial (gostei 59,78, 80,00, 76,00,

77,67 % dos provadores). O farelo de crambe pode ser incluso até 135 g/kg de MS na DTP sem

afetar o desempenho produtivo, eficiência na utilização de nutrientes em vacas leiteiras e

preferência de queijo por consumidores não treinados. Segundo capítulo: Utilizamos a abordagem

x

meta-analítica para avaliar os efeitos da substituição de diferentes fontes de proteína por dietas de

brassicas sobre a produção de leite e na utilização de nutrientes de vacas leiteiras, de 37 artigos

revisados por pares. O farelo de canola (FC) foi a principal fonte de brassica. Os efeitos foram

comparados pelas diferenças médias brutas (DMB) entre as dietas com FC e fontes de proteínas

(controle) e ponderados pela variância inversa usando modelos de efeito aleatório. O nível de

heterogeneidade foi analisado por estatística I2 (baixa ≤ 25%, moderada = 26 a 50% e alta> 50%).

Em geral, o uso de FC como fonte de proteína aumentou o consumo de MS (DMR = 0,22 ± 0,12

kg de MS/dia, P <0,01; n = 79; I2 = 9,1%) e ingestão de proteína bruta (PB) = (DMR, 14 ± 0,07

kg PB/dia, P <0,01; n = 33; I2 = 21,1%), ambos com baixa heterogeneidade, mas não afetaram a

digestibilidade do trato total da matéria orgânica (P = 0,50; n = 12; I2 = 29,2 %). No geral, o uso

de FC aumentou a produção de leite (DMR = 0,69 ± 0,35 kg/dia, P <0,01; n = 88; I2 = 74,9%),

mas seu efeito depende da comparação entre as fontes proteínas: o FC versus o FS não houve efeito

a produção de leite (DMR = 0,23 ± 0,66 kg/dia, P = 0,50; n = 33), mas a produção de leite aumentou

com a substituição de DDG por FC (DMR = 2,03 ± 1,67 kg/dia; P <0,01; n = 13) e de outras fontes

de proteína por FC (DMR = 0,82 ± 0,43 kg/dia; P <0,01; n = 42). No geral, o uso de FC não afetou

o teor de proteína do leite (P = 0,08; n = 60; I2 = 19,5%) e teor de gordura do leite

(P = 0,20; n = 60; I2 = 16,9%), mas FC aumentou a produção de proteína do leite

(DMR = 0,02 ± 0,01 kg/dia, P <0,01; n = 60; I2 = 0%). O uso de FC reduziu o teor nitrogênio

ureico no leite (N) (DMR = - 0,98 ± 0,31 mg/dL; P <0,01; n = 22; I2 = 32,2%) e aumentou a

eficiência de consumo N no leite (DMR = 0,22% N do leite/consumo de N ± 0,07 mg/dL; P ≤

0,05; n = 34; I2 = 0%), ambos com baixa heterogeneidade. Concluímos que FC é fonte de proteína

semelhante ao FS e é mais eficaz do que o DDG e outras fontes (farelo de algodão, farelo de milho

e farinha de girassol) para vacas leiteiras em lactação. De modo geral os farelos de brassicas (farelo

de crambe e farelo de canola pode ser incluídos em dietas de vacas leiteiras em substituição á

fontes proteicas sem afetar no desempenho produtivo, eficiência de utilização de nutrientes.

Palavras-Chave: farelo de canola, farelo de crambe, metanálise

xi

ABSTRAT

MOURA, D. C. Brassica meal for dairy cows. 2018. 65f. Thesis (PhD in Animal Science),

Faculdade de Agronomia e Medicina Veterinária, Universidade Federal de Mato Grosso, Cuiabá,

2018.

This thesis is divided into 2 chapters. The objective of this study was to investigate the effects of

inclusion of crambe meal (CM, 382.4 g crude protein/kg dry matter (DM), 450 mg

glucosinolates/kg DM) in the total partial diet (pTMR, 0, 45, 90 and 135 g/kg DM), on the

productive performance, nutrient utilization efficiency in dairy cows and cheese preference for

untrained consumers. Twelve dairy cows were used: eight crossbred Holstein × Zebu cows (456 ±

91 kg body weight) and four Jersey cows (384 ± 32.29 kg body weight) distributed in three

simultaneous 4 × 4 Latin squares with four experimental periods of 21 days each. The pTMR were

isonitrogenated (130 g CP/kg DM) and offered ad libitum between milking from 7:00 am and 6:30

pm. Cows between the hours of 19:00 in the afternoon until 6:30 in the morning remained in

pastures Panicum Maximum cv. Mombaça (564 g neutral detergent fiber/kg MS and 90.4 g CP/kg

DM). Milk yield and pTMR intake were recorded from day 15th to day 21st of each experimental

period. The milk samples were collected on days 17th and 18th of each experimental period. Fecal

samples from each animal were collected between 17th and 21st to estimate fecal excretion (using

titanium dioxide as an external indicator) and for pasture consumption (using indigestible neutral

detergent insoluble fiber after 288 ruminal in situ incubation). The inclusion of CM did not affect

pTMR intake (P = 0.173, 11.47 ± 0.20 kg DM/day), forage intake (P = 0.185, 0.90 ± 0.07 kg

DM/day), CP intake (P = 0.481, 1.49 ± 0.01 kg CP/day), organic matter digestibility (P = 0.254,

0.749 (P = 0.545, 0.747 ± 0.02 g/g), microbial protein synthesis (P = 0.348, 0.83 ± 0.08 kg/d),

milk yield (P = 0., 64.2 ± 1.3 g/d), milk yield (P = 0.462; 13.29 ± 0.24 kg/d), nitrogen (N) milk

(P = 0.566; 64.2 ± 1.3 g/d), milk urea-N (P = 0.178; 10.6 ± 0.94 mg/dL), N urinary excretion (P =

0.717; 90.9 ± 1.9 g/d), N milk efficiency (P = 0.622; 0.268 ± 0.01 g N milk/g N intake) hepatic

function IU/mL (GGT 32.05 ± 2.94, ALT 15.98 ± 0.44 and AST 48.02 ± 5.71), cheese yield (0.21

± 0.01 kg/kg milk) and sensory analysis (I liked 59.78 , 80.00, 76.00, 77.67% of the tasters).

Crambe meal can be up to 135 g/kg DM in pTMR without affects productive performance,

efficiency of nutrient utilization in dairy cows and cheese preference for untrained consumers. We

used meta-analytical approach to evaluate the effects of replacement of different sources of protein

by brassicas meals on milk production and nutrient utilization of dairy cows, from 37 peer-review

papers. Canola meal (CM) was unique brassica source founded. The effects were compared by

xii

raw mean differences (RMD) between CM diet and control treatment means and weighted by

inverse variance using random-effect models. Heterogeneity level was analyzed by I2 statistic (low

≤ 25%; moderate = 26 to 50%; and high > 50%). In overall, use of CM as protein source increased

DM intake (RMD = 0,22 ± 0.12 kg DM/d; P < 0.01; n = 79; I2 = 9.1%) and crude protein (CP)

intake (RMD = 0,14 ± 0.07 kg CP/d; P < 0.01; n = 33; I2 = 21.1%), both with low heterogeneity,

but it did not affect organic matter total-tract digestibility (P = 0.50; n = 12; I2 = 29.2%). In overall,

use of CM increased milk yield (RMD = 0.69 ± 0.35 kg/d; P < 0.01; n = 88; I2 = 74.9%), but its

effect depends on protein sources comparation: CM versus SBM did not increase milk yield (RMD

= 0.23 ± 0.66 kg/d; P = 0.50; n = 33), but milk yield was increased with replacement of DDG by

CM (RMD = 2.03 ± 1.67 kg/d; P < 0.01; n = 13) and of other protein sources by CM (RMD =

0.82 ± 0.43 kg/d; P < 0.01; n = 42). In overall, CM use did not affect milk protein content (P =

0.08; n = 60; I2 = 19.5%) and milk fat content (P = 0.20; n = 60; I2 = 16.9%), but CM increased

milk protein yield (RMD = 0,02 ± 0.01 kg/d; P < 0.01; n = 60; I2 = 0%). Use of CM reduced milk

urea nitrogen (N) (RMD = - 0,98 ± 0.31 mg/dL; P < 0.01; n = 22; I2 = 32.2%) and increase N

intake milk efficiency (RMD = 0.22% N milk/N intake ± 0.07 mg/dL; P ≤ 0.05; n = 34; I2 = 0%),

both with low heterogeneity. We concluded that CM is similar protein source to SBM and it is

more effective than DDG and other sources (cottonseed meal, corn gluten meal and sunflower

meal) to lactating dairy cows.

Key words: canola meal, crambe meal, meta-analysis

xiii

SÚMARIO

1 - INTRODUÇÃO GERAL ............................................................................................. 1

2- REFERÊNCIA BIBLIOGRAFICA .............................................................................. 6

Capitulo 1- Crambe meal in diets for dairy cows ............................................................ 10

INTRODUCTION ........................................................................................................... 11

MATERIALS AND METHODS .................................................................................... 12

RESULTS ........................................................................................................................ 19

DISCUSSION ................................................................................................................. 20

CONCLUSIONS ............................................................................................................. 22

REFERENCES ................................................................................................................ 22

Capitulo 2 - Meta-analysis of the use of brassicas meals in diets for dairy cows ........... 33

INTRODUCTION ........................................................................................................... 34

MATERIALS AND METHODS .................................................................................... 35

RESULTS AND DISCUSSION ..................................................................................... 37

CONCLUSIONS ............................................................................................................. 39

REFERENCES ................................................................................................................ 40

1

1 - INTRODUÇÃO GERAL

As Brassicaceae são compostas por 51 gêneros e 218 espécies, algumas das quais são

espécies importantes de culturas cultivadas como vegetais, forragens e oleaginosas

(Sabharwal et al., 2006). Após a extração de oleaginosas são produzidos produtos com potencial

de uso na alimentação dos ruminantes como fonte de proteína. O farelo de canola, farelo de

crambe e farelo de carinata são as principais fontes de brassicas inclusas na alimentação animal,

mas seu uso depende dos níveis de glicosinolatos (Tripathi e Mishra, 2007; Moura et al., 2017).

O crambe (Crambe abyssinica H.) representa uma fonte alternativa e promissora de óleo

vegetal para produção de biodiesel conhecida como couve etíope. É uma oleaginosa da família

Brassicaceae e também conhecidas como da família das crucíferas, da qual fazem parte plantas

como a mostarda (Brassica campestris L.), a canola (Brassica napus L.) e a nabiça

(Raphanus raphanistrum L.). O crambe é nativo da região do Mediterrâneo sendo e também

cultivado na África, Ásia, Europa, Estados Unidos, México e América do Sul

(Fontana et al.,1998; Souza et al., 2009).

As plantas de crambe são herbáceas anuais, com altura entre 70 e 90 cm, com ciclo

anual curto (90 dias), ramifica-se próxima ao solo para formar galhos (trinta ou mais), os quais

se ramificam, formando galhos terciários. Suas folhas são ovais e assimétricas. As flores são

brancas, numerosas pequenas e estão agrupadas em cachos (Opliger et al., 1991; Fontana et

al.,1998; Souza et al., 2009). Os frutos são uma siliquia de forma esférica inicialmente verde,

tornando-se amarelo com a maturidade e distribuído por todos os galhos da planta. Cada siliquia

possui uma semente de cor verde ou marrom esverdeado com diâmetro entre 0,8 a 2,5 mm

(Desai et al., 1997).

A cultura do crambe é tolerante a seca e a geadas em grande parte de seu

desenvolvimento, cultivada entre a safra de verão e inverno, podendo ser implantada e cultivada

de forma mecanizada, utilizando implementos da cultura soja (Pitol et al., 2010). Para Brito

(2009) o ponto importante de crambe é a baixa ocorrência de pragas, o que diminui os custos

de sua produção.,

A cultura de crambe foi utilizada pela primeira vez em 1933, na Estação Botânica

Boronez, na antiga União das Republicas Socialistas Soviéticas (Mastebroek et al., 1994). As

pesquisas com crambe e a sua produção comercial se intensificaram a partir dos anos 80, após

2

sua introdução nos Estados Unidos da América, no Reino Unido e em alguns países da Europa,

como a Itália, França e Portugal. Contudo, as áreas plantadas nesses países não aumentaram

expressivamente, devido a competição por área com as principais culturas de safra, como o

milho, a soja e o trigo, sendo que nessas regiões não foi possível cultivar o crambe em safrinhas

(período de entressafra compreendido entre culturas principais e iniciada após a cultura de

verão). Dessa maneira, o crambe vem crescendo muito em outros países, como a Austrália, a

África do Sul, o Paraguai e o Brasil (Pitol et al., 2010).

A introdução da crambe no Brasil ocorreu na década de 90 por meio da introdução de

cultivares vindo do México, os quais foram selecionados por pesquisadores da Fundação Mato

Grosso do Sul, em 1995, originando o primeiro cultivar de crambe (FMS Brilhante) no país,

entretanto, somente a partir de 2003 com o lançamento do Programa Nacional de Biodiesel

(PNB) despertou interesse ao cultivo (Pitol et al., 2010; Pilau et al., 2011). De acordo com Pitol

et al. (2010), a produtividade de semente de crambe no Brasil é de 1.000 a 1.500 kg/ha, com

potencial de produção de 450 kg de óleo/ha. No Brasil central, nas regiões Centro-oeste e

Sudeste, o crambe pode ser cultivado como cultura de “safrinha”, no período de fevereiro a

abril, após as culturas de verão (Fundação MS, 2009).

Estudos realizados na estação de pesquisa da Fundação do Mato Grosso do Sul (2007),

em Maracajú – MS, destacaram como vantagens do crambe tolerância à seca, à geada e depois

de estabelecida, elevado teor de óleo (34% a 38%), precocidade (florescimento aos 35 dias e a

colheita aos 85/90 dias) (Jaspar et al., 2010).

Segundo Carlsson (1983), o óleo produzido não é comestível, porém possui grande

utilidade para matéria prima industrial (lubrificante, inibidor da corrosão, matéria-prima para a

produção de borracha sintética e plástico), pois apresenta cerca de 60% de ácido erúcico

(C22:2). O ácido erúcico é um ácido graxo monoinsaturado de cadeia longa que pode provocar

lesões cardíacas quando ingeridos (Air, 1997). Assim, este óleo não concorre com óleos

destinados ao setor alimentício.

