Long-term oral drenching of crude glycerol to primiparous dairy cows in early lactation

G Model ANIFEE-12922; No. of Pages 9

ARTICLE IN PRESS Animal Feed Science and Technology xxx (2013) xxx–xxx

Contents lists available at SciVerse ScienceDirect

Animal Feed Science and Technology journal homepage: www.elsevier.com/locate/anifeedsci

Long-term oral drenching of crude glycerol to primiparous dairy cows in early lactation Marko Kass a,b,∗ , Tiia Ariko a , Jaak Samarütel a , Katri Ling a , Hanno Jaakson a , Tanel Kaart b,c , David Arney a , Olav Kärt a,b , Meelis Ots a,b a b c

Department of Nutrition and Animal Products Quality, Estonian University of Life Sciences, Kreutzwaldi 46, 51006 Tartu, Estonia Bio-Competence Centre of Healthy Dairy Products, Kreutzwaldi 1, 51014 Tartu, Estonia Department of Animal Genetics and Breeding, Estonian University of Life Sciences, Kreutzwaldi 46, 51006 Tartu, Estonia

a r t i c l e

i n f o

Article history: Received 19 January 2012 Received in revised form 12 April 2013 Accepted 10 June 2013 Available online xxx Keywords: Crude glycerol Oral drenching Milk production Blood metabolites Early lactation Primiparous dairy cow

a b s t r a c t The objective of this experiment was to study the long-term effects of supplemental oral drenching of crude glycerol (CG) on feed intake, milk yield and composition, plasma metabolites and energy balance indices of primiparous dairy cows in early lactation. Fourteen primiparous Holstein dairy cows were paired according to body weight (BW) and body condition score (BCS) at calving, and allocated at random to either a treatment or a control group. Both groups were fed a basal diet, containing 11.4 MJ kg−1 DM metabolisable energy and 103 g/kg DM metabolisable protein, twice a day. Silage (ad libitum) and a pelleted concentrate mixture with mineral mix were fed separately during the three six-day periods from days 4 to 21 post partum. An oral drench of 500 ml of CG mixed with lukewarm water was fed supplementally before the morning feedings to the treatment groups, while the control group were given no oral drench. Blood samples were collected on two consecutive days at the end of each six-day period before administration of glycerol. Results showed that long-term administration of CG increased silage intake (P<0.001), and total dry matter intakes (DMI; P=0.023). Oral drenching with glycerol increased milk yields (P<0.01), and milk lactose content (P<0.001). Supplemental glycerol had no effect on estimates for milk energy, milk constituent ratios or milk acetone content. No differences in BCS changes or on concentrations of plasma glucose, insulin, and ␤-hydroxybutyrate (BHB) were found between the two groups. Dairy cows given oral glycerol had lower concentrations of plasma urea (P=0.005) during the post partum period. There was a treatment and time interaction effect on the non-esterified fatty acids (NEFA) concentration (P=0.048), although this was complex, and the only period of significant differences was in the first six-day period, when NEFA concentration was higher (P=0.025) in the control group. The data indicate that prolonged oral drenching of supplemental CG in the early lactation period improves total DMI, and therefore has a positive effect on milk yield of primiparous dairy cows in this period. © 2013 Elsevier B.V. All rights reserved.

Abbreviations: ADF, ADF expressed inclusive of residual ash; BCS, body condition score; BW, body weight; CG, crude glycerol; DIM, days in milk; DMI, dry matter intake; DM, dry matter; BHB, ␤-hydroxybutyrate; aNDF, NDF assayed with a heat stable amylase and expressed inclusive of residual ash; NEB, negative energy balance; NEFA, non-esterified fatty acids; TMR, total mixed ration. ∗ Corresponding author at: Department of Nutrition and Animal Products Quality, Estonian University of Life Sciences, Kreutzwaldi 46, 51006 Tartu, Estonia. Tel.: +372 731 3478; fax: +372 731 3477. E-mail address: [email protected] (M. Kass). 0377-8401/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.anifeedsci.2013.06.004

Please cite this article in press as: Kass, M., et al., Long-term oral drenching of crude glycerol to primiparous dairy cows in early lactation. Anim. Feed Sci. Tech. (2013), http://dx.doi.org/10.1016/j.anifeedsci.2013.06.004

G Model ANIFEE-12922; No. of Pages 9

ARTICLE IN PRESS M. Kass et al. / Animal Feed Science and Technology xxx (2013) xxx–xxx

2

1. Introduction Early lactation is the most critical period of lactation, associated with reduced dry matter intakes, and increased energy and glucose demands for milk secretion (Grummer, 1993). As the energy intake does not meet the energy requirements a negative energy balance may occur, and hence fatty acid mobilisation from adipose tissue increases. Transition dairy cows have increased requirements for glucose, by the mammary gland, to support lactose synthesis (Overton and Waldron, 2004). In practice, several glucogenic substances are thought to increase the supply of glucose and therefore improve the metabolic status of the periparturient cow (Ingvartsen, 2006). Several studies have shown glycerol to be a potential glucogenic feed additive, increasing the proportion of propionic acid in the rumen of dairy cows (DeFrain et al., 2004; Linke et al., 2004; Carvalho et al., 2011). Elevation of glucose concentration alone (Goff and Horst, 2001), or together with insulin concentration (Linke et al., 2004), have been reported after drenching large amounts of glycerol via an oesophageal pump (Goff and Horst, 2001) or orally (Linke et al., 2004). Earlier studies (Johnson, 1951; Fisher et al., 1971) have also indicated that glycerol can be used as an appetite stimulant, thus alleviating the potential risk of ketosis and also improving milk yield and blood glucose concentrations. To study the effect of glycerol supplementation on dairy cow performance different administration strategies have been used. Glycerol has been fed to transition cows (DeFrain et al., 2004; Ogborn, 2006), early lactation cows (Chung et al., 2007; Wang et al., 2009), and mid-lactation dairy cows (Khalili et al., 1997; Donkin et al., 2009) to improve DMI and milk performance. Glycerol has been either fed top-dressed (Chung et al., 2007; Lomander et al., 2012a), added to the total mixed ration (TMR; DeFrain et al., 2004; Wang et al., 2009) or orally drenched (Goff and Horst, 2001; Ogborn, 2006) to dairy cows to alleviate the symptoms of negative energy balance (NEB) or improve fertility (Lomander et al., 2012b) at transition period or in early lactation. Multiparous Holstein dairy cows are more vulnerable to metabolic disorders, as they mobilise significantly more body energy in early lactation (Friggens et al., 2007), therefore more attention has been paid to them. It is nevertheless important to know the effects on younger cows, who additionally partition their energy for growth, and this experiment was designed to focus on such animals in the post partum period. There are limited data in the literature regarding the long-term oral drenching of glycerol for a period longer than five days, and effects on primiparous cows. The hypotheses were that long-term oral drenching of glycerol improves lactation performance and indices of energy status of primiparous dairy cows. The objectives of this experiment were to study the effects of orally administered crude glycerol on feed intake, milk yield and composition, indices of energy status and blood parameters of primiparous dairy cows throughout the first three weeks of lactation.

2. Materials and methods 2.1. Animals and experimental design The study was carried out on Eerika Experimental Farm (Märja 61406, Estonia) of the Estonian University of Life Sciences. Fourteen primiparous Holstein dairy cows were involved in the study, from days four to 21 of lactation; which was divided into three six-day periods. The cows were tethered in individual stalls, bedded on rubber mats, and they were milked and sampled in their stalls. Stratified random sampling was used to pair cows, according to body weight and body condition score (using the system proposed by Edmonson et al., 1989) at calving, into either a treatment or a control group (Fig. 1). The mean BWs (mean ± SD) were 557.6 ± 38.0 kg for the control and 551.3 ± 42.1 kg for the treatment group cows at the beginning of the experiment (P=0.50, pairwise t-test); the mean BCSs at parturition were 3.46 ± 0.09 and 3.46 ± 0.20 (P=1.00, pairwise t-test), respectively. Experimental procedures were performed as licensed under the regulations of the Ethics Board of the Estonian Ministry of Agriculture for experiments with live animals.

Fig. 1. Measurement and sample collection days of different parameters. BCS, body condition score; DIM, days in milk.

Please cite this article in press as: Kass, M., et al., Long-term oral drenching of crude glycerol to primiparous dairy cows in early lactation. Anim. Feed Sci. Tech. (2013), http://dx.doi.org/10.1016/j.anifeedsci.2013.06.004

G Model ANIFEE-12922; No. of Pages 9

ARTICLE IN PRESS M. Kass et al. / Animal Feed Science and Technology xxx (2013) xxx–xxx

3

Table 1 Ingredients and mean chemical composition of the basal diet. g/kg in DM Silage Concentrate mixturea Dairy cow mineral mixb Salt Mean chemical composition Organic matter Crude protein Ether extract Neutral detergent fibre (aNDF) Acid detergent fibre (ADF) Calcium Phosphorus Metabolisable energyc (MJ/kg DM)

495 495 7 3 920 179 47 351 228 9.7 4.9 11.4

aNDF, NDF assayed with a heat stable amylase and expressed inclusive of residual ash; ADF expressed inclusive of residual ash. a Concentrate mixture: rapeseed cake 27.0%, wheat 24.0%, wheat bran 10.1%, maize 9.0%, corn feed 8.5%, soybean meal 8.0%, sliced fodder beet 6.0%, barley 5.0%, salt 0.9%, palm oil 0.8%, limestone 0.5%, Premix Ko 0.2%. b Dairy cow mineral mix: Ca – 175 g/kg, P – 40 g/kg, NaCl – 200 g/kg, Na – 80 g/kg, Mg – 50 g/kg. c ME – metabolisable energy calculated according to Oll (1995).