O processo de extração de óleo de crambe gera produtos (farelos ou tortas) com elevado

teor proteico e com grande potencial de uso na alimentação animal em substituições às fontes

tradicionais, como farelo de soja e de algodão.

Entretanto tornando-se necessário, o estudo mais detalhado do perfil aminoacídico do

farelo de crambe para poder substituir o farelo de soja em momentos de escassez ou preço em

3

alta no mercado, sem que este fornecimento de aminoácidos não seja prejudicado. Em estudos

realizados por Liu et al. (1993) e Anderson et al. (1993), verificaram que tanto o teor de

metionina como o de cistina é maior no farelo de crambe do que no farelo de soja.

Pesquisadores norte-americanos observaram que a substituição integral do farelo de soja

pelo farelo de crambe in natura (10% de inclusão na dieta), com teor de glicosinolatos de

(56 mmol/kg de MS ou 22 g de equivalente sirigin/kg de MS – peso molecular do sirigin de

397) não afetaram o crescimento e características de carcaça de bovinos, bem como o

desempenho reprodutivo e função da tireóide em vacas de corte gestantes e não lactantes

(Anderson et al., 1993; Anderson et al., 2000).

No Brasil, Mizubuti et al. (2011) observaram que a torta de crambe apresenta

degradabilidade ruminal e digestibilidade intestinal da proteína semelhantes ao do farelo de

soja. Conava et al. (2015) verificou que a substituição do farelo de soja pela torta de crambe

(21% de inclusão na dieta, base da matéria seca; teores de glicosinolatos não informados)

reduziu o consumo e digestibilidade da dieta em ovinos, em razão do aumento excessivo do

teor de lipideos na dieta de 7,5% de extrato etéreo (acima do limite padrão de 6%, base da

matéria seca), comprometendo a digestão da fibra.

Diante do exposto, verifica-se ainda há necessidade de maiores investigações dos efeitos

do farelo de crambe e outras brassicas na alimentação de vacas de leite.

A canola (Brassica napus L. var oleífera) é uma oleaginosa e cultura alternativa de

inverno (Zimmermann, 2005). Foi desenvolvida a partir do melhoramento genético da colza,

obtida do cruzamento Brassica napus (colza) e Brassica campestris (mostarda) passando ser

chamada de canola (Canola Council of Canada, 2009). O termo canola significa a abreviação

internacional para Canadian Oil Low Acid, grão com baixos níveis de ácido erúcico no óleo

(menos que 2%) e baixos níveis de glicosinolatos no farelo (menos que 3 mg/g) (Bell, 1984;

Bett et al., 1999; Newkirk, 2009).

No Brasil cultiva-se apenas canola de primavera, da espécie Brassica napus L. var.

oleífera, que início a implantação na década de 80 no estado do Paraná (PR) e em 2003 em

Goiás (GO) sendo esta uma ótima opção nos sistemas de rotação de culturas, auxiliando na

quebra do ciclo de diversas doenças e pragas das gramíneas e leguminosas, isso ocorre pelo

fato de ser uma cultura da família de crucíferas. (Tomm, 2000; Tomm, 2006).

4

A canola é uma planta anual com o hábito de crescimento indeterminado, com sistema

radicular pivotante e ramificação lateral significativa. O caule é herbáceo, ereto, com tamanho

variável de 0,5 a 1,7 m. As folhas inferiores da planta são pecioladas e formam uma roseta.

Após a elongação do caule, as folhas emitidas são lanceoladas e abraçam parcialmente a haste.

As flores, agrupadas em cachos, são pequenas e amarelas, formadas por quatro pétalas dispostas

em cruz, seis estames e pistilo. A duração do período de floração varia conforme com a cultivar.

Os frutos são síliquas com cerca de 6 cm de comprimento. Os números de grãos e o

comprimento das síliquas, varia dependendo a cultivar. (García, 2007).

A semente de canola contém aproximadamente 40,5% de óleo, o que a torna uma das

oleaginosas com maior teor de óleo disponível para a produção de biodiesel (Lardy, 1993). Os

principais ácidos graxos que compõe o óleo são em sua maioria, oléico (C18: 1), linoléico

(C18:2) e linolênico (C18:3) (Sanches 1997).

Após a extração do óleo é gerado o farelo de canola. De acordo com Queiroz (2008), o

grão de canola é moído, esmagado e prensado para extração do óleo. Posteriormente para

retirada do óleo remanescente é adicionado solvente (hexano) e tostado e moído gerando o

farelo de canola.

Hoje, o farelo de canola é a segunda mais consumida no mundo (12% de participação

de mercado), abaixo de apenas a farelo de soja (71% de participação no mercado) (SoyStats,

2018).

O farelo de canola apresenta teores médios de 38,0% de PB, 5,2% de amido; 21,2% de

FDN e 17,2% de FDA e 1,5% de EE (Bell, 1993). O mesmo apresenta aproximadamente 35%

de proteína não degradável no rúmen (NRC, 2001). O farelo de canola apresenta um perfil de

aminoácidos semelhante ao do farelo de soja, porém com teor inferior de lisina e superior de

metionina + cistina (NRC 2001). Piepenbrink and Schingoethe (1998) corroboram afirmando

que o perfil de aminoácidos do farelo de canola e muito semelhante ao encontrado no leite de

bovinos. Assim havendo interesse crescente em balancear as dietas de vacas leiteiras visando

atender as exigências de aminoácidos.

O cultivo de Carinata está sendo implantada na Dakota do Sul, Dakota do Norte,

Minnesota, Montana e na Flórida (EUA) como potencial para cultura de inverno e nova matéria-

prima para a produção de biocombustíveis (Lawrence e Anderson, 2015). Esta oleaginosa

5

pertence a das família Brassica onde estão incluidas as culturas alimentares mais comuns

repolho, couve de Bruxelas, couve-flor, couve, colza / canola e brócolis (Moser, 2010).

Após a extração do óleo, é gerado o farelo de carinata que está havendo um grande

interesse para ser inclusão na alimentação animal, principalmente pelo alto teor proteína 35-

40% na matéria seca, boa fonte de proteína degradável do rúmen (70% de proteína bruta

degradada no rúmen) e digestibilidade total comparável à das farinhas de soja (farelo de soja

96% versus farelo de carinata 94,4%) (Lawrence e Anderson, 2015).

O SDSU Dairy e Food Science Department testou recentemente os efeitos da

alimentação de carinata no desempenho de crescimento de novilhas leiteiras, com nível de

inclusão de 10 % da MS total da dieta em comparação DDGS (grãos secos de destilaria com

solúveis) e verificou que a carinata pode ser inclusa até 10% da dieta, pois apresentou

desempenho de crescimento semelhante em comparação com novilhas alimentadas com dietas

com DDGS (Rodriguez-Hernandez e Jill Anderson, 2016).

Este foi um dos primeiros estudos realizados sobre a alimentação de carinata para

alimentação de bovinos leiteiros. Assim novas pesquisas são necessárias para testar os efeitos

carinata na alimentação de vacas em comparação com outras fontes de proteína

Os capítulos foram formatados de acordo com as normas da Journal of Dairy Science.

6

2- REFERÊNCIA BIBLIOGRAFICA

AIR Crambe abyssinica, a comprehensive program – Workshop – Part 4 – Utilization.

Summary information. AIR3-CT94-2480, 1997. Disponível em:

(http://www.biomatnet.org/secure/Air/F709.htm). Acesso em: 17/09/2013.

ANDERSON, V. L.; CATON, J. S. KIRSCH, J. D.; REDMER, D. A. Effect of crambe

meal on performance, reproduction, and thyroid hormone levels in gestating and lactating beef

cows. Journal Animal Science, v.78, p.2269-2274, 2000.

ANDERSON, V. L.; SLANGER, W. D. B.; BERG, P. T. Crambe meal is equivalent to

soybean meal for backgrounding and finishing beef steers. Journal Animal Science, v.71,

p.2608-2613, 1993.

BELL, J. M. Nutrients and toxicants in rapeseed meal: A review. Journal Animal

Science, v. 58, p.996-1010, 1984.

BELL, J. M. Factors affectrngthe nutritional value of canola meal: A review Canadian

Journal of Animal Science. 73: 679-691, 1993.

BETT, V.; SANTOS, G. T.; AROEIRA, L. J. M.; PETIT, H. V.; DIAS, P. G.; LEGGI,

T. C. S. S.; PERON, K. F.; ZEOULA, L. M. Desempenho e digestibilidade in vivo de cordeiros

alimentados com dietas contendo canola em grão integral em diferentes formas. Revista

Brasileira de Zootecnia, v.28, n.4, p.808-815, 1999.

BRITO, D. M. C. Aspecto do metabolismo de plantas de crambe (Crambe

abyssinica) submetidas a diferentes doses de nitrogênio visando à produção de óleo para

biodiesel. 2009. 58p. Dissertação (Mestrado em química, química agrária). Instituto de ciências

exatas, Universidade Federal rural do Rio de Janeiro, Seropédica, RJ, 2009.

CANOLA COUNCIL OF CANADA. 2009. Canola meal nutrient composition.

Disponível em: https://cigi.ca/wp-content/uploads/2011/12/2009-Canola_Guide.pdf. Acesso

em 01/03/2018.

CANOVA, E. B.; BUENO, M. S.; MOREIRA, H. L. et al. Crambe cake (crambe

abyssinica hochst) on lamb diets. Ciência e Agrotecnologia, v.39, n.1, p. 75-81, 2015.

CARLSON, K. D.; TOOKEY, H .L. Crambe Meal as a protein source for feeds. Journal

of the American Oil Chemists’ Society. v. 60. n. 12, 1983.

DESAI, B. B.; KOTECHA, P. M.; SALUNKHE, D. K. Seeds handbook: biology,

production processing and storage. Marcel Dekker,, 627 p., 1997.

7

FONTANA, F.; LAZZERI, L.; MALAGUTI, L.; GALLETTI, S. Agronomic

characterisation of some Crambe abyssinica genotypes in a lacality of the Po Valley. European

Journal of Agronomy , v.9, p.117-126, 1998.

FUNDAÇÃO MATO GROSSO DO SUL. Crambe FMS Brilhante. Maracajú, 2009.

Disponível em: <http://www.fundacaoms.org.br/page.php?34>. Acesso em: 13/09/2012.

GARCÍA, E. R. Manual de produción canola. Puebla: Secretaria de Desarrollo Rural

Del Estado de Puebla, 2007.

JASPER, S. P.; BIAGGIONI, M. A. M.; SILVA, P. R. A.; SEKI, A. S.; BUENO, O. C.

análise energética da cultura do crambe (Crambe abyssinica Hochst) produzida em plantio

direto. Engenharia Agrícola, v.30, n.3, p.395-403, 2010.

LARDY, G. P.; CATLETT, G. E.; KERLEY, M. S. Determination of the ruminal

escape value and duodenal amino acid flow of rapeseed meal. Journal of Animal Science, v.

71, n.11, p.3096-3104, 1993.

LAWRENCE, R.; ANDERSON, J. 2015. Farinha de Camelina e carinata: fontes

potenciais de proteína para o gado leiteiro. Disponível:

http://igrow.org/livestock/dairy/camelina-meal-carinata-potential-protein-sources-for-dairy-

cattle/. Acesso em: 01/03/2018.

LIU, Y. G.; ZHOU, M. Q.; LIU, M. L. A survey of nutrients and toxic factors in

commercial rapeseed meal in China and evaluation of detoxification by water extraction.

Animal Feed Science and Technology, v.45, n.3, p.257-270, 1994.

MASTEBROEK, H. D.; WALLENBURG, S. C.; VAN SOEST, L. J. M. Variation for

agronomic characteristics in crambe (Crambe abyssinica hochst. Ex Fries). Industrial crops

and products. v. 2, p 129-136, 1994.

MIZUBUTI, I, Y.; RIBEIRO, E. L. A.; PEREIRA, E. S.; PINTO, A. P.; FRANCO, A.

L. C.; SYPERRECK, M. A.; DORÉA, J. R. R.; CUNHA, G. E.; CAPELARI, M. G. M.;

MUNIZ, E. B. Cinética de fermentação ruminal in vitro de alguns co-produtos gerados na

cadeia produtiva do biodiesel pela técnica de produção de gás. Ciências Agrárias, v. 32,

suplemento 1, p. 2021-2028, 2011.

MOSER, B. R. Camelina (Camelina sativa L.) oil as a biofuels feedstock: Golden

opportunity or false hope?. Lipid Technology, 22: 270-273, 2010.

8

MOURA, D. C.; FONSECA, T. S.; SOARES, S. R.; SILVA, H. M.; VIEIRA, G.V. J.;

BOTINI, L. A. A.; SINHORIN, P.; OGUNADE, I. M.; OLIVEIRA A. S. Crambe meal

subjected to chemical and physical treatments in sheep feeding. Livest Sci, v.203, p.136-140,

2017.

NRC. Nutrient Requirements of Dairy Cattle. 7th rev. ed. Natl. Acad. Sci., Washington,

DC. 2001

NEWKIRK, R. Canola Meal: Feed Industry Guide. 4th ed. Canadian International

Grains Institute, Winnipeg, MB, Canada, 2009.

OPLINGER, E. S.; OELKE, E. A.; KAMINSKI, A. R.; PUTNAM, D. H.; TEYNOR,

T. M.; DOLL, J. D.; KELLING, K. A.; DURGAN, B. R.; NOETZEL, D. M. Crambe.

Departamento de Agronomia e ciência do solo. In: faculdade de ciências biológicas e de

extensão agrícola cooperativa. Universidade de Wisconsin, Madison, 1991.

PIEPENBRINK, M. S.; SCHINGOETHE, D. J. Ruminal degradation, amino acid

composition, and estimated intestinal digestibilities of four protein supplements. Journal Dairy

Science, v.81, p.454–461, 1998.

PILAU, F. G.; BATTISTI, R.; SOMAVILLA, L.; SCHWERZ, L. Temperatura basal,

duração do ciclo e constante térmica para a cultura do crambe. Bragantia, Campinas, v. 70, n.

4, p.958-964, 2011.

PITOL, C.; ROSCOE, R. Introdução e melhoramento do crambe no brasil. In:

FUNDAÇÃO MS. Tecnologia e produção: crambe 2010. Maracajú: FUNDAÇÃO MS. p.4 6,

2010.