2.2. Diet and feeding The diet (Table 1) was composed according to current Estonian feeding recommendations based on BW and expected milk yield, comprising silage, pelleted concentrate mixture and a mineral mix providing 11.4 MJ kg−1 DM metabolisable energy and 103 g/kg DM metabolisable protein. Cows were fed individually twice a day at 07:45 and 17:30 h, in their stalls. At each feeding the cows were initially offered concentrate mixture with the mineral mix. After 1 h the uneaten concentrate mixture was collected, removed, and weighed. Grass and red clover mixture silage (70% and 30%), containing 9.4 MJ kg−1 DM metabolisable energy and 80 g/kg DM metabolisable protein, was then fed ad libitum. Silage refusals were removed before the subsequent feeding and weighed. Based on these data, daily DMIs for concentrate mixture, silage and total DMI were calculated. The cows in the treatment group were given 500 ml (573 g) of crude glycerol (82.6% glycerol, 9.3% salts, 7.1% water, 0.6% crude fat, 0.4% methanol), once a day just before morning feeding. The glycerol was mixed with 500 ml of lukewarm water (36 ± 2 ◦ C) to minimise the drenching time and hence the inconvenience for the animal. The control cows were treated in exactly the same way, but were given no oral drench. Cows had fresh water continuously accessible throughout the experimental period. 2.3. Sample collection and analysis Silage samples were collected twice in each six-day period (Fig. 1). Each batch of concentrate mixture and mineral mix feed was analysed. Chemical analyses of concentrate mixture and silage samples were made using established methods (AOAC, 2005). Dry matter content was determined by heating a feed sample for 2 h at +130 ◦ C to constant weight. Analysis for ether extract content was performed by petroleum ether extraction with the Soxtec System 2043 Extraction Unit (FOSS, Hillerød, Denmark). Crude protein content was analysed by the Kjeldahl method with a Kjeltec 2300 analyser (FOSS, Hillerød, Denmark). The neutral detergent fibre (aNDF) and acid detergent fibre (ADF) concentrations were determined with a fibre analyzer ANKOM220 (ANKOM Technology, Fairport, USA) using methods described by Van Soest et al. (1991). Calcium content in feeds was determined flame-photometrically and phosphorus spectrophotometrically (AOAC, 2005). Glycerol content of CG was measured by HPLC (Agilent 1200 Series Refractive Index Detector, Agilent Technologies, Santa Clara, USA), using a Zorbax carbohydrate column (250 mm × 4.6 mm (i.d.) with 0.5 ␮m film thickness; Agilent Technologies, Santa Clara, USA) by the adaption of the recognised method OIV MA-E-AS311-03-Sucres:2009 (OIV, 18 Rue D’Aguesseau, 75008 Paris, France). Water content was analysed by gravimetric analysis at 102 ◦ C. Methanol content was measured with the Agilent 7890A GC System using a capillary column CP-WAX 57CB (50 mm × 320 ␮m × 0.2 ␮m; Agilent Technologies, Santa Clara, USA) by the method EÜ2870/2000:III (Estonian Accreditation Centre) and crude fat by the Schmid-Bondzynski-Ratslaff reference method. Milk yields were recorded daily (08:00 h and 18:00 h) using in-line milk metres (Tru-Test HI, Tru-Test Ltd., New Zealand). Individual milk samples for the analysis of milk composition were taken twice a day in each period (Fig. 1). Milk samples from one day were combined proportionally, according to within-day differences in milk yields between morning and evening milkings, and kept at +4 ◦ C until analysis on the following morning. The milk fat, protein, and lactose contents were measured using infrared spectrometer MilkScan 4300 (FOSS Electric A/S, Hillerød, Denmark). Milk acetone was analysed by an Agilent 7890A gas chromatograph (Agilent Technologies Inc, USA) using 4% Carbowax 20M, matrix 80/120 Carbopack B-DA column (Sigma–Aldrich, St. Louis, USA). Blood samples were collected on the same days as the milk samples (Fig. 1). Bloods were taken, before the administration of glycerol, from the coccygeal vein and collected into 10 ml heparinised vacuum tubes (Terumo® Venoject® , VT-100SHL, Please cite this article in press as: Kass, M., et al., Long-term oral drenching of crude glycerol to primiparous dairy cows in early lactation. Anim. Feed Sci. Tech. (2013), http://dx.doi.org/10.1016/j.anifeedsci.2013.06.004

G Model ANIFEE-12922; No. of Pages 9

ARTICLE IN PRESS M. Kass et al. / Animal Feed Science and Technology xxx (2013) xxx–xxx

4

Terumo Europe N.V., Belgium). Plasma was separated immediately after blood collection at the on-farm laboratory and was kept at −22 ◦ C till analysis. Concentrations of metabolites in blood plasma were analysed spectrophotometrically (Helios ␤, Unicam Ltd., Cambridge, United Kingdom) using standard test kits (Human Gesellschaft für Biochemica und Diagonostica GmbH, Wiesbaden, Germany) for glucose and urea, and test kits from Randox Laboratories Ltd. (Ardmore, United Kingdom) for NEFA and BHB. Blood insulin concentration was measured radio-immunologically (1470 Wallac Wizard Gamma Counter, Perkin Elmer Life and Analytical Sciences, Inc., Waltham, Massachusetts, USA) using 125I radioimmunoassay test kits (CoatA-Count Insulin). The inter- and intra-assay coefficients of variation of the methods were below 6%. 2.4. Calculation and statistical analysis Estimates for milk energy were calculated from the means of two consecutive days’ milk compositional records. Milk energy (MJ/kg) was calculated as: 4.183 × {(0.0929 × milk fat g/kg) + (0.0547 × milk protein g/kg) + (0.0395 × milk lactose g/kg)}, according to the NRC (2001). BCS change was calculated as the difference between the mean values at 4 and 5 DIM to the mean values at 20 and 21 DIM. Feed efficiency of each animal was calculated for each experimental day, dividing milk yield (kg/d) values by total DMI (kg/d). Statistical analyses were performed with reference to the experimental design of the three equal six-day periods (Fig. 1). The CG drenching (treatment) and time effects of DMI, milk yield and composition, BCS variables and blood parameters were analysed using the linear mixed model of the form:Yijkl =  + Ai + Bi + (A×B)ij + Ck + εijkl ,where Yijkl – dependent variable,  – model intercept, Ai – fixed effect of period, Bj – fixed effect of the treatment group, (A × B)ij – fixed effect of period and treatment group interaction, Ck – random animals’ pair effect, εijkl – residual error term. The models also considered that the correlation between repeated measurements of the same cow did not converge, as this effect was already covered by the pair effect. All values in the text and tables are presented as least square means. Statistical significance was declared at P ≤ 0.05, with trends noted at P ≤ 0.10. All statistical analyses were performed with SAS software (version 9.1; SAS Institute, 2003). 3. Results 3.1. Dry matter intake and BCS Long-term oral drenching of CG had a positive effect on intake parameters (Table 2). Glycerol treatment in early lactation increased total DMI (P=0.023), in particular the silage DMI increased (P=0.001). As the treatment cows showed a tendency for lower concentrate mixture DMI (P=0.066) their mean silage/concentrate mixture intake ratio was higher compared to the control cows (P=0.017). When considering the effect of the experimental periods (of six days), the total DMI was greater for the treatment group than the control group (P=0.018) in the second period of experiment. Silage DMI was different between control and treatment cows in all three periods. There were no difference in BCS change between the groups; the loss of body condition up to DIM 21 was 0.39 units for both groups.

Table 2 Effect of long-term drenching of crude glycerol on feed intake and feed efficiency. Traits

Period

Total, kg/d

1 2 3

Silage, kg/d

1 2 3

Concentrate mixture, kg/d

Treatment

SEM

P-value

Effects’ P-values

Control

Glycerol

Time

T×T

11.3a 12.5b 13.9ab

11.5a 13.5b 14.4ab

0.80

0.67 0.018 0.24

0.023

<0.001

0.38

6.49ab 6.34a 7.01b

7.47 7.30 7.66

0.43

0.001 0.001 0.024

<0.001

0.035

0.67

1 2 3

4.79a 6.15b 6.88a

3.99a 6.19b 6.72b

0.55

0.007 0.88 0.57

0.066

0.001

0.10

Silage/concentrate ratio

1 2 3

2.02a 1.16b 1.07b

2.60a 1.43b 1.24b

0.28

0.02 0.27 0.48

0.017

0.001

0.47

Feed efficiency

1 2 3

2.21 2.27 2.15

2.28 2.19 2.19

0.07

0.99 0.33 0.54

0.75

0.35

0.36

Treatment

T × T, treatment and time interactions; SEM, standard error of the means; DMI, dry matter intake. Least square means within a column and trait with different superscripts (a–c) differ (P<0.05).

Please cite this article in press as: Kass, M., et al., Long-term oral drenching of crude glycerol to primiparous dairy cows in early lactation. Anim. Feed Sci. Tech. (2013), http://dx.doi.org/10.1016/j.anifeedsci.2013.06.004

G Model ANIFEE-12922; No. of Pages 9

ARTICLE IN PRESS M. Kass et al. / Animal Feed Science and Technology xxx (2013) xxx–xxx

5

3.2. Milk yield and composition Milk yields were greater for the treatment cows (P=0.001; Table 3). Considering experimental periods, milk yields were different between the groups during the second (P=0.048) and third periods (P=0.011). Glycerol treatment also had a positive effect on milk lactose content (P=0.020), and there was a treatment × time interaction (P=0.021) for an increase in milk lactose content. This increase in lactose content was significant for both groups over time. The milk fat and protein contents, somatic cell scores, milk acetone, the milk fat/protein and fat/lactose ratios were not affected by treatment (Table 3). A decrease in milk protein content within the control group over time was significant over the whole experimental period (P=0.001). There was a tendency in the glycerol treatment for lower milk urea content (P=0.074). Table 3 Effect of oral drenching of crude glycerol on milk yield, milk energy yield and milk composition in early lactation. Traits

Period

Treatment

SEM

Time

T×T

0.13 0.048 0.011

0.001

<0.001

0.78

0.13

0.76 0.36 0.38

0.39

0.45

0.61

0.924 0.935 0.992

0.07

0.78 0.62 0.21

0.39

0.56

0.55

1.18a 1.39b 1.55c

0.09

0.44 0.49 0.54

0.23

<0.001

0.99

93.3a 102.8ab 107.9b

7.58

0.88 0.38 0.33

0.32

0.06

0.67

Glycerol

1 2 3

24.7a 27.7b 29.4c

25.9a 29.3b 31.5c

1.78

Fat

1 2 3

1.39 1.39 1.42

1.35 1.51 1.53

Protein

1 2 3

0.938 0.909 0.927

Lactose

1 2 3

1.13a 1.34b 1.51c

Energy (MJ/d)

1 2 3

Composition, g/kg Fat

94.2 97.1 101.5

Effects’ P-values Treatment

Control Yield, kg/d Milk

P-value

1 2 3

55.9a 50.2b 46.3b

52.5 51.1 47.8

0.24

0.25 0.76 0.59

0.86

0.003

0.43

Protein

1 2 3

37.5a 32.3b 30.3b

36.6a 32.5b 31.3c

0.07

0.29 0.78 0.24

0.81

<0.001

0.28

Lactose

1 2 3

45.2a 47.7b 49.3c

46.7a 48.1a 49.1b

0.05

0.001 0.34 0.68

0.02

<0.001

0.02

Energy (MJ/kg)

1 2 3

3.78a 3.48b 3.31c

3.65a 3.52ab 3.40b

0.09

0.23 0.67 0.45

0.99

<0.001

0.34

Fat/protein ratio

1 2 3

1.51 1.56 1.53

1.44 1.58 1.54

0.08

0.56 0.84 0.93

0.87

0.40

0.83

Fat/lactose ratio

1 2 3

1.24a 1.06b 0.94b

1.13a 1.06a 0.97b

0.05

0.09 0.93 0.60

0.52

<0.001

0.25

SCS

1 2 3

4.46a 3.68ab 2.74b

4.21a 3.97a 2.84b

0.51

0.66 0.60 0.86

0.88

0.001

0.79

Acetone, ␮mol/l

1 2 3

127 113 60.4

73.1 104 65.5

45.3

0.33 0.87 0.93

0.54

0.46

0.73

Urea, mg/l

1 2 3

231 233 245

225 210 230

15.3

0.57 0.11 0.29

0.07

0.31

0.71

T × T, treatment and time interactions; SEM, standard error of the means; SCS, somatic cell score (SCS = log2 (SCC/100,000) +3). Least square means within a column and trait with different superscripts (a–c) differ (P<0.05).