QUEIROZ, M. A. A. Desempenho, características da carcaça e parâmetros metabólicos

de cordeiros recebendo rações ricas em amido e fontes protéicas. 2008, 156 p. Tese (Doutorado

em Agronomia) Escola Superior de Agricultura Luiz de Queiroz, Piracicaba, 2008.

RODRIGUEZ-HERNANDEZ, K.; ANDERSON, J. 2016. Disponível em:

http://igrow.org/livestock/dairy/carinata-meal-potential-as-a-feedstuff-for-growing-dairy-

heifers/. Acesso em: 01/2018.

SANCHES, S. 1997. 47 p. Ácidos graxos em três gerações de sementes de canola

(Brassica napus L.) nas variedades CTC-4 e ICIOLA-4 e de oito variedades de canola

produzidas no PR. Maringá: UEM. Dissertação (Mestrado) – Universidade Estadual de

Maringá, Departamento de Química, 1997.

9

SOUZA, A. D. V; FAVARO, S. P.; ITAVO, L. C. V.; ROSCOE, R. Caracterização

química de sementes e tortas de pinhão-manso, nabo-forrageiro e crambe. Revista Pesquisa

Agropecuário Brasileira. v.44, n.10, p.1328-1335, 2009.

SOYSTATS. 2018. The American Soybean Association. Disponível em:

http://soystats.com/international-world-protein-meal-consumption/. Acesso em: 20/02/2018.

TOMM, G. O. Situação atual e perspectivas da canola no Brasil. Passo Fundo: Embrapa

Trigo, 2000. (Embrapa Trigo. Comunicado técnico online, 58). Disponível em:

http://www.cnpt.embrapa.br/biblio/p_co58.htm. Acesso em: 15/01/2018.

TOMM, G. O. Canola: alternativa de renda e benefícios para os cultivos seguintes.

Revista Plantio Direto, v.15, n.94, p.4-8, 2006.

TRIPATHI, M. K.; MISHRA, A. S. 2007.Glucosinolates in animal nutrition: A review.

Animal Feed Science Technology, v.132 p.1–27, 2007.

ZIMMERMANN J. Cultivo da Canola como alternativa de safrinha no Distrito Federal.

Boletim técnico. UPISFaculdades Integradas, Departamento de Agronomia, Planaltina,

Distrito Federal, 2005.

10

Crambe meal in diets for dairy cows 1

Abstract: We evaluated the effects crambe meal (CM; 382.4g crude protein (CP)/kg dry matter 2

(DM); 450 mg glucosinolates/kg DM) inclusion in partial total mixed ration (pTMR; 0, 45, 90 and 3

135 g/kg DM), on productive performance, efficiency of nutrient utilization in dairy cows and 4

cheese preference for untrained consumers. Twelve dairy cows were used: eight crossbred 5

Holstein × Zebu cows (456 ± 91 kg body weight) and four Jersey cows (384 ± 32.29 kg body 6

weight) distributed in three simultaneous 4 × 4 Latin squares with four experimental periods of 21 7

days each. The pTMR were isonitrogenated (130 g CP/kg DM) and offered ad libitum between 8

milking from 7:00 am and 6:30 pm. Cows between the hours of 19:00 in the afternoon until 6:30 9

in the morning remained in pastures Panicum Maximum cv. Mombasa (564 g neutral detergent 10

fiber/kg MS and 90.4 g CP/kg DM). Milk yield and pTMR intake were recorded from day 15th to 11

day 21st of each experimental period. The milk samples were collected on days 17th and 18th of 12

each experimental period. Fecal samples from each animal were collected between 17th and 21st to 13

estimate fecal excretion (using titanium dioxide as an external indicator) and for pasture 14

consumption (using indigestible neutral detergent insoluble fiber after 288 ruminal in situ 15

incubation). Spot urine samples were collected after feeding (3 h) on the 20th day of each period 16

by manual stimulation of the vulva. The inclusion of CM did not affect pTMR intake (P = 0.173, 17

11.47 ± 0.20 kg DM/day), forage intake (P = 0.185, 0.90 ± 0.07 kg DM/day), CP intake (P = 0.481, 18

1.49 ± 0.01 kg CP/day), organic matter digestibility (P = 0.254, 0.749 (P = 0.545, 0.747 ± 0.02 19

g/g), microbial protein synthesis (P = 0.348, 0.83 ± 0.08 kg/d), milk yield (P = 0., 64.2 ± 1.3 g/d), 20

milk yield (P = 0.462; 13.29 ± 0.24 kg/d), nitrogen (N) milk (P = 0.566; 64.2 ± 1.3 g/d), milk 21

urea-N (P = 0.178; 10.6 ± 0.94 mg/dL), N urinary excretion (P = 0.717; 90.9 ± 1.9 g/d), N milk 22

efficiency (P = 0.622; 0.268 ± 0.01 g N milk/g N intake) hepatic function IU/mL (GGT 32.05 ± 23

2.94, ALT 15.98 ± 0.44 and AST 48.02 ± 5.71), cheese yield (0.21 ± 0.01 kg/kg milk) and sensory 24

11

analysis (I liked 59.78 , 80.00, 76.00, 77.67% of the tasters). Crambe meal can be up to 135 g/kg 25

DM in pTMR without affects productive performance, efficiency of nutrient utilization in dairy 26

cows and cheese preference for untrained consumers 27

Key words: brassica; byproducts; intake; glucosinolates 28

29

1 - Introduction 30

Crambe (Crambe abyssinica H) represents alternative source of vegetable oil to produce 31

biodiesel and other industrial use (Souza et al., 2009). Crambe meal (CM) is a byproduct obtained 32

after oilseed extraction and contains 370 to 430 g crude protein (CP)/ kg dry matter (DM) 33

depending on the cultivar, method of oil extraction (Mizubuti et al., 2011; Moura et al., 2017). 34

However, glucosinolates is a potential limiting for use in animal diet (Tripathi e Mishra, 2007). 35

Moura et al. (2017) observed that CM provided for Animal Nutrition Industry in Brazil had low 36

glucosinolates level, and that its use did not affect intake, total-tract digestibility, ruminal microbial 37

protein synthesis and nitrogen (N) balance in ruminant. 38

Crambe meal and soybean meal (SBM) exhibit similar intestinal digestibility the protein 39

(Lambert et al., 1970; Mendonça et al., 2015a; Mendonça et al., 2015b; Moura et al., 2017). The 40

methionine in CM contents are higher than SBM, but CM has low lysine level 41

(Anderson et al., 1993, Liu et al., 1993). 42

Therefore, there is evidence that CM could be an alternative protein source to SBM to 43

lactating dairy cows. However, we did not found studies about use of CM on diet to dairy cows, 44

and so it is necessary to elucidate its effects on nutrient utilization efficiency and dairy cow 45

performance. We hypothesized that inclusion SBM with CM did not affect the efficiency of 46

nutrient utilization and performance the dairy cows. Therefore, we evaluated the effects crambe 47

meal (CM; 382.4g crude protein (CP)/kg dry matter (DM); 450 mg glucosinolates/kg DM) 48

inclusion in partial total mixed ration (pTMR; 0, 45, 90 and 135 g/kg DM), on productive 49

12

performance, efficiency of nutrient utilization in dairy cows and cheese preference for untrained 50

consumers. 51

2 - Materials and methods 52

Care and handling of the animals used in the current study were conducted as outlined in 53

the guidelines of the Universidade Federal de Mato Grosso Institutional Animal Care and Use 54

Committee (IACUC# 23108718743/2016-11). 55

This experiment was conducted at the commercial dairy farm (Dona Hermínia Fazenda, 56

Sinop, MT, Brazil; 55o20`19.802`` O and 11o49`50.988`` S; altitude 344 m above sea level) and 57

Dairy Cattle Research Lab at the Universidade Federal de Mato Grosso - Sinop (55o28`51.532`` 58

O and 11o51`49.517`` S; altitude 378 m above sea level), from 11th of August to 7th of november 59

2014 (dry to rain transition), totaling 84 experimental days. CM was provided by Camaruru Ltda 60

(Itubiaria, Goiás, Brazil). 61

Animals, experimental design and pasture management 62

Twelve cows eight Holstein × Zebu dairy cows (456 ± 91 kg body weight (BW) and 73 ± 63

64 days in milk) and four Jersey (384 ± 32.29 kg body weight (BW) and 73 ± 64 days in milk) 64

cows were blocked by days in milk and randomly assigned with squares to treatment sequences in 65

tree replicated 4 x 4 Latin squares (1 Latin square Jersey and 2 Latin square Holstein × Zebu dairy 66

cows). Treatment sequences within Latin squares were balanced for carryover effects in 67

subsequent four periods (Williams, 1949). Each period lasted 21 d and consisted of 14 d for diet 68

adaption and 7 d for data and samples collection. At the end of the experiment all, all cows less 69

than 150 d of gestation. 70

Cows were fed on pasture plus pTMR system. The cows received four isonitrogenous 71

pTMR (130 ± 0.10 g crude protein/kg DM) containing four levels inclusion of CM 72

(0, 45, 90 and 135 g/kg DM). The chemical composition of the feeds is presented in Table 1. The 73

13

proportion and chemical composition of the pTMR are described in Table 2. Each cow individually 74

received pTMR ad libitum between the morning and afternoon milking in a tie-stall barn, totality 75

16 h of feeding. The pTMR were fed four daily at 0700, 0900, 1200 am and 0300 pm Amounts of 76

pTMR offered to the cows were adjusted daily to allow refusals equal to 5 to 10% of intake. The 77

DM content from weekly composites of the silages and concentrated mixture were used to adjust 78

as-fed pTMR composition to maintain constant dietary ingredient over the trial. After the last 79

milking (6:00 pm), the cows went to 1.3 acre of pasture of Panicum maximum cv. Mombaça, with 80

access to water. 81

Sampling and Experimental procedures 82

The average sward was determined by measuring the height at pre-grazing and post-grazing 83

by 15 measurements in each paddock. The forage mass in pre-grazing was determined using a 84

square of 0.25 m2, with two measurements per paddock. After collection, the components were 85

separated by leaf, green stem and dead material, and were weighed and stored (-15oC) for 86

subsequent pre-drying, grinding and chemical analysis. During the collection period (7 d) the 87

pasture intake was sampled daily by simulated grazing. Immediately, the samples were stored (-88

15oC). At the end of collection period (d 7) pasture sample by simulated grazing were removed 89

from the freezer, thawed at room temperature, and blended manually to obtain a composite sample 90

per each period. The composite sample of pasture (simulated grazing) was predried in a forced-air 91

oven at 55°C for 72 h. 92

Were sampled individual ingredients of concentrate mixture in each mixture preparation 93

(21 d) and kept in a freezer (−15°C) for subsequent pre-drying, ground and chemical analysis. 94

Daily intake of pTMR was determined by the difference between the weight of DM offered and 95

refused. Twice daily were weighed corn silage, concentrate mixture offered and pTMR refusals 96

(removed) for each cow. Twice daily were sampled (approximately 100 g) of the offered diet and 97

refusals and stored in plastic bags, labeled, and kept in a freezer (−15°C). At the end of collection 98

14

period (d 7), corn silage and refusal samples of each animal were removed from the freezer, thawed 99

at room temperature, and blended manually to obtain a composite sample per animal for each 100

period. The composite sample of corn silage and refusal were predried in a forced-air oven at 55°C 101

for 72 h. 102

Titanium dioxide (TiO2) was used as external marker to estimate the fecal excretion 103

(Titgemeyer et al., 2001) and indigestible NDF (iNDF (indigestible neutral detergent fiber)); after 104

288 hours of in situ rumen) was used as internal marker to estimate intake. After morning milking, 105

each cow received 15 g of TiO2 during the 16th to 21st d of each period. Feces were directly 106

collected from the rectum once daily at 0800, 1000, 1200, 1400 and 1600 h, from 16th to 21st day 107

of each period, and stored (-15oC) for subsequent predrying, grinding and chemical analysis. After 108

predrying and grinding, a single composite fecal sample was obtained per cow per period. 109

The dry matter intake (DMI) from pasture (DMIpasture) per cow was estimated as: 110

DMIpasture (kg/d) = [(FE × iNDFf) – iNDFc intake]/iNDFp 111

where: FE, the fecal excretion (kg/d); iNDFf, iNDF in feces (kg/kg DM); iNDFc intake, iNDF 112

concentrate intake (kg/d) = supplement DM intake (kg/d) × iNDF in suppplement (kg/kg DM); 113

iNDFp, iNDF in pasture by simulated grazing (kg/kg DM). 114

Feces, feed and pasture samples were dried in a forced-ventilation oven at 55 ºC for 72 115

hours, ground in a Wiley mill (Tecnal Equipamentos para Laboratório, Piracicaba, São Paulo, 116

Brazil) with 1-mm screen for chemical analysis and 2-mm screen for ruminal incubation in situ 117

(iNDF). 118

Cows were mechanically milked twice daily (0600 a.m and 0500 p.m.) and milk yields 119

were recorded daily from the 15th to the 21st of each period. Milk samples from a.m. and p.m. 120

milking were collected on 18th and 19th d of each period. Composite samples were prepared daily 121

according to milk production and two aliquots ware taken from each composite sample: the first 122

aliquot (50 mL) forwarded to analyze fat, protein and lactose in milk using Mast Classic milk 123

15

analyzer (Milkotester LTD, Model: LM2, Serial: 11624); the second aliquot was deproteinized 124

with trichloroacetic acid (20 mL of milk mixed with 10 mL of trichloroacetic acid 250 g/L), filtered 125

(Whatman #1 filter paper) and stored (-15 ºC) for subsequent analysis of allantoin and urea. Milk 126

yield was adjusted to 40 g fat/kg as NRC (2001). Body weights and body condition score (1 to 5; 127

Ferguson et al. 1994) were measured 21th d of each period. 128

Blood samples were collected in EDTA Vacutainer® tubes from coccygeal vein, 3 h after 129

a.m. supplement feeding on 19th d of each period. Blood samples were immediately centrifuged 130

(2,300 x g, for 15 minutes, room temperature) and plasma was stored (-15oC) for subsequent 131

analysis of urea nitrogen and enzymes gamma glutamyl transpeptidase, alanine 132

aminotransaminase and aspartate aminotransaminase. 133

Spot urine samples were obtained at approximately 3 h supplement post feeding on 20th d 134

of each period by mechanical stimulation of the vulva. The urine samples were filtered and aliquots 135

of 10 mL were immediately diluted into 40 mL of 0.072 N H2SO4 and stored (-15°C) for 136

subsequent analysis of nitrogen, urea, allantoin, uric acid and creatinine. Immediately before the 137

analysis, the urine samples for each cow/period were thawed and centrifuged (2,300 x g, for 15 138

minutes, room temperature). The balance of nitrogenous compounds (N) was obtained by 139

calculating the difference of intake N, feces N, urine N and milk N. 140

Estimation of rumen microbial protein synthesis 141

The microbial protein synthesis in the rumen (MPS, kg/d) was calculated as a function of 142

purine absorbed (PA; mmol/d): 143

MPS (g/d) = [(70 × PA) / (0.83 × 0.116 × 1000] × 6.25 144

where: 70 is the N content in purines (mg N/mmol); 0.83 is the intestinal digestibility of microbial 145

purines microbial purine digestibility; and 0.116 is the purine-N:total bacterial-N 146

(Chen & Gomes, 1992). 147

16

The PA was calculated from total excretion of purine derivatives (EPD), where EPD is sum 148

of urinary allantoin and uric acid excreted, and allantoin secreted in milk, as: 149

EPD (mmol/d) = 0.85 × PA + 0.442 × BW0.75 150

where: 0.85 is efficiency of intestinal absortion of purines (Verbic et al., 1980) and 0.442 × BW0.75 151

the contribution to endogenous excretion of purines obtained from dairy cows (Oliveira, 2014). 152

Urine volume was estimated using creatinine concentration as a marker and assuming a daily 153

creatinine excretion of 24.05 mg/kg of BW (Chizzotti et al., 2008). 154

Chemical analysis 155

The GIs (glucosinolates) were extracted of the CM with methanol and quantified by high-156

performance liquid chromatography (C18 5 µm 250 mm x 4.6 mm I.D. column, from Vertical 157

Chromatography Co, Nonthaburi, Thailand). The GIs were detected at 233 nm and quantified 158

using sinigrin (Sinigrin hydrate, FLUKA Sigma, St. Louis MO) as an external standard (standard 159

curve: 1000, 500, 400, 300, 200, 100, 50 and 5 mg / L). The results were expressed as sinigrin 160

equivalents (mg / kg DM). The sum of the peak areas observed between 5 and 18 minutes were 161

considered as GIs total (Lee et al., 2006) 162

Samples of feeds concentrate, pasture, refusals, and feces were analyzed for the 163

concentrations of dry matter (DM method no. 934.01), organic matter (OM method no. 942.05), 164

crude protein (CP method no. 954.01), ether extract (EE, method no. 920.39) according to the 165

AOAC (1990). Neutral detergent fiber (NDF) was determined using heat stable amylase without 166

sodium sulfite and corrected for residual ash (Mertens, 2002) and N (Licitra et al., 1996) 167

(aNDFom), with an Ankom® fiber analyzer (Ankom technology, Fairport, NY). Neutral detergent 168

insoluble N (NDIN) was measured according to Licitra et al. (1996). The levels of non-fibrous 169

carbohydrates corrected for ash and protein (NFC) were calculated as: NFC = 100 - [(CP - CP 170

from urea + urea in the supplement) + aNDFom + EEt + ashes] (Hall 2000). Fecal titanium 171

concentration was obtained as Myers et al. (2004). Indigestible NDF of feeds, refusals and feces 172

17

was obtained after in situ ruminal incubation in polyester bag (Ankon®, Ankon®, filter bag 57, 173

Ankom technology, Fairport, NY, USA) for 264 h (Casali et al., 2008) 174

Analyses of blood plasma GGT, ALT and AST were performed by optimized ultraviolet 175

kinetic method (Schumann et al., 2002 a, b) using commercial kits (Labtest Diagnostica S.A., 176

Lagoa da Santa, Minas Gerais, Brazil). Urea in plasma and milk samples was measured by 177

enzymatic colorimetric assay, with commercial kits (Urea CE Ref. 27, Labtest Diagnostica S.A., 178

Lagoa da Santa, Minas Gerais, Brazil). Urinary uric in urine samples was determined using the 179

enzymatic-Trinder method with commercial kits (Ácido úrico Liquiform Ref. 73, Labtest 180

Diagnostica SA, Lagoa da Santa, Minas Gerais, Brazil). Allantoin in milk and urine samples was 181

determined colorimetrically as Young and Conway (1942). 182

In situ ruminal degradability 183

For in situ evaluation we used a half-breed cattle, cannulated in the rumen, weighing 500 184

kg and an average age of 36 months. The cattle fed on forage grass Panicum Maximum cv Massai 185

and concentrated feed containing corn, soybean meal and 300 g/kg crambe meal, which was given 186

2 kg of feed once a day in the morning (0800 am) following recommendations (Nocek, 1988). 187

Water and commercial mineral mixture (Matsuda ® 805 FOS) were administered ad libitum. To 188

determine the degradability were used in nylon bags measuring 10x20 cm, with porosity of 50 189

micrometers, which was added 4 g of dried sample (pasture, corn silage, corn, soy meal and crambe 190

meal) in air respecting 20 mg/cm2 (Nocek, 1988). The samples were incubated in the rumen, 191

sequentially, to be taken together at the end of the incubation period, being used times 0, 2, 4, 8, 192

16, 24, 36, 48 and 72 h (NRC, 2001). 193

Immediately after incubation in the rumen, the bags were immersed in cold water 194

(± 0 ° C) and then washed manually with tap water at room temperature until it was clear. The 195

soluble fractions (time zero of incubation) were determined by the same procedures, but without 196

ruminal incubation, washed only under running water. After washing the bags were taken to 197

18

dryness in forced ventilation oven at a temperature of 65 ° C for 72 h, then placed in a desiccator 198

for 30 minutes and weighed to determine the concentrations of DM 199

(method no. 934.01) and CP (method no. 954.01), according to the AOAC (1990). 200

The effective degradability (ED) of DM and CP were estimated using the asymptotic 201

growth model of the first order as Orskov and McDonald (1979) described by the function: Yt = a 202

+ b * (1-e -kd * t); wherein: Yt = fraction degraded at time "t" (%); a = soluble fraction (%); b = 203

potentially degradable insoluble fraction (%); b = kd degradation rate (h-1); and independent 204

variable t = time (h). 205

Cheese production and sensory analysis 206

Milk samples (1 L cow/period) were pasteurized by heating at 65 ° C for 30 minutes. After 207

cooling the milk to 37 ° C was added 0.4 ml of calcium chloride and 0.9 mL of rennet per 1 L of 208

milk. After 40 minutes standing, yielded the 'mass' of cheese was cut into cubes, remaining at rest 209

for 3 minutes to whey removal, the "mass" was moved slowly for 20 minutes. Was added 3.5% by 210

weight of milk by cheese sodium chloride (NaCl), which was added as 500 g and 24 h after turning 211

made. Thereafter the cheeses were unpacked, heavy, cut into small squares. After three days, about 212

¼ of the cheese was used to determine the crude protein content (N x 6.38) modified by the 213

Kjeldahl method (AOAC, 1995), moisture content by direct drying in an oven at 105 ° C, the rest 214

was used for sensory analysis. 215

The cheeses of each cow/period were subjected to sensory test with untrained consumers. 216

with an average of 10/cheese/period, totaling 30 reviews/level CM of inclusion. Each person 217

received a paper containing radio liked and disliked and guidance on how to perform the test. 218

Statistical analysis 219

Data were analyzed as three simultaneous 4 x 4 Latin squares, using a mixed model 220

(Littell et al., 1997) through the PROC MIXED procedure of SAS (SAS Institute, 1999-2000): 221

Yijkl = µ + Si + Pj + Ck(i) + Tl + STil + eijkl, 222

19

where: Yijkl is the dependent variable; µ is the overall mean; Si is the random effect of square i; Pj 223

is the random effect of period; Ck(i) is the random effect of cow k (within Latin square i); Tl is the 224

fixed effect of treatment l (four CM level on supplement); STil is random effect of interaction 225

between square I and treatment l; and eijkl = random error associated with each observation, 226

assumed NID (0, σ2). Linear, quadratic and cubic effect of CM level on pTMR were tested by 227

partitioning freedom degrees for diet into single freedom degrees variable corresponding to linear, 228

quadratic and cubic effects. Significance was declared at P ≤ 0.05. All reported values were as 229

least square means. 230

Cubic effects were not significant. Thus, the P-values for those components are not 231

presented in Tables 4–6 232

3 - Results 233

Crambe meal inclusion did not affect pTMR intake (P = 0.173), pasture intake (P = 0.185), 234

total DM intake (P = 0.481), OM intake (P = 0.420), EE intake (P = 0.216), CP intake (P = 0.481), 235

aNDFrom intake (P = 0.078) and TDN intake ((P = 0.262) (Table 4). In addition, CM inclusion 236

did not affect total-tract digestibility of the DM (P = 0.229), OM (P = 0.254), aNDFrom (P = 237

0.451), NFC (P = 0.255), TDN (P = 0.120), ruminal microbial protein (P = 0.338) and serum 238

concentrations of GGT (P = 0.529), ALT (P = 0.654) and AST (P = 0.432). Increased levels of 239

CM in pTMR linearly reduced NFC intake (P = 0.002) and it linearly increased EE digestibility 240

(P = 0.052) (Table 4). 241

Crambe meal inclusion in pTMR did not affect body weight (P = 0.379), body condition 242

score (P = 0.693), milk yield (P = 0.462) and milk lactose (P = 0.139), protein (P = 0.626) and fat 243

(P = 0.191) contents, and feed efficiency (P = 0.800) (Table 5). Crambe meal inclusion inclusion 244

did not affect cheese DM (P = 0.484) and CP (P = 0.847), cheese yield (P = 0.314) and cheese 245

preference for untrained consumers (P = 0.299) (Table 5). 246

20

Crambe meal inclusion did not affect Plasma urea-N (P = 0.258), milk urea-N (P = 0.178), 247

urinary N excretion (P = 0.717), N balance (P = 0.598) and N-efficiency to lactation (P = 0.10) 248

(Table 6). 249

4 - Discussion 250

The average stocking rate was 12.24 AU/ha and the total available forage mass was 251

8380.66 ± 669.50 kg DM/ha, composed of 2421.54 ± 595.24 kg of foliar blade/kg of DM, 4904.22 252

± 1011.82 kg of stalk/kg of MS (Figure 1). 253

Low-glucosinolates CM inclusion in pTMR (up to 135 g/kg DM) does not affect diet 254

intake, diet total-tract digestibility, ruminal microbial protein synthesis, hepatic function, milk 255

yield and composition, N dietary efficiency of crossbreed Holstein × Zebu cows, and cheese 256

preference for untrained consumers. Therefore, our hypothesis that CM is similar protein source 257

to SBM for lactating dairy cows was confirmed. 258

The absence of effects on intake and digestibility probably occurred due to low 259

glucosinolates content of CM used (450 mg/kg DM), and confirm our previous studies (Moura et 260

al., 2017). Brassicas meal are classified with low glucosinolates contents when they present values 261

between 2800 and 13000 mg/kg DM (Tripathi and Mishara, 2007, adapted). However, the 262

reduction in NFC intake can be explained by the increase in the inclusion of crambe meal and 263

reduction of corn and soybean meal in the diet, which resulted in lower starch content in diets with 264

a higher inclusion level of crambe meal. The causes of increase in EE digestibility with CM is 265

unclear, but as CM had high content of erucic acid (22:1 ω-9), its may have improve fatty acid 266

intestinal digestibility in relation SBM (rich in unsaturated C:18). 267

The absence of effects on ruminal microbial protein synthesis was due no CM effect no 268

intake and digestibility, that did not affect N and energy availability for ruminal microbial growth. 269

Therefore, absence of CM effects on milk production and milk composition is a response to intake, 270

21

digestibility and ruminal microbial protein synthesis, because energy and metabolizable protein 271

are main nutritional drivers of lactation (NRC, 2001). Absence of effects on the milk composition 272

and milk N- efficiency can be explained by the composition of the diets (isoproteic and energetic), 273

as well as the similarity of the amino acid profile of crambe meal, to soybean meal (Anderson et 274

al., 1993). 275

With rupture and contact of the enzyme myrosinase with glucosinolates, a hydrolysis 276

occurs through the cleavage of the glucose in its connection with the sulfur atom, releasing the 277

isothiocyanates, thiocyanates and nitriles, potentially causing liver damage and other organs 278

(Halkier and Gershenzan, 2006). The major enzymes for liver function indicators are gamma 279

glutamyltranspeptidase (GGT), alanine aminotransaminase (ALT), aspartate aminotransaminase 280

(AST). Although the observed values of ALT and AST were not influenced by diets, they were 281

outside the recommended reference range for species 22 to 28 IU/L for ALT, 60 to 280 IU/L for 282

AST (Radostites et al., 2002). These results were observed for all inclusion levels even for a diet 283

without inclusion of CM. In addition to levels, we must consider factors such as an animal species, 284

race, age, sex, breeding systems can interfere with levels, hepatic enzymes, without being 285

associated with pathological factors (Gregory, 1995). However, the GGT activity the mean values 286

of all treatments remained within the physiological standard (25-50 IU/L) reference values 287

observed by Gregory et al. (1999). 288

The effects found for composition and yield of cheeses can have occurred due to the 289

absence of effects on the milk composition of cows fed with CM inclusion. The absence of effects 290

on cheese preference indicate that CM with low glucosinolates level does not have chemical 291

composts that affect cheese sensorial characteristic in relation to typical diets with SBM. 292

293

22

5 - Conclusions 294

Crambe meal with low glucosinolates levels can be included in pTMR (up 135 g/kg DM) 295

without affects productive performance, efficiency of nutrient utilization in Jersey and Holstein × 296

Zebu crossbreed dairy cows with 132 kg of milk and cheese preference for untrained consumers. 297

298

Acknowledgments 299

Family Peron (Dairy Farm Dona Herminia) to provide the structure, Fundação de Amparo à 300

Pesquisa do Estado de Mato Grosso for financial support (FAPEMAT, project number 301

483724/2011 PRONEM 006/2011; Chair: André Soares de Oliveira), Caramuru Alimentos S.A 302