Please cite this article in press as: Kass, M., et al., Long-term oral drenching of crude glycerol to primiparous dairy cows in early lactation. Anim. Feed Sci. Tech. (2013), http://dx.doi.org/10.1016/j.anifeedsci.2013.06.004

G Model ANIFEE-12922; No. of Pages 9

ARTICLE IN PRESS M. Kass et al. / Animal Feed Science and Technology xxx (2013) xxx–xxx

6

Table 4 Effect of crude glycerol drenching on selected blood plasma metabolites and insulin in the early lactation. Traits

Period

Glucose, mg/dl

1 2 3

Insulin, ml U/l

1 2 3

3.96a 4.80ab 6.28b

BHB, mmol/l

1 2 3

1.02a 0.88ab 0.63b

NEFA, ␮mol/l

1 2 3

Urea, mg/dl

1 2 3

Treatment Control 76.2a 77.9ab 84.8b

1077a 891a 634b 29.9a 32.6ab 34.3b

SEM

P-value

Glycerol 78.7 77.1 82.7

Effects’ P-values Treatment

Time

T×T

2.79

0.52 0.82 0.58

0.94

0.031

0.68

3.84 3.90 5.49

0.89

0.90 0.34 0.39

0.26

0.008

0.81

0.98 1.01 0.88

0.11

0.77 0.33 0.06

0.15

0.033

0.31

0.025 0.39 0.48

0.68

<0.001

0.048

0.06 0.13 0.12

0.005

0.001

0.97

854ab 975a 702b 26.7a 30.0ab 31.6b

81.9

1.66

T × T, treatment and time interactions; SEM, standard error of the means; BHB, ␤-hydroxybutyrate; NEFA, non-esterified fatty acids. Least square means within a column and trait with different superscripts (a–c) differ (P<0.05).

Fig. 2. Effect of long-term oral drenching of CG on plasma non-esterified fatty acids (NEFA) concentrations.

3.3. Blood parameters Long-term oral drenching of crude glycerol had no effect on plasma concentrations of glucose, insulin, or BHB over the whole experimental period (Table 4). The treatment group cows had lower NEFA concentrations in period 1 (P=0.025), and there was an overall treatment × time interaction (P=0.048) for a decrease in the plasma NEFA concentration (Fig. 2). Blood plasma urea concentrations were also affected by glycerol administration; the treatment cows had lower values than the control cows (P=0.005). 4. Discussion Prolonged oral drenching of crude glycerol before morning feeding to primiparous cows increased total DMI during the first three weeks of the post partum period. As was also found with a similar amount of glycerol mixed to TMR fed in early lactation to multiparous cows (Bodarski et al., 2005). Several other studies with multiparous cows have reported either no stimulative (DeFrain et al., 2004; Chung et al., 2007; Donkin et al., 2009; Wang et al., 2009) or depressive (Ogborn, 2006; Osborne et al., 2009) effects of glycerol on appetite in early lactation. Some authors (Chung et al., 2007; Wang et al., 2009) have assumed that these different outcomes could be related to the impurity of the product. Crude glycerol contains water, methanol and salts (Thompson and He, 2006), which might limit DMI. Although, even impure forms of glycerol mixed into the diet can be fed to dairy cows up to very high levels (10% DM) (Schröder and Südekum, 1999; Kass et al., 2012). Moreover, Werner Omazic et al. (2011) showed that refined and crude glycerol fed for four weeks post partum did not depress silage or total DMI compared to control group cows. These results accord with the findings of the current study, showing no adverse effect of unrefined glycerol on intake of diet components. Although, it is difficult to generalise the effect of glycerol on feed Please cite this article in press as: Kass, M., et al., Long-term oral drenching of crude glycerol to primiparous dairy cows in early lactation. Anim. Feed Sci. Tech. (2013), http://dx.doi.org/10.1016/j.anifeedsci.2013.06.004

G Model ANIFEE-12922; No. of Pages 9

ARTICLE IN PRESS M. Kass et al. / Animal Feed Science and Technology xxx (2013) xxx–xxx