(Itumbiara, Goiás, Brazil) for providing crambe meal, Conselho Nacional de Desenvolvimento 303

Científico e Tecnológico (CNPq, Brazil) and Coordenação de Aperfeçoamento de Pessoal de Nível 304

Superior (CAPES; Brazil) for scholarships provided author D. C. Moura. 305

REFERENCES 306

Anderson, V. L., W. D. B. Slanger, and P. T. Berg. 1993. Crambe meal is equivalent to soybean 307

meal for backgrounding and finishing beef steers. J. Animal Sci. 71:2608-2613. 308

AOAC International. 1990. Official Methods of Analysis, 15th ed. AOAC International, Arlington. 309

AOAC International. 1995. Official methods of analysis of the Association of Official Analitycal 310

Chemists (method 991.20). Arlington. 311

Chen, X. B., and M. J Gomes. 1992. Estimation of Microbial Protein Supply to Sheep and Cattle 312

Based on Urinary Excretion of Purine Derivatives—An Overview of Technical Details. 313

International Feed Research Unit. Rowett Research Institute, Aberdeen, UK (occasional 314

publication). 21. 315

23

Chizzotti, M. L., S. C., Valadares Filho, R. F. D., Valadares, F. H. M. Chizzotti, and L.O. Tedeschi, 316

2008. Determination of creatinine excretion and evaluation of spot urine sampling in Holstein 317

cattle. Liv. Sci. 113: 218-225. 318

Casali, A. O., E. Detmann., S. C., S. C. Valares Filho, J. C. Pereira, L. T. Henriques, S. G. Freitas, 319

M. F. Paulino. 2008. Influência do tempo de incubação e do tamanho de partículas sobre os 320

teores de compostos indigestíveis em alimentos e fezes bovinas obtidos por procedimentos in 321

situ. R. Bras. Zootec. 37:335-342. 322

Ferguson, J. D., D.T Galligan, and N. Thomsen, 1994. Principal descriptors of body condition 323

score in holstein Cows. J. Dairy Sci. 77: 2695-2703. 324

Gregory, L.Valores padrões de referência de parâmetros bioquímicos séricos utilizados na 325

avaliação das funções hepática e renal de bovinos, da raça Jersey, criados no Estado de São 326

Paulo. Influência de fatores etários, sexuais e da infecção pelo Vírus da Leucose dos Bovinos. 327

São Paulo: Faculdade de Medicina Veterinária e Zootecnia da USP, 1995. 161p. (Dissertação, 328

Mestrado). 329

Gregory, L. E. H. Birgel Junior, R. M.S Mirandola, W. P. Araujo, and E. H. Birgel. 1999. Valores 330

de referência da atividade enzimática da aspartato-aminotransferase e da gama-331

glutamiltransferase em bovinos da raça Jersey. Influência dos fatores etários, sexuais e da 332

infecção pelo vírus da leucose dos bovinos. Arq. Bras. Med. Vet. Zootec. 51:6. 333

Halkeer, B. A., J. Gershenzon. 2006. Biology and biochemistry of glucosinalates. Annual Review 334

of plant Biology. 57:303-333. 335

Hall, M. B. 2000 Neutral detergent-soluble carbohydrates. Nutritional relevance and analysis. 336

Gainesville: University of Florida, 76p. 337

Lambert, J. L., D. C. Clanton, A. Woff, and G. C. Mustakas. Crambe meal protein and hulls in 338

beef cattle rations. J. Anim. Sci, v.31, n.3, p.601-607, 1970. 339

340

24

Lee, K. C., Man, W. C., Wan, C., Albert, W. M. L., Zhong, Z. Z., Zhi, H. J., Zongwei, C. 2006. 341

Determination of glucosinolates in traditional Chinese herbs by high-performance liquid 342

chromatography and electrospray ionization mass spectrometry. Anal. Bioanal. Chem. 343

386:2225–2232. 344

Liu, Y.G., A. Steg, V. A. Hinde, 1993. Crambe meal: a review of nutrition, toxicity and effect of 345

treatments. Anim. Feed Sci. Technol, 41:133-147. 346

Licitra, G., T. M. Hernandez, and P. J Van Soest. 1996. Standardization of procedures for nitrogen 347

fractionation of ruminant feeds. Anim. Feed Sci. Technol. 57: 347-358. 348

Littell, R. D., P. R. Henry, and C. B. Ammerman. 1997. Statistical analysis of repeated measures 349

data using SAS procedures. J. Anim. Sci.,76:1216-1231. 350

Mertens, D.R. 2002. Gravimetric determination of amylase-treated neutral detergent fiber in feeds 351

with refluxing in beaker or crucibles: collaborative study. J. AOAC Int. 85: 1217–1240. 352

Mendonça, B. P. C., R. P. Lana, E. Detmann, R. H. T. B. Goes and T. R. Castro. 2015a. Uso do 353

farelo e da torta de crambe na alimentação de bezerros em crescimento. Arq. Bras. Med. Vet. 354

Zootec. 67: 583-590. 355

Mendonça, B. P. C., R. P Lana, E. Detmann, R. H. T. B. Goes, and T. R. Castro. 2015b. Torta de 356

crambe na terminação de bovinos de corte em confinamento. Arq Bras Med Vet Zoo, 67: 583-357

590. 358

Mizubuti, I, Y., E. L. A. Ribeiro. E. S. Pereira, A. P. Pinto, A. L. C. Franco, M. A. Syoerreck, J. 359

R. R. Dórea, G. E. Cunha, M. G. M. Capelari, E. B. Muniz. 2011. Cinética de fermentação 360

ruminal in vitro de alguns co-produtos gerados na cadeia produtiva do biodiesel pela técnica 361

de produção de gás. Semina: Ciênc. Agrár. 32: 2021-2028, 2011. 362

Moura, D. C., T. S. Fonseca, S. R. Soares, H. M. Silva, G.V. J. Vieira, L. A. Botini, A. P. Sinhorin, 363

I. M. Ogunade, A. S. Oliveira. 2017. Crambe meal subjected to chemical and physical 364

treatments in sheep feeding. Livest Sci, 203:136-140. 365

25

Myers, W. D., P. A. Ludden, V. Nayigihugu, and B. W. Hess. 2004. Technical Note: A procedure 366

for the preparation and quantitative analysis of samples for titanium dioxide. J. Anim. Sci., 82, 367

179-183. 368

National Research Council. 2001. Nutrient Requirements of Dairy Cattle, 7th rev. ed. Natl. Acad 369

Press, Washington, DC. 370

Nocek, J. E., and J. B.Russel. 1988. Protein and energy as an integrated system, relationship of 371

ruminal protein and carbohydrate availability to microbial synthesis and milk production. J. 372

Dairy Sci. 71:2070-2107. 373

Oliveira A. S. 2014. Endogenous fraction of purine derivatives in dairy cows: meta-analysis. Proc. 374

Aust. Soc. Anim. Prod. 30:181. 375

Orskov, E., I. Mcdonald, 1979. The estimation of protein degradability in the rumen from 376

incubation measurements weighted according to rate of passage. J. Agri. Sci. 92:.499-503. 377

Radostitis, O. M., C. C. Gay, D. C. Blood,. and K. W. Hinchcliff. 2002. Clinica Veterinaria: Um 378

tratado de doencas dos bovinos, ovinos, suinos, caprinos e equinos. 9 ed. Rio de Janeiro: Ed. 379

Koogan. Page 1737. 380

Souza, A. D. V., S. P Favaro, L. C. V. Itavo, and R. Roscoe. 2009. Caracterização química de 381

sementes e tortas de pinhão-manso, nabo-forrageiro e crambe. Pesq. agropec. bras. 44:1328-382

1335. 383

Schumann, G., R. Bonora, F. Ceriotti, G. Ferard, C. A. Ferrero, P. F. Franck, F. J. Gella, W. 384

Hoelzel, P. J. Jorgensen, T. Kanno, A. Kessner, R. Klauke, N. Kristiansen, J. M. Lessinger, 385

T.P. Linsinger, H. Misaki, M. Panteghini, J. Pauwels, F. Schiele, H. G. Schimmel, G. 386

Weidemann, and L. Siekmann, 2002a. IFCC primary reference procedures for the 387

measurement of catalytic activity concentrations of enzymes at 37 ◦C. International Federation 388

of Clinical Chemistry and Laboratory Medicine. Part 4. Reference procedure for the 389

26

measurement of catalytic concentration of alanine aminotransferase. Clin. Chem. Lab. Med. 390

40:718–724. 391

Schumann, G., R. Bonora, F. Ceriotti, G. Ferard, C. A. Ferrero, P. F. Franck, F. J Gella, W. 392

Hoelzel, P. J. Jorgensen, T. Kanno, A. Kessner, R. Klauke, N. Kristiansen, J. M. Lessinger, 393

T.P. Linsinger, H. Misaki, M. Panteghini, J. Pauwels, F. Schiele, Schimmel, H. G. Weidemann, 394

and L. Siekmann, 2002b. IFCC primary reference procedures for the measurement of catalytic 395

activity concentrations of enzymes at 37 ◦C. International Federation of Clinical Chemistry and 396

Laboratory Medicine. Part 5. Reference procedure for the measurement of catalytic 397

concentration of aspartate aminotransferase. Clin. Chem. Lab. Med. 40:725–733. 398

Titgemeyer, E.C., C. K Armendariz, D. L. Bindel, R. H. Greenwood, and C. A Loest. 2001. 399

Evaluation of titanium dioxide as a digestibility marker for cattle. J. Anim. Sci., 79:1059-1063. 400

Tripathi, M. K., A. S. Mishra. 2007.Glucosinolates in animal nutrition: A review. Anim. Feed Sci. 401

Technol. 132:1–27. 402

Verbic, J., X. B. Chen, N. A. McLeod, and E. R. Orskov. 1990. Excretion of purine derivatives by 403

ruminants. Effect of microbial nucleic acid infusion on purine derivative excretion by steers. 404

J. Agric. Sci. Camb. 114:243–248. 405

Williams EJ (1949). Experimental designs balanced for the estimation of residual effects of 406

treatments. Proc Natl Acad Sci U S A. 2:149. 407

Young, G. and C. F. Conway. 1942. On the estimation of allantoin by the rimini-schryver reaction. 408

142:839-853. 409

410

411

412

413

414

27

415

416

Figure 1 - Available pasture mass of Panicum maximum cv grass Mombaça in the experimental 417

periods 418

419

420

421

422

423

424

425

426

427

428

429

430

431

432

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

1st period 2nd period 3rd period 4th period

Pas

ture

Mas

s (K

g D

M/a

cre)

Stem

Foliar blade

28

433

434

435

436

Table 1. Chemical composition of Panicum maximum cv. Mombaça obtained by simulated grazing 437

and ingredients used in the partial total mixed ration supplements 438

Itensa Feedsa

Pasture CS CG SM CM

Dry matter (DM), g/kg 403.3 287.0 897.5 915.2 915.7

Organic matter, g/kg DM 924.1 956.8 984.1 930.9 928.0

Ether extract, g/kg DM 14.1 15.4 26.7 18.1 30.1

Crude protein, g/kg DM 90.4 66.1 92.2 510.0 381.8

NDIPb, g/kg N 276.2 212.9 75.6 30.5 159.6

ADIPc, g/kg N 54.5 80.2 19.2 26.2 30.1

aNDFomd , g/kg DM 563.5 532.4 96.1 114.6 333.5

Non-fibrous carbohydrates, g/kg DM 256.0 344.3 769.1 288.3 182.7

Lignin, g/kg DM 38.6 35.6 7.5 4.6 101.5

NDF indigestiblee, g/kg DM 128.2 153.0 17.2 7.7 262.6

Glucosinolatesf, mg/kg DM - - 450.0

aPasture; CS, corn silage; CM, corn grain ground; SM, soybean meal; CM, and crambe meal. 439 bNDIP, neutral detergent insoluble protein. 440 cADIP, acid detergent insoluble protein. 441 daNDFom, neutral detergent insoluble fiber corrected for ash and N. 442 eObtained after 264 hours in situ rumen incubation. 443 f sirigin equivalents 444 445

446

447

448

449

450

451

452

29

453

454

455

456

Table 2. Ingredients and chemical composition of the partial total mixed ration (pTMR) with 457

different inclusions of crambe meal. 458

Ingredients CM in pTMR (g/kg DM)

0 45 90 135

Ingredient composition, g/kg DM

Corn silage 700 700 700 700

Corn grain ground 183.0 166.5 150.0 133.5

Soybean meal 102.0 73.5 45.0 16.5

Crambe meal (CM) 0 45.0 90.0 135.0

Urea/ammonium sulfate 9:1 4.5 4.5 4.5 4.5

Mineral mixturea 10.5 10.5 10.5 10.5

Chemical composition

Dry matter (DM), g/kg as fed 459.0 459.0 459.0 459.0

Organic matter, g/kg DM 945.0 944.0 943.0 942.0

Ether extract, g/kg DM 17.0 18.0 18.0 19.0

Crude protein, g/kg DM 127 128 129 131

NDIPb, g/kg CP 100 116 132.0 148.0

ADIPc, g/kg CP 42.0 43.0 44.0 44.0

aNDFomd, g/kg DM 402.0 412.0 422.0 432.0

Non-fibrous carbohydrates, g/kg DM 406.0 393.0 380.0 368.0

NDF indigestiblee, g/kg DM 111.1 120.0 129.0 138.0

Lignin, g/kg DM 27.0 31.0 35.0 40.0

Rumen degradable protein, g/kg DM 118.0 121.0 123.0 126.0

Glucosinolatesf, mg/kg DM nd 20.25 40.50 60.75`

aCommercial mixture mineral (guarantee levels per kg of product: 160g of calcium; 80g of 459

phosphorus; 12g of sulfur; 114 g of sodium; 15g of magnesium; 100mg of cobalt; 700mg of 460

copper; 100 mg of iodine; 15g of manganese; 30 mg of selenium; 2500mg of zinc; 3000mg (max) 461

fluorine). 462 bNDIP, neutral detergent insoluble protein CP. 463

30

cADIP, acid detergent insoluble protein CP. 464 daNDFom, neutral detergent insoluble fiber corrected for ash and N. 465 eObtained after 288 hours in situ rumen incubation. 466 f equivalent sirigin 467

Table 3. Effect crambe meal (CM) inclusion in partial total mixed ration (pTMR) on intake, total-468

tract digestibility and ruminal microbial protein synthesis (MPS) of grazing dairy cows 469

Item CM in pTMR (g/kg DM)

SEDb P-valuec

0 45 90 135 L Q

Intake, kg/day

pTMR dry matter (DM) 11.68 11.60 11.30 11.31 0.679 0.173 0.848

Pasture 0.835 0.856 0.914 0.992 0.272 0.185 0.726

Total DM 12.51 12.46 12.22 12.33 0.8062 0.481 0.729

Organic matter 11.91 11.85 11.61 11.71 0.765 0.420 0.734

Ether extract 0.203 0.210 0.216 0.212 0.012 0.216 0.325

Crude protein 1.48 1.49 1.50 1.50 0.056 0.481 0.627

aNDFoma 5.35 5.41 5.42 5.71 0.414 0.078 0.353

Non-fibrous carbohydrates 4.83 4.69 4.43 4.29 0.278 0.002 0.951

Total digestible nutrient 9.14 9.01 8.80 9.34 0.617 0.754 0.262

pTMR intake:total DM, g/g 0.933 0.934 0.927 0.920 0.020 0.1349 0.547

Digestibility, g/g

Dry matter 0.715 0.717 0.715 0.746 0.016 0.229 0.389

Organic matter 0.740 0.745 0.746 0.767 0.015 0.254 0.609

Ether extract 0.756 0.733 0.782 0.818 0.023 0.052 0.224

Crude protein 0.735 0.740 0.750 0.773 0.017 0.094 0.545

aNDFoma 0.726 0.720 0.726 0.744 0.018 0.451 0.483

Non-fibrous carbohydrates (NFC) 0.756 0.766 0.760 0.789 0.017 0.255 0.578

Total digestible nutrient, g/g DM 0.782 0.768 0.780 0.815 0.020 0.120 0.121

MPS, kg / dia 0.880 0.771 0.735 0.922 0.217 0.887 0.338

g MPS / kg TDN intake 99.82 90.22 90.14 99.13 20.76 0.978 0.621

Plasma, IU/mL

Gamma glutamyl transpeptidase 32.47 30.52 35.13 31.18 5.967 0.930 0.592

Alanine aminotransaminase 16.40 15.52 16.40 15.34 2.125 0.654 0.937

31

Aspartate aminotransaminase 50.33 46.58 47.41 44.64 9.458 0.432 0.913 aaNDFom, neutral detergent insoluble fibre corrected for ash and N. 470 bSED = standard error of the least squares means. 471 cProbability of linear (L) or quadratic (Q) effect of CM level in pTMR. 472 473 Table 5. Effect of the crambe meal (CM) inclusion in partial total mixed ration (pTMR) on 474

productive performance of grazing dairy cows 475

Item

CM in pTMR

(g/kg DM) SEDa P-valueb

0 45 90 135 L Q

Body weigth, kg 483.52 466.52 475.91 471.40 49.64 0.399 0.379

Body condition score (1 a 5) 2.78 2.77 2.76 2.80 0.253 0.833 0.693

Milk yield, kg/d 13.16 13.63 13.10 13.25 0.849 0.793 0.462

4.0% fat correted milk, kg/d 14.25 14.65 13.70 14.24 0.887 0.563 0.852

Milk lactose, g/kg 48.16 47.78 47.44 47.49 0.637 0.139 0.516

Milk crude protein, g/kg 30.97 30.86 30.77 30.90 0.410 0.778 0.626

Milk fat, g/kg 45.81 45.13 43.67 45.07 3.096 0.302 0.191

Milk lactose, kg/d 0.634 0.651 0.617 0.628 0.038 0.874 0.113

Milk crude protein, kg/d 0.407 0.420 0.402 0.406 0.024 0.550 0.573

Milk fat, kg/d 0.600 0.614 0.565 0.594 0.040 0.531 0.770

Feed efficiency, kg/kg 1.15 1.18 1.14 1.16 0.078 0.853 0.800

Yield and sensory analysis the cheese

Dry matter (DM), g/kg 35.96 36.34 36.92 35.94 1.668 0.903 0.484

Crude protein kg/d 5.70 5.56 5.74 5.72 0.733 0.874 0.847

Yield kg / kg the milk 0.227 0.222 0.216 0.208 0.021 0.314 0.928

Like, % consumers 59.78 80.00 76.73 77.68 9.113 0.239 0.299

Disliked, % consumers 40.22 20.00 23.27 22.32 9.113 0.239 0.299 aSED = standard error of the least squares means. 476 bProbability of linear (L) or quadratic (Q) effect of CM level in pTMR. 477

478

479

480

481

482

32

483

484

485

Table 6. Effect of the crambe meal (CM) inclusion in partial total mixed ration (pTMR) on nitrogen 486

(N) efficiency of grazing dairy cows 487

Item

CM in Ptmr

(g/kg DM) SEDa P-valueb

0 45 90 135 L Q

Plasm urea-N, mg/dL 10.98 10.80 11.68 10.75 0.370 0.889 0.258

Milk urea-N, mg/dL 11.86 10.18 10.86 9.70 2.103 0.178 0.767

Urinary urea-N, g/d 20.08 24.68 25.50 28.58 11.15 0.587 0.943

Urinary urea-N:total N, g/g 0.519 0.466 0.474 0.351 0.082 0.169 0.646

Intake N, g/d 236.60 239.19 240.64 240.01 8.952 0.481 0.627

Feces N, g/d 62.30 62.50 60.13 54.37 5.083 0.195 0.481

Urinary N, g/d 91.70 89.27 89.40 93.30 11.53 0.899 0.717

Milk N, g/d 63.94 65.95 62.91 63.91 3.914 0.566 0.673

Balance N, g/d 20.09 24.68 25.50 28.58 11.16 0.587 0.943

N efficiency, g/g intake N

Feces N 0.264 0.259 0.254 0.222 0.018 0.100 0.405

Urine N 0.383 0.366 0.368 0.389 0.041 0.914 0.622

Milk N 0.271 0.274 0.261 0.264 0.013 0.100 0.920

Balance N 0.084 0.106 0.112 0.117 0.046 0.598 0.856

aSED = standard error of the least squares means. 488 bProbability of linear (L) or quadratic (Q) effect of CM level in pTMR. 489

490

491

492

493

494

495 496

497 498 499

33

Meta-analysis of the use of brassicas meals in diets for dairy cows 500

501

ABSTRACT: We used meta-analytical approach to evaluate the effects of replacement of 502

different sources of protein by brassicas meals on milk production and nutrient utilization of 503

dairy cows, from 37 peer-review papers. Canola meal (CM) was unique brassica source 504

founded. The effects were compared by raw mean differences (RMD) between CM diet and 505

control treatment means and weighted by inverse variance using random-effect models. Control 506

protein sources (28.6 ± 6.9 kg milk/d) were soybean meal (SBM; 57%), dried destillers grains 507

(DDG; 21%), corn gluten meal (7%), cottonseed meal (5%), sunflower meal (2%) and other 508

protein sources (9%). Heterogeneity level was analyzed by I2 statistic (low ≤ 25%; moderate = 509

26 to 50%; and high > 50%). In overall, use of CM as protein source increased DM intake 510

(RMD = 0,22 ± 0.12 kg DM/d; P < 0.01; n = 79; I2 = 9.1%) and crude protein (CP) intake 511

(RMD = 0,14 ± 0.07 kg CP/d; P < 0.01; n = 33; I2 = 21.1%), both with low heterogeneity, but 512

it did not affect organic matter total-tract digestibility (P = 0.50; n = 12; I2 = 29.2%). In overall, 513

use of CM increased milk yield (RMD = 0.69 ± 0.35 kg/d; P < 0.01; n = 88; I2 = 74.9%), but 514

its effect depends on protein sources comparation: CM versus SBM did not increase milk yield 515

(RMD = 0.23 ± 0.66 kg/d; P = 0.50; n = 33), but milk yield was increased with replacement of 516

DDG by CM (RMD = 2.03 ± 1.67 kg/d; P < 0.01; n = 13) and of other protein sources by CM 517

(RMD = 0.82 ± 0.43 kg/d; P < 0.01; n = 42). In overall, CM use did not affect milk protein 518

content (P = 0.08; n = 60; I2 = 19.5%) and milk fat content (P = 0.20; n = 60; I2 = 16.9%), but 519

CM increased milk protein yield (RMD = 0,02 ± 0.01 kg/d; P < 0.01; n = 60; I2 = 0%). Use of 520

CM reduced milk urea nitrogen (N) (RMD = - 0,98 ± 0.31 mg/dL; P < 0.01; n = 22; I2 = 32.2%) 521

and increase N intake milk efficiency (RMD = 0.22% N milk/N intake ± 0.07 mg/dL; P ≤ 0.05; 522

n = 34; I2 = 0%), both with low heterogeneity. We concluded that CM is similar protein source 523

34

to SBM and it is more effective than DDG and other sources (cottonseed meal, corn gluten meal 524

and sunflower meal) to lactating dairy cows. 525

Key words: canola meal, effect sizes, heterogeneity 526

INTRODUCTION 527

528 Brassicaceae is composed of 51 genera and 218 species, which some are important 529

species of crops cultivated as vegetables, forages and oilseeds (Sabharwal et al., 2006). After 530

oilseed extraction are produced products with potential of use in the animal as protein source. 531

Colza, canola, crambe and carinata are main brassicas meals used in animal nutrition, but its 532

use depends of glucosinolates levels (Tripathi and Mishra, 2007; Moura et al., 2017). 533

Glucosinolates are goitrogenic compounds that reduce the availability of iodine to the 534

animal and diminish the synthesis of thyroxine (Grenet and Journet, 1971). A lower intake and 535

animal performance were also reported in diets containing brassica meal with high 536

glucosinolates levels (Papas et al., 1978; Thomke, 1981; Tripathi et al., 2001). Therefore, only 537

lower glucosinolates brassicas meals (< 30 μmol/g oil-free meal) are considered a suitable 538

feedstuff for livestock (Tripathi and Mishra, 2007). 539

Canola is a term specifically to identify colza varieties containing less than 2% erucic 540

acid in the oil portion and lower glucosinolates level on meals (Bell, 1984; Newkirk, 2009). 541

Canadian plant breeders were the first to develop canola cultivars in 1970’s year 542

(Stefansson and Kondra, 1975). Today, canola meal is the second oilseed meal consumed in the 543

world (12% market share), below only of the soybean meal (71% market share) 544

(SoyStats, 2018). 545

Have been reported that canola meal (CM) was similar to soybean meal (SBM; Brito 546

and Broderick, 2007; Huhtanen et al., 2011; Matineau et al., 2013), but more effective protein 547

source to lactating dairy cows that cottonseed meal (Brito and Broderick, 2007) and dry 548

35

distillated grain (DDG; Swanepoel et al., 2014), due differences on protein intestinal 549

digestibility and first-limiting AA profile to milk production (i.e. Lys, Met and His). In 550

previous meta-analysis were confirmed that replacement of SBM by CM did not affect milk 551

yield (Huhtanen et al., 2011; Matineau et al., 2013), but replacement of other sources 552

(not specified) improved milk production (Matineau et al., 2013). However, is not clear yet 553

which protein source the CM may be more effective to milk production. 554

We hypothesis is that CM use in replacement to SBM does not affect dairy cows 555

performance, but its use in replacement to DDG, cottonseed meal and other oilseeds meal 556

reduces milk production of dairy cows. Therefore, we used a meta-analytical approach to 557

evaluate the effects of replacing of different sources of protein by brassicas meals on milk 558

production and nutrient utilization by dairy cows. 559

560

MATERIALS AND METHODS 561

Literature Search 562

Papers were identified by searching for peer-reviewed manuscripts that were published 563

in English using online manuscript retrieval databases [Web of Science 564

(https://login.webofknowledge.com), PubMed (https://www.ncbi.nlm.nih.gov/pubmed), 565

Google Scholar (http://www.scholar.google.com/), ScienceDirect 566

(http://www.sciencedirect.com/)]. Up to 274 publications were retrieved using search terms 567

including “brassica,” “brassica aleracea,” “brassica rapa” “canola meal,” “rapessed meal,” 568

“ crambe meal,” “canerita meal,” “dairy cows. Of the papers that were retrieved, only those that 569

satisfied the predetermined inclusion criteria were included in the analysis. 570

Inclusion Criteria 571

A flowchart explaining the process of study identification and selection for analyzing 572

the effects of replacement of different protein sources by brassica meal is shown in Figure 1. 573

36

The study selection criteria were (1) publication in English in a peer-reviewed journal, (2) use 574

of concurrent negative control and treatment groups, (3) use of lactating dairy cows, (4) use of 575

at least one Brassica meal as a dietary treatment, (5) presentation of least squares means and 576

standard errors of the means for DMI our milk yield. The study exclusion criteria were: (1) did 577

not involve feeding a TMR, (2) involved a study that was not randomized. We only included 578

peer-reviewed publications in the study because the peer review process is a proxy for assessing 579

the quality of studies (Weisz et al., 1995). 580

Data Extraction 581

Based on the inclusion criteria, 37 articles were reviewed by selected pairs and ranked 582

by the first author, publication reference, genetic type, forage in diet (% DM), brassica meal 583

type, glucosinolates mg/kg DM, replaced culture, days in milk (begin experiment), number 584

cows, experimental design, type of diet (pasture, TMR, partial TMR) , days experiment, diet 585

composition, milk yield of cows, number of replicates, significance, and variances (SE, SD) 586

were extracted for the following response variables for control and brassicas treatments: 587

nutrient intake, diet total-tract digestibility, feed efficiency, milk yield, milk composition and 588

nitrogen metabolism. 589

Statistical Analysis 590

Meta-analysis was conducted using the metafor package of R Software 591

(Viechtbauer, 2010) version 3.2.3. The effects of different sources of protein by brassica meal 592

were evaluated by examining the raw mean differences (RMD) between brassica meal and 593

inoculated treatment means (effect size), which were weighted by the inverse of the variance in 594

the respective studies using the method proposed by DerSimonian and Laird (1986) for a 595

random effect model. When were not reported sufficient information to use variance 596

(i.e SEM or SD), we weighted the observations by replication number per treatment. 597