7

intake, the results of the present study are supported by other studies (Bodarski et al., 2005; Werner Omazic et al., 2011), which observed a positive effect of feeding larger amounts of glycerol over a longer delivery period. This may be as a result of the ruminal environment adapting to glycerol feeding with time and therefore the deleterious effect of disturbance of the ruminal environment is mitigated by the supply of a glucogenic precursor. In addition to dissimilarities associated with the method of feeding glycerol (administration, quantity, delivery period, purity etc.), there might be other issues, which might affect the feed intake such as an elevated concentration of plasma NEFA (Ingvartsen and Andersen, 2000). The increase in DMI had a consequential effect on milk yield. Earlier studies with the short-term drenching of glycerol have shown contrary results. Ogborn (2006) detected no effect on milk yield when drenching 500 ml/d of glycerol for the first five DIM. Similar findings to that were reported when either drenching or feeding 300 ml/d top dressed with TMR around parturition (Kupczynski et al., 2012). Although, Goff and Horst (2001) observed that administrating large volumes (up to 3 l/d) of glycerol increased milk yield 1.8–2.7 kg. Incorporation of a relatively similar amount of glycerol, as in the current study, into the TMR has been shown to have a positive effect on milk yield during the first three weeks of lactation (Bodarski et al., 2005; Lomander et al., 2012a). However, the response of milk yield to glycerol when it is either orally administrated or mixed to the TMR remains unclear. Milk lactose content was affected by the treatment, and there was a treatment x day interaction for an increase in the lactose content of the milk. The higher milk lactose content could have been driven by the fate of glycerol in the rumen. Glycerol is largely fermented to propionate or directly absorbed, and to a lesser extent escapes rumen fermentation (Rémond et al., 1993). It could therefore increase the availability of glucose for the animal to synthesise additional lactose in the mammary gland. Indeed, the higher milk yield of treatment cows in the current study could have been driven by osmotic activity of lactose. The milk urea content tended to decrease with oral glycerol treatment, which is in agreement with DeFrain et al. (2004). It is possible that glycerol supplies more energy to the rumen microorganisms, therefore they synthesise more microbial protein, and the N available in the rumen will be used more efficiently. Moreover, supplemental glycerol decreased blood plasma concentrations of urea in the current study. Osman et al. (2008) reported a similar decrease in blood urea concentrations during the first 4 h post-drenching, on DIM 7 and 14. As this effect did not carry over the 8-h period post drenching (Osman et al., 2008), it is difficult to compare with our results where blood samples were collected before the morning feeding. It is important to consider that blood urea concentration is related to its sampling time, as peak values occur several hours after feeding (Gustafsson and Palmquist, 1993). To evaluate the negative energy balance several indirect indicators were used: the blood metabolites NEFA and BHB (Herdt, 2000), body condition score (Edmonson et al., 1989) and milk composition (Heuer et al., 1999). In this study the mean BCS of cows before calving was 3.46, which is in accord with the range of optimal condition score for pre partum primiparous Estonian Holstein cows (Samarütel et al., 2006). Glycerol administration had no affect on mean BCS, nor on BCS changes over the experimental period. The results for BCS losses of 0.39 indicate moderate lipid mobilisation and negative energy balance in both groups. Recent other studies are in agreement, that glycerol feeding either in the transition period (DeFrain et al., 2004; Osborne et al., 2009; Carvalho et al., 2011) or in early lactation (Chung et al., 2007; Osman et al., 2010) has no significant effect on BCS. It is considered that BCS measurements, as indirect indicators of energy balance, were probably not sensitive enough to evaluate energy deficiency in such a short period (Samarütel et al., 2008). Heuer et al. (1999) have also pointed out that the first test day milk yield and the milk fat to protein ratio were more reliable indicators of disease, fertility, and milk yield than was BCS loss. Milk acetone content has been related to enhanced ketogenesis and enhanced fat mobilisation (Holtenius and Holtenius, 1996), but no treatment effect on milk acetone was found in the current study. Reist et al. (2002) found that fat/lactose ratio and fat/protein ratio are more precise measures than the milk acetone content in predicting energy balance in early lactation. In the current study, neither of these ratios of milk components were affected by glycerol administration, but the mean milk fat/protein ratios were over 1.5, indicating an energy deficiency (Pehrson, 1996; Heuer et al., 1999) in both groups. With the exception of blood plasma urea and NEFA levels, there was no effect of treatment on the concentrations of blood plasma metabolites including insulin. This is in agreement with Ogborn (2006) who found that short-term oral drenching had no effect on concentrations of blood glucose or BHB. However, several studies have indicated that orally administrated glycerol can be effective in elevating blood plasma glucose levels (Osman et al., 2010), where it stays elevated for 8 h postdrenching, returning to baseline values within 24 h (Goff and Horst, 2001; Linke et al., 2004) which was the timeframe from glycerol drenching to blood sampling in the current study. However, glycerol added to drinking water (Osborne et al., 2009) or mixed into TMR (DeFrain et al., 2004; Carvalho et al., 2011) tended to decrease blood glucose levels in the post partum period. These findings could be related to the fact that oral administration of glycerol is more effective to elevated glucose levels in blood compared to adding it to feed (Linke et al., 2004). Supplemental glycerol over a long period post partum had no affect on blood insulin concentrations in early lactation, which is consistent with previous reports (DeFrain et al., 2004; Chung et al., 2007; Osman et al., 2008, 2010). Similarly to monitoring glucose, the time of blood sampling could play an important role in determining the affect on blood insulin concentrations of glycerol administration method, and this might explain the contrary findings of Linke et al. (2004). An interaction of treatment and time on blood NEFA concentration was detected in the current study. In the first six-day period after calving plasma NEFA concentration in the treatment group was significantly lower than in the control group. The decline on NEFA levels for the control group was deeper over all three periods compared to the treatment cows. DeFrain et al. (2004) found a similar decrease in blood NEFA concentrations on day seven in early lactation. Osman et al. (2010) Please cite this article in press as: Kass, M., et al., Long-term oral drenching of crude glycerol to primiparous dairy cows in early lactation. Anim. Feed Sci. Tech. (2013), http://dx.doi.org/10.1016/j.anifeedsci.2013.06.004

G Model ANIFEE-12922; No. of Pages 9

ARTICLE IN PRESS M. Kass et al. / Animal Feed Science and Technology xxx (2013) xxx–xxx