37

Between-study variability (i.e., heterogeneity of effect size) was evaluated using the chi-598

squared (Q) test and the I2 statistic, which measures the percentage of variation due to 599

heterogeneity (Higgins et al., 2003). An I2 value less than 25% indicated low heterogeneity, 600

whereas values between 25 to 50% denoted moderate heterogeneity and those above 50 high 601

heterogeneity (Higgins et al., 2003). Publication bias was assessed using funnel plots 602

(Light and Pillemer, 1984) and was tested for funnel plot asymmetry 603

(indicative of publication bias) by Egger’s regression method between RMD and SE (Egger et 604

al., 1997). We adopted a level of P = 0.05 of probability for significance and P = 0.10 of 605

probability for trend. 606

607

RESULTS AND DISCUSSION 608

We founded only CM as brassica source used in diets to lactating dairy cows. Therefore, 609

we evaluated only the effects of CM on milk production and nutrient utilization of dairy cows. 610

Raw mean differences to feed efficiency and N-milk efficiency were weight by replication 611

number per treatment and the others response variables by inverse of the variance. Protein 612

source of the control treatments were soybean meal (57%), dried destillers grains (21%), corn 613

gluten meal (7%), cottonseed meal (5%), sunflower meal (2%) and other protein sources (9%). 614

Experimental design use in the studies were Latin square (53.3%), randomized block (27.8%), 615

changeover (14.4%) and not reported experimental design (4.4%). Total mixed ration was uses 616

in 96.2% of studies. 617

Overall, the replacement of several protein sources for CM did not, organic matter (OM) 618

total-tract digestibility (P = 0.50), ruminal pH (P = 0.50) and propionate concentration 619

(P = 0.36) (Table 1). However, CM use in comparation with all protein sources evaluated 620

reduced CP total-tract digestibility (P < 0.01) and it increased (P < 0.01) DM, OM, CP and 621

NDF intakes, and ruminal acetate concentration (P = 0.03) (Table 1). Overall, the replacement 622

38

of several protein sources for CM increased milk yield (P < 0.01), milk protein yield (P < 0.01), 623

tended to increase protein milk content (P = 0.08), but it did not affect milk fat content 624

(P = 0.20) and yield (P = 0.24), and feed efficiency (P > 0.05). As result of the increase in milk 625

yield and N dietary efficiency, CM use in comparation with all protein sources evaluated 626

reduced milk urea-N (P < 0.01) and increased milk N efficiency (P < 0.05) (Table 1). 627

Low and medium heterogeneity (I2 statistic < 25 and 26-50%) were observed for all 628

response variables, except for milk yield (I2 = 74.5%) (Table 1). There was no evidence of 629

funnel plot asymmetry (P > 0.10) for any response (Figure 2), indicating that publication bias 630

was not evident. We identified that protein source used to compare with CM was a main 631

heterogeneity source to milk yield response. Replacement of SBM by CM did not increase milk 632

yield (RMD = 0.23 ± 0.66 kg/d; P = 0.50; n = 33; Figure 3), but milk yield was increased with 633

replacement of DDG by CM (RMD = 2.03 ± 1.67 kg/d; P < 0.01; n = 13; Figure 4) and of other 634

protein sources (cottonseed meal, corn gluten meal and sunflower meal) by CM 635

(RMD = 0.82 ± 0.43 kg/d; P < 0.01; n = 42; Figure 5). 636

We confirmed our hypothesis that CM use in replacement to SBM does not affect dairy 637

cows performance, but its use in replacement to DDG, cottonseed meal and other oilseeds meal 638

reduces milk production of dairy cows. Similar milk production response of CM with SBM 639

may be attributed to similar ruminal protein degradation, intestinal digestibility and/or better 640

first limiting essential amino acids (EAA) profile to lactating (i.e. Lys, Met and His) (NRC, 641

2001). There is evidence that DGG in diet reduced intestinal digestibility and N retention in 642

ruminant, due higher ADIN (acid detergent insoluble nitrogen) and lower Lys than SBM 643

(Mjoun et al., 2010; Stotzer, 2017). As CM has similar protein value that DDG, its suggest that 644

CM has higher intestinal digestibility or Lys content than DDG. Similarly, higher milk response 645

of the CM versus other sources (cottonseed meal, corn gluten meal and sunflower meal) may 646

39

be attributed high ruminal protein degradation, intestinal digestibility and/or better first limiting 647

EAA profile to lactation (i.e. Lys, Met and His). 648

Although we identified reduction on CP total-tract digestibility with CM versus all 649

sources, the reduction on MUN and increase on N-milk efficiency suggest that CM increase 650

anabolic use of AA to lactating and reduce AA losses by catabolism process. In the meta-651

analysis of Huhtanen et al. (2011) and Martineau et al. (2013) also reported positive responses 652

to DMI when CM replaced other dietary proteins (mainly SBM). The increase in DMI can be 653

attributed to the increase nutrient demand, where the more balanced supply of AA increases 654

lactation performance and, consequently, increases energy demand, which stimulates feed 655

intake (NRC, 2001). The increase in rumen acetate concentration when CM replaced other 656

protein sources can be due increase of NDF intake. 657

CONCLUSIONS 658

Canola meal is a similar protein source compared to SBM to lactating dairy cows. 659

However, CM is more effective than DDG and other sources (cottonseed meal, corn gluten 660

meal and sunflower meal) to lactating dairy cows because it increases milk yield and N 661

efficiency to lactation. Possibly this increase in milk production is more associated with an 662

improvement in the balance between AA and energy, but is necessary to elucidate the causes 663

of higher N efficiency to lactating of the CM to compare DDG and other protein sources. 664

665

ACKNOWLEDGMENTS 666

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) and 667

Coordenação de Aperfeçoamento de Pessoal de Nível Superior (CAPES; Brazil) for 668

scholarships provided author D. C. Moura. Núcleo de Pesquisa em Pecuária de Leite 669

40

responsibility A. S. Oliveira (Universidade Federal de Mato Grosso, Campus Universitário de 670

Sinop) 671

REFERENCES 672

Bell, J. M. 1984. Nutrients and toxicants in rapeseed meal: A review. J. Anim. Sci. 58:996–673 1010. 674

675 Brito, A. F., G. A. Broderick, and S. M. Reynal. 2007. Effects of different protein supplements 676

on omasal nutrient flow and microbial protein synthesis in lactating dairy cows. J. Dairy 677 Sci. 90:1828–1841. 678

679 DerSimonian, R., and N. Laird. 1986. Meta-analysis in clinical trials. Control. Clin. Trials 680

7:177–188. 681 682 Egger, M., G.D. Smith, M. Schneider, and C. Minder. 1997. Bias in meta-analysis detected by 683

a simple, graphical test. BMJ 315:629– 634. 684 685 Grenet, N., and M. Journet. 1971. Rapeseed oil meal in animal feeding. III. Influence of the 686

processing method and the proportion of oil meal in concentrate fed to dairy cows on feed 687 intake, milk production and composition. Ann. Zootech. 20:437–449. 688

689 Higgins, J. P. T., S. G. Thompsom, J. J. Deeks, and D. G. Altman. 2003. Measuring 690

inconsistency in meta-analysis. BMJ 327:557–560. 691 692 Huhtanen, P., M. Hetta, and C. Swensson. 2011. Evaluation of canola meal as a protein 693

supplement for dairy cows: A review and a metaanalysis. Can. J. Anim. Sci. 91:529–543. 694 695 Light, R. J., and D. B. Pillemer. 1984. Summing Up: The Science of Reviewing Research. 696

Harvard University Press, Cambridge, MA. 697 698 Martineau, R., D. R. Ouellet, and H. Lapierre. 2013. Feeding canola meal to dairy cows: A 699

meta-analysis on lactational responses. J. Dairy Sci. 96:1701–1714 700 701 Mjoun, K., K. F. Kalscheur ,1 A. R. Hippen, and D. J. Schingoethe. 2010. Ruminal 702

degradability and intestinal digestibility of protein and amino acids in soybean and corn 703 distillers grains products. J Dairy Sci. 93:4144-4154. 704

705 Moura, D. C., T. S. Fonseca, S. R. Soares, H. M. Silva, F. J. G. Vieira, L. A. Botini, A. P. 706

Sinhorin, I. M. Ogunaded and A. S. Oliveira. 2017. Crambe meal subjected to chemical and 707 physical treatments in sheep feeding. Livest Sci. 203:136-140. 708

709 Newkirk, R. 2009. Canola Meal: Feed Industry Guide. 4th ed. Canadian International Grains 710

Institute, Winnipeg, MB, Canada. 711 712 NRC. 2001. Nutrient Requirements of Dairy Cattle. 7th rev. ed. Natl. Acad. Sci., Washington, 713

DC. 714 715

41

Papas, A., J. R. Ingalls, and P. Cansfield. 1978. Effects of Tower and 1821 rapeseed meals and 716 Tower gums on milk yield, milk composition and blood parameters of lactating dairy cows. 717 Can. J. Anim. Sci. 58:671–679. 718

719 Sabharwal, V., M. S. Negi, N. Wilson and M. Lakshmikumaran. Analysis of the 5s rDNA nor-720

transcribed spacer region of Brassica and allied genera. 2006. IV International Symposium 721 on Brassicas and XIV Crucifer Genetics Workshop. Acta Hortic. 706, 83-98. 722

723 Stefansson, B. R. and Z. P. Kondra. 1975. Tower summer rape. Can J. Plant Sci. 55:343–344. 724 725 Stotzer, E. S. Grãos destilados de milho e sorgo na alimentação de ovinos, Sinop: Universidade 726

Federal do Mato Grosso UFMT, 2017. 33p. (Dissertação, Mestrado). 727 728 Thomke, S. 1981. Review of rapeseed meal in animal nutrition: Ruminant animals. J. Am. Oil 729

Chem. Soc. 58:805–810. 730 731 Tripathi, M. K., I. S. Agrawal, S. D. Sharma and A. S. Mishra 2001. Effect of substitution of 732

soybean meal with treated or untreated high glucosinolate mustard (Brassica juncea) meal 733 on intake, digestibility, growth performance and body composition of calves. Anim. Feed 734 Sci. Technol. 94: 137-146. 735

736 Tripathi, M. K. and A. S. Mishra. 2017. Prospects and Problems of Dietary Glucosinolates in 737

Animal Feeding. J Adv Dairy Res. 5:3. 738 739 Tripathi, M.K. and A. S. Mishra. 2007. Glucosinolates in animal nutrition: A review. Anim. 740

Feed Sci. Technol.132:1–27. 741 742 Viechtbauer, W. 2010. Conducting meta-analysis in R with the metaphor package. J. Stat. 743

Softw. 36:1–48. 744 745 Weisz, J. R., B. Weiss, S. S. Han, D. A. Granger, and T. Morton. 1995. Effects of psychotherapy 746

with children and adolescents revisited: A meta-analysis of treatment outcome studies. 747 Psychol. Bull. 117:450–468. 748

749 SoyStats. 2018. The American Soybean Association. Accessed on Feb 20, 2018. 750

http://soystats.com/international-world-protein-meal-consumption/ 751 752 Swanepoel, N., P. H. Robinson, and L. J. Erasmus. 2014. Determining the optimal ratio of 753

canola meal and high protein dried distillers grain protein in diets of high producing 754 Holstein dairy cows. Anim. Feed Sci. Technol.189:41-53. 755

756 757 758 759 760 761 762 763 764 765

42

Table 1. Effect replacement of several protein sources for canola meal on the performance and 766 metabolism of dairy cows 767

Item Control1

mean (SD) N2

RMD3 (95% CI) Heterogeneity4

Random effect P-value P-value I2 (%)

DMI, kg/d 20.94 (3.38) 79 0.22 (0.09, 0.34) <0.01 0.25 9.06

OM intake, kg/d 18.66 (2.61) 12 0.56 (0.23, 0.90) <0.01 0.23 21.74

CP intake, kg/d 3.36 (0.46) 10 0.14 (0.08, 0.21) <0.01 0.08 41.02

NDF intake, kg/d 7.52 (0.82) 14 0.40 (0.28, 0.52) <0.01 0.13 30.90

DM digestibility, % 62.04 (19.54) 20 -0.45 (-0.96, 0.05) 0.08 0.06 35.00

OM digestibility, % 72.27 (3.82) 12 0.20 (0.38, 0.78) 0.50 0.16 29.23

CP digestibility, % 62.17 (21.87) 12 -1.44 (-2.03, -0.86) <0.01 0.33 11.35

NDF digestibility, % 49.57 (12.52) 12 3.20 (2.34, 4.07) <0.01 0.81 0.0

Acetate, mmol/L 59.30 (6.29) 19 0.53 (0.05, 1.01) 0.03 0.86 0.0

Propionate, mmol/L 18.58 (1.98) 19 0.18 (-0.20, 0.57) 0.36 0.85 0.0

pH ruminal 6.38 (0.27) 18 0.02 (-0.06, 0.01) 0.23 0.19 21.96

Milk yield, kg/d 28.61 (6.95) 88 0.69 (0.32, 1.06) <0.01 <0.01 74.95

Milk yield 4%, kg/d 26.09 (8.13) 60 0.82(0.58, 1.05) <0.01 0.54 0.0

Feed efficiency 1.38 (0.19) 79 0.001 (0.00, 0.002) >0.05 0.80 0.0

Milk fat, % 3.78 (0.61) 67 0.03 (-0.07, 0.01) 0.20 0.12 16.86

Milk fat yield, kg/d 1.06 (0.29) 66 0.008 (-0.005, 0.02) 0.24 0.21 11.60

Milk protein, % 3.29 (0.24) 74 -0.01 (-0.03. 0.00) 0.08 0.08 19.52

Milk protein yield, kg/d 0.93 (0.21) 49 0.02 (0.007, 0.02) <0.01 1.00 0.0

Milk lactose, % 4.80 (0.20) 52 0.0006 (-0.01, 0.01) 0.93 0.86 0.0

Milk lactose yield, kg/d 1.45 (0.34) 45 0.02 (0.01, 0.05) <0.01 0.99 0.0

MUN, mg / dL 11.78 (2.75) 22 -0.98 (-1.29, -0.67) <0.01 0.07 32.27

N efficiency5, % 28.12 (4.88) 34 0.22 (0.20, 0.25) <0.05 0.70 0.00 2N = number of comparisons between canola meal and all different protein sources (complete data set is available 768

in Supplementary file S1). 769 3RMD = raw mean difference between brassica meal and different protein sources. 770 4I2 = proportion of total variation of size effect estimates that is due to heterogeneity; P-value to χ2 (Q) test of 771

heterogeneity. 772 5 Milk N intake in proportion of N intake. 773

774

775

776 777 778 779

43

780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804

805 806 807 808 809 810 811 812 Figure 1. Flow diagram showing the inclusion criteria for selection of the studies used to 813 perform the meta-analysis of the effects of substitution of different sources of protein by 814 brassica meal. 815 816 817 818 819 820 821 822 823 824