8

have also reported that 400 ml of oral glycerol, tended to decrease blood NEFA concentration, and a linear decrease in NEFA concentrations was found with increasing glycerol in the TMR (Wang et al., 2009). However, in the current study, the effect of glycerol administration on reducing the NEFA concentration remains unclear. 5. Conclusion Supplemental oral drenching of 500 ml of crude glycerol to primiparous Holstein dairy cows up to 21 days in lactation improved silage intake and total DMI. Long-term crude glycerol drenching had a positive effect on milk yield and milk lactose content. NEFA concentrations during the first experimental period were lower in glycerol group, but during the whole experiment its concentration declined extensively for control cows. The energy status, estimated from the other indirect measures, showed no effect of the orally administrated glycerol. Based on these results it is concluded that longterm oral drenching with crude glycerol of primiparous cows postcalving could be an acceptable additional energy source for dairy cows in early lactation. Conflict of interest None. Acknowledgement We are grateful to Mrs Ülle Lätt and Mr Siim Suitsmart for technical assistance and to other colleagues for fruitful discussions. We also thank the staff of Eerika Experimental Farm for their cooperation. The research leading to these results was co-financed by the European Community’s Regional Development Fund within the framework of the Competence Centre Programme of Enterprise Estonia (Lasnamäe 2, Tallinn 11412, Estonia) under project no. EU30002 carried out by the Bio-Competence Centre of Healthy Dairy Products (Kreutzwaldi 1, Tartu 51014, Estonia). References AOAC, 2005. Official Methods of Analysis of AOAC International, 18th ed. Association of Official Analytical Chemists International, Gaithersburg, MD, USA. Bodarski, R., Wertelecki, T., Bommer, F., Gosiewski, S., 2005. The changes of metabolic status and lactation performance in dairy cows under feeding TMR with glycerin (glycerol) supplement at periparturient period. Electron. J. Pol. Agric. Univ. Anim. Husb. 8 (4), 22–30. Carvalho, E.R., Schmelz-Roberts, N.S., White, H.M., Doane, P.H., Donkin, S.S., 2011. Replacing corn with glycerol in diets for transition dairy cows. J. Dairy Sci. 94, 908–916. Chung, Y.-H., Rico, D.E., Martinez, C.M., Cassidy, T.W., Noirot, V., Ames, A., Varga, G.A., 2007. Effects of feeding dry glycerin to early postpartum Holstein dairy cows on lactational performance and metabolic profiles. J. Dairy Sci. 90, 5682–5691. DeFrain, J.M., Hippen, A.R., Kalscheur, K.F., Jardon, P.W., 2004. Feeding glycerol to transition dairy cows: effects on blood metabolites and lactation performance. J. Dairy Sci. 87, 4195–4206. Donkin, S.S., Koser, S.L., White, H.M., Doane, P.H., Cecava, M.J., 2009. Feeding value of glycerol as a replacement for corn grain in rations fed to lactating dairy cows. J. Dairy Sci. 92, 5111–5119. Edmonson, A.J., Lean, I.J., Weaver, L.D., Farver, T., Webster, G., 1989. A body condition scoring chart for Holstein dairy cows. J. Dairy Sci. 72, 68–78. Fisher, L.J., Erfle, J.D., Sauer, F.D., 1971. Preliminary evaluation of the addition of glucogenic materials to the rations of lactating cows. Can. J. Anim. Sci. 51, 721–727. Friggens, N.C., Berg, P., Theilgaard, P., Korsgaard, I.R., Ingvartsen, K.L., Løvendahl, P., Jensen, J., 2007. Breed and parity effects on energy balance profile through lactation: evidence of genetically driven body energy change. J. Dairy Sci. 90, 5291–5305. Goff, J.P., Horst, R.L., 2001. Oral glycerol as an aid treatment of ketosis/fatty liver complex. J. Dairy Sci. 84 (Suppl. (1)), 153 (abstract). Grummer, R.R., 1993. Etiology of lipid-related metabolic disorders in periparturient dairy cows. J. Dairy Sci. 76, 3882–3896. Gustafsson, A.H., Palmquist, D.L., 1993. Diurnal variation of rumen ammonia, serum urea, and milk urea in dairy cows at high and low yields. J. Dairy Sci. 76, 475–484. Heuer, C., Schukken, Y.H., Dobbelaar, P., 1999. Postpartum body condition score and results from the first test day milk as predictors of disease, fertility, yield, and culling in commercial dairy herds. J. Dairy Sci. 82, 295–304. Herdt, T.H., 2000. Variability characteristics and test selection in herd-level nutritional and metabolic profile testing. Vet. Clin. North Am. Food Anim. Pract. 16, 387–403. Holtenius, P., Holtenius, K., 1996. New aspects of ketone bodies in energy metabolism of dairy cows: a review. J. Vet. Med. A. 43, 579–587. Ingvartsen, K.L., 2006. Feeding- and management-related diseases in the transition cow: physiological adaptations around calving and strategies to reduce feeding-related diseases. Anim. Feed Sci. Technol. 126, 175–213. Ingvartsen, K.L., Andersen, J.B., 2000. Integration of metabolism and intake regulation: a review focusing on periparturient animals. J. Dairy Sci. 83, 1573–1597. Johnson, R.B., 1951. New methods of treating ketosis. a preliminary report. North Am Vet. 32, 327–332. Kass, M., Ariko, T., Kaart, T., Rihma, E., Ots, M., Arney, D., Kärt, O., 2012. Effect of replacement of barley meal with crude glycerol on lactation performance of primiparous dairy cows fed a grass silage-based diet. Livest. Sci. 150, 240–247. Khalili, H., Varvikko, T., Toivonen, V., Hissa, K., Suvitie, M., 1997. The effects of added glycerol or unprotected free fatty acids or a combination of the two on silage intake, milk production, rumen fermentation and diet digestibility in cows given grass silage based diets. Agric. Food Sci. Finland 6, 349–362. Kupczynski, R., Bodarski, R., Janeczek, W., Kuczaj, M., 2012. Effect of glycerol and propylene glycol supplementation on metabolic parameters and production performance in periparturient period of dairy cows. Agric. J. 7, 88–94. Linke, P.L., DeFrain, J.M., Hippen, A.R., Jardon, P.W., 2004. Ruminal and plasma responses in dairy cows to drenching or feeding glycerol. J. Dairy Sci. 87 (Suppl. 1), 343 (Abstract). Lomander, H., Frössling, J., Ingvartsen, K.L., Gustafsson, H., Svensson, C., 2012a. Supplemental feeding with glycerol or propylene glycol of dairy cows in early lactation – Effects on metabolic status, body condition, and milk yield. J. Dairy Sci. 95, 2397–2408. Lomander, H., Gustafsson, H., Frössling, J., Ingvarsten, K.L., Larsen, T., Svensson, C., 2012b. Effect of supplemental feeding with glycerol or propylene glycol in early lactation on the fertility of Swedish dairy cows. Reprod. Dom. Anim. 47, 988–994. National Research Council, 2001. Nutrient Requirements of Dairy Cattle, 7th ed. National Academy Press, Washington, DC, USA.

Please cite this article in press as: Kass, M., et al., Long-term oral drenching of crude glycerol to primiparous dairy cows in early lactation. Anim. Feed Sci. Tech. (2013), http://dx.doi.org/10.1016/j.anifeedsci.2013.06.004

G Model ANIFEE-12922; No. of Pages 9

ARTICLE IN PRESS M. Kass et al. / Animal Feed Science and Technology xxx (2013) xxx–xxx

9

Ogborn, K.L., 2006. Effects of Method of Delivery of Glycerol on Performance and Metabolism of Dairy Cows during the Transition Period. Cornell Univ., Ithaca, New York, USA (MS Thesis). Oll, Ü., 1995. Feeding Requirements for Livestock with Feed Tables. Tartu, Estonia., pp. 186 (in Estonian). Osborne, V.R., Odongo, N.E., Cant, J.P., Swanson, K.C., McBride, B.W., 2009. Effects of supplementing glycerol and soybean oil in drinking water on feed and water intake, energy balance, and production performance of periparturient dairy cows. J. Dairy Sci. 92, 698–707. Osman, M.A., Allen, P.S., Mehyar, N.A., Bobe, G., Coetzee, J.F., Koehler, K.J., Beitz, D.C., 2008. Acute metabolic responses of postpartal dairy cows to subcutaneous glucagon injections, oral glycerol, or both. J. Dairy Sci. 91, 3311–3322. Osman, M.A., Allen, P.S., Bobe, G., Coetzee, J.F., Abuzaid, A., Koehler, K.J., Beitz, D.C., 2010. Chronic metabolic responses of postpartal dairy cows to subcutaneous glucagon injections, oral glycerol, or both. J. Dairy Sci. 93, 3505–3512. Overton, T.R., Waldron, M.R., 2004. Nutritional management of transition dairy cows: strategies to optimize metabolic health. J. Dairy Sci. 87 (Suppl.), E105–E119. Pehrson, B., 1996. Milk analysis as an indicator of the nutritional and disease status of dairy cows. In: Garnsworthy, P.C., Wiseman, J., Haresign, W. (Eds.), Recent Advances in Animal Nutrition 1996. Nottingham University Press, Nottingham, UK, pp. 113–133. Reist, M., Erdin, D., von Euw, D., Tschuemperlin, K., Leuenberger, H., Chilliard, Y., Hammon, H.M., Morel, C., Philipona, C., Zbinden, Y., Kuenzi, N., Blum, J.W., 2002. Estimation of energy balance at the individual and herd level using blood and milk traits in high-yielding dairy cows. J. Dairy Sci. 85, 3314–3327. Rémond, B., Souday, E., Jouany, J.P., 1993. In vitro and in vivo fermentation of glycerol by rumen microbes. Anim. Feed Sci. Technol. 41, 121–132. Samarütel, J., Ling, K., Jaakson, H., Kaart, T., Kärt, O., 2006. Effect of body condition score at parturition on the production performance, fertility and culling in primiparous Estonian Holstein cows. Vet. Med. Zoot. 36, 69–74. Samarütel, J., Waldmann, A., Ling, K., Jaakson, H., Kaart, T., Leesmäe, A., Kärt, O., 2008. Relationships between luteal activity, fertility, blood metabolites and body condition score in multiparous Estonian Holstein dairy cows under different management. J. Dairy Res. 75, 485–490. Institute, S.A.S., 2003. SAS Online DOC. Version 9.1. SAS Institute Inc, Cary, NC. Schröder, A., Südekum, K.-H., 1999. Glycerol as a by-product of biodiesel production in diets for ruminant. In: Wratten, N., Salisbury, P.A. (Eds.), New Horizons for an Old Crop. Proceeding of 10th International Rapeseed Congress. Canberra, Australia. The Regional Institute Ltd, Gosford, New South Wales, Australia (paper no 241). Thompson, J.C., He, B.B., 2006. Characterization of crude glycerol from biodiesel production from multiple feedstocks. Appl. Eng. Agric. 22, 261–265. Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber and non-starch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583–3597. Wang, C., Liu, Q., Yang, W.Z., Huo, W.J., Dong, K.H., Huang, Y.X., Yang, X.M., He, D.C., 2009. Effects of glycerol on lactation performance, energy balance and metabolites in early lactation Holstein dairy cows. Anim. Feed Sci. Technol. 151, 12–20. Werner Omazic, A., Bertilsson, J., Tråvén, M., Holtenius, K.,2011. Crude vs. refined glycerol supplementation of dairy cows in early lactation – effects on dry matter intake, lactation performance and metabolism. Advances in Animal Biosciences. In: Proceedings of the 8th International Symposium on the Nutrition of Herbivores (ISNH 8), Vol. 2. Cambridge University Press, p. pp424.

Please cite this article in press as: Kass, M., et al., Long-term oral drenching of crude glycerol to primiparous dairy cows in early lactation. Anim. Feed Sci. Tech. (2013), http://dx.doi.org/10.1016/j.anifeedsci.2013.06.004