Records identified by prior search WEB of SCIENCE

“Dairy Cow, “Canola meal”, “Crambe meal”, “Brassica”, “Brassica aleracea”, “Brassica rapessed”, “Rapessed meal” and “Canerita

meal” (n = 274)

Records automatically excluded for “duplicate” and “no article (n = 81)

Records after excluded for “duplicate” and “no article (n = 193)

Records manually excluded (n =156)

Studies include in meta-analysis to performance and efficiency utilization

(n = 37)

44

825

826

827 828

Figure 2. Funnel plots showing the effects of replacing different sources of protein for brassica 829

meal on the performance of dairy cows. The horizontal line indicates the raw mean difference 830

(RMD) estimate and the vertical line indicates their corresponding standard errors (SE). P-value 831

refers to the test for funnel plot asymmetry by Egger`s regression method between RMD and 832

SE. Funnel plot asymmetry is indicative of publication bias. 833

834

835

836

837

Milk Fat (%) Milk Protein (%)

Milk yield (kg/d)

DMI (kg/d)

P = 0.52 n = 79

P = 0.10 n = 88

P = 0.67 n = 67

P = 0.99 n = 74

DM digestibility (%)

P = 0.86 n = 20

45

838

Figure 3. Forest plot showing the effects of replacement soybean meal by canola meal on milk 839

yield (kg/d) of dairy cows. The x-axis shows the raw mean difference (RMD); diamonds to the 840

left of the solid line represent a reduction in the measure, whereas diamonds to the right of the 841

line indicate an increase. Each diamond represents the mean size effect for that study, and the 842

size of the diamond reflects the relative weighting of the study to the overall size effect estimate 843

with larger diamonds representing greater weight. The lines connected to the diamond 844

represents the upper and lower 95% confidence interval for the size effect. The dotted vertical 845

line represents the overall size effect estimate. The diamond at the bottom represents the mean 846

response across the studies, and the solid vertical line represents a mean difference of zero or 847

no effect. 848

849

Overall (I2 =74.9%, P = 0.50)

RMD, kg/d

100% 0.23 [-0.42, 0.87]

46

850

851

Figure 4. Forest plot showing the effects of replacement dry distillated grains by canola meal 852

on milk yield (kg/d) of dairy cows. The x-axis shows the raw mean difference (RMD); 853

diamonds to the left of the solid line represent a reduction in the measure, whereas diamonds to 854

the right of the line indicate an increase. Each diamond represents the mean size effect for that 855

study, and the size of the diamond reflects the relative weighting of the study to the overall size 856

effect estimate with larger diamonds representing greater weight. The lines connected to the 857

diamond represents the upper and lower 95% confidence interval for the size effect. The dotted 858

vertical line represents the overall size effect estimate. The diamond at the bottom represents 859

the mean response across the studies, and the solid vertical line represents a mean difference of 860

zero or no effect. 861

862

863

864

865

Overall (I2 = 18.0%, P < 0.01)

RMD, kg/d

47

866

867

Figure 5. Forest plot showing the effects of replacement other protein sources (cottonseed meal, 868

corn gluten meal and sunflower meal) grain by canola meal on milk yield (kg/d) of dairy cows. 869

The x-axis shows the raw mean difference (RMD); diamonds to the left of the solid line 870

represent a reduction in the measure, whereas diamonds to the right of the line indicate an 871

increase. Each diamond represents the mean size effect for that study, and the size of the 872

diamond reflects the relative weighting of the study to the overall size effect estimate with larger 873

diamonds representing greater weight. The lines connected to the diamond represents the upper 874

and lower 95% confidence interval for the size effect. The dotted vertical line represents the 875

overall size effect estimate. The diamond at the bottom represents the mean response across the 876

studies, and the solid vertical line represents a mean difference of zero or no effect. 877

Overall (I2 = 67.0%, P < 0.01)

RMD, kg/d

100% 0.82 [0.39, 1.24]

48

Appendix. List of references used in meta-analysis 878

879

Acharya, I. P., D. J. Schingoethe, K. F. Kalscheur, and D. P. Casper. 2015. Response of lactating 880 dairy cows to dietary protein from canola meal or distillers' grains on dry matter intake, 881 milk production, milk composition, and amino acid status. Can. J. Anim. 95:267-279. 882

883 Brito, A. F. and G. A. Broderick. 2007. Effects of Different Protein Supplements on Milk 884

Production and Nutrient Utilization in Lactating Dairy Cows1. J. Dairy Sci. 90:1816-1827. 885 886 Broderick, G. A., A. P. Faciola, and L. E. Armentano. 2015. Replacing dietary soybean meal 887

with canola meal improves production and efficiency of lactating dairy cows. J. Dairy Sci. 888 98:5672-5687. 889

890 Choi, C. W., A. Vanhatalo, S. Ahvenjärvi, and P. Huhtanen. 2002. Effects of several protein 891

supplements on flow of soluble non-ammonia nitrogen from the forestomach and milk 892 production in dairy cows. Anim. Feed Sci. Technol. 102:15-33. 893

894 Christen, K. A., D. J. Schingoethe, K. F. Kalscheur, A. R. Hippen, K. K. Karges, and M. L. 895

Gibson. 2010. Response of lactating dairy cows to high protein distillers grains or 3 other 896 protein supplements. J. Dairy Sci. 93:2095-2104. 897

898 DePeters, E. J. and D. L. Bath. 1986. Canola Meal Versus Cottonseed Meal as the Protein 899

Supplement in Dairy Diets. J. Dairy Sci.69:148-154. 900 901 Emanuelson, M., K. Å. Ahlin, and H. Wiktorsson. 1993. Long-term feeding of rapeseed meal 902

and full-fat rapeseed of double low cultivars to dairy cows. Livest Sci. 33:199-214. 903 904 Fisher, L. J. and D. S. Walsh. 1976. Substitution of rapeseed meal for soybean meal as a source 905

of protein for lactating cows. Can. J. Anim.56:233-242. 906 907 Franke, K., U. Meyer, H. Wagner, H. O. Hoppen, and G. Flachowsky. 2009. Effect of various 908

iodine supplementations, rapeseed meal application and two different iodine species on the 909 iodine status and iodine excretion of dairy cows. Livest Sci. 125:223-231. 910

911 Gidlund, H., M. Hetta, S. J. Krizsan, S. Lemosquet, and P. Huhtanen. 2015. Effects of soybean 912

meal or canola meal on milk production and methane emissions in lactating dairy cows fed 913 grass silage-based diets. J. Dairy Sci.98:8093-8106. 914

915 Ingalls, J. R. and H. R. Sharma. 1975. Feeding of Bronowski, Span and commercial rapeseed 916

meals with or without addition of molasses or flavor in rations of lactating cows. Can. J. 917 Anim. 55:721-729. 918

919 Khorasani, G. R., G. De Boer, B. Robinson, and J. J. Kennelly. 1994. Influence of Dietary 920

Protein and Starch on Production and Metabolic Responses of Dairy Cows. J. Dairy 921 Sci.77:813-824. 922

923 Kokkonen, T., M. Tuori, V. Leivonen, and L. Syrjälä-Qvist. 2000. Effect of silage dry matter 924

content and rapeseed meal supplementation on dairy cows. 1. Milk production and feed 925 utilisation. Anim. Feed Sci. Technol. 84:213-228. 926

49

Kokkonen, T. M. Tuori and L. SyrjaÈlaÈ-Qvist. 2000.Effect of silage dry matter content and 927 rapeseed meal supplementation on dairy cows 2. Rumen fermentation and digesta passage rate. 928 Anim. Feed Sci. Technol. 84: 229-242. 929 930 Laarveld, B. and D. A. Christensen. 1976. Rapeseed Meal in Complete Feeds for Dairy Cows. 931

J. Dairy Sci. 59:1929-1935. 932 933 Maesoomi, S. M., G. R. Ghorbani, M. Alikhani, and A. Nikkhah. 2006. Short Communication: 934

Canola Meal as a Substitute for Cottonseed Meal in Diet of Midlactation Holsteins. J. Dairy 935 Sci. 89:1673-1677. 936

937 Maiga, H. A., D. M. Harris, M. E. Meyer, C. R. Dahlen, and M. L. Bauer. 2011. Effect of 938

pelleted high-oil canola meal from on-farm biodiesel production on rumen fermentation in 939 lactating Holstein dairy cows. Prof. Anim. Sci. 27:29-34. 940

941 Maxin, G., D. R. Ouellet, and H. Lapierre. 2013. Effect of substitution of soybean meal by 942

canola meal or distillers grains in dairy rations on amino acid and glucose availability. J. 943 Dairy Sci. 96:7806-7817. 944

945 McClean, C. and B. Laarveld. 1991. Effect of somatotropin and protein supplement on thyroid 946

function of dairy cattle. Can. J. Anim. 71:1053-1061. 947 948 McDonnell, R. P. and M. V. Staines. 2017. Replacing wheat with canola meal and maize grain 949

in the diet of lactating dairy cows: Feed intake, milk production and cow condition 950 responses. J. Dairy Sci. 84:240-247. 951

952 Moate, P. J., S. R. O. Williams, C. Grainger, M. C. Hannah, E. N. Ponnampalam, and R. J. 953

Eckard. 2011. Influence of cold-pressed canola, brewers grains and hominy meal as dietary 954 supplements suitable for reducing enteric methane emissions from lactating dairy cows. 955 Anim. Feed Sci. Technol. 166–167:254-264. 956

957 Mulrooney, C. N., D. J. Schingoethe, K. F. Kalscheur, and A. R. Hippen. 2009. Canola meal 958

replacing distillers grains with solubles for lactating dairy cows. J. Dairy Sci. 92:5669-5676. 959 960 Murphy, J. J., J. F. Connolly, and G. P. McNeill. 1995. Effects on cow performance and milk 961

fat composition of feeding full fat soyabeans and rapeseeds to dairy cows at pasture. Livest. 962 Prod. Sci. 44:13-25. 963

964 Oba, M., G. B. Penner, T. D. Whyte, and K. Wierenga. 2010. Effects of feeding triticale dried 965

distillers grains plus solubles as a nitrogen source on productivity of lactating dairy cows. 966 J. Dairy Sci. 93:2044-2052. 967

968 Paula, E. M. G. A. Broderick, M. A. C. Danes, N. E. Lobos, G. I. Zanton, and A. P. Faciola. 969

2018. Effects of replacing soybean meal with canola meal or treated canola meal on ruminal 970 digestion, omasal nutrient flow, and performance in lactating dairy cows. J. Dairy Sci. 971 101:1–12. 972

973 Puhakka, L., S. Jaakkola, I. Simpura, T. Kokkonen, and A. Vanhatalo. 2016. Effects of 974

replacing rapeseed meal with fava bean at 2 concentrate crude protein levels on feed intake, 975

50

nutrient digestion, and milk production in cows fed grass silage–based diets. J. Dairy Sci. 976 99:7993-8006. 977

978 Razzaghi, A., R. Valizadeh, A. A. Naserian, M. D. Mesgaran, A. J. Carpenter, and M. H. 979

Ghaffari. 2016. Effect of dietary sugar concentration and sunflower seed supplementation 980 on lactation performance, ruminal fermentation, milk fatty acid profile, and blood 981 metabolites of dairy cows. J. Dairy Sci. 99:3539-3548. 982

983 Robinson, P. H. 1996. Evaluation of a seal by-product meal as a feedstuff for dairy cows. Anim. 984

Feed Sci. Technol. 63:51-62. 985 986 Robinson, P. H. and J. J. Kennelly. 1988. Influence of intake of rumen undegradable protein on 987

milk production of late lactation Holstein cows. J. Dairy Sci. 71:2135-2142. 988 989 Robinson, P. H., G. R. Khorasani, and J. J. Kennelly. 1994. Forestomach and Whole Tract 990

Digestion in Lactating Dairy Cows Fed Canola Meal Treated with Variable Levels of Acetic 991 Acid. J. Dairy Sci. 77:552-559. 992

993 Sanchez, J. M. and D. W. Claypool. 1983. Canola Meal as a Protein Supplement in Dairy 994

Rations1, 2. J. Dairy Sci. 66:80-85. 995 996 Sharma, H. R., J. R. Ingalls, and J. A. McKirdy. 1977. Effects of feeding a high level of Tower 997

rapeseed meal in dairy rations on feed intake and milk production. Can. J. Anim. 57:653-998 662. 999

1000 Swanepoel, N., P. H. Robinson, and L. J. Erasmus. 2014. Determining the optimal ratio of 1001

canola meal and high protein dried distillers grain protein in diets of high producing 1002 Holstein dairy cows. Anim. Feed Sci. Technol.189:41-53. 1003

1004 Vicente, G. R., J. A. Shelford, R. G. Peterson, and C. R. Krishnamurti. 1984. Effects of feeding 1005

canola-meal-protected-tallow or soybean-meal-protected-tallow in the low-roughage diet of 1006 dairy-cows in early lactation. Can. J. Anim. Sci. 64:81-91. 1007

1008 Vincent, I. C., R. Hill, and R. C. Campling. 1990. A note on the use of rapeseed, sunflower and 1009

soyabean meals as protein sources in compound foods for milking cattle. Anim. Prod. 1010 50:541-543. 1011

1012 Weisbjerg, M. R., M. K. Larsen, L. Hymøller, M. Thorhauge, U. Kidmose, J. H. Nielsen, and 1013

J. B. Andersen. 2013. Milk production and composition in Danish Holstein, Danish Red, 1014 and Danish Jersey cows supplemented with saturated or unsaturated fat. Livest Sci. 155:60-1015 70. 1016

1017 Weiss, W. P., D. J. Wyatt, D. H. Kleinschmit, and M. T. Socha. 2015. Effect of including canola 1018

meal and supplemental iodine in diets of dairy cows on short-term changes in iodine 1019 concentrations in milk. J. Dairy Sci. 98:4841-4849. 1020

1021 1022