Variation of starch and fat in the diet affects metabolic status and oxidative stress in ewes

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Small Ruminant Research 74 (2008) 123–129

Variation of starch and fat in the diet affects metabolic status and oxidative stress in ewes S. Sgorlon a , G. Stradaioli a , G. Gabai b , B. Stefanon a,∗ b

a Dipartimento di Scienze Animali, via delle Scienze, 208-33100 Udine, Italy Dipartimento di Scienze Sperimentali Veterinarie, Complesso Agripolis v.le dell’Universit`a, 16-35020 Legnaro (Padova), Italy

Received 6 February 2007; received in revised form 2 April 2007; accepted 12 April 2007 Available online 29 May 2007

Abstract The effect of a rapid variation of dietary starch and fat on metabolic and oxidative status in lactating ewes was investigated. Fifteen sheep at the end of pregnancy were assigned to three experimental groups and fed until 32 days from lambing a complete diet (22% starch and 3% fat on DM basis). At day 33, a group of ewes continued to receive the control diet (CTR), while the HST (high starch) group received a complete diet with 28% starch and 2.6% fat, and the HFA (high fat) group a diet with 16% starch and 5.4% fat, the diets were formulated to be iso-energetic. Blood and milk samples were collected before the change of diets (20 days), just after (35 days) and after 50 days from lambing. The HFA diet lead to a higher milk fat content compared to the CTR and HST diets, but the average daily gain of lambs during the trial was similar. The inclusion of starch in the diet increased blood glucose, insulin and lactate and reduced ␤-hydroxybutyrate (BHBA) and pH, while HFA reduced lactate and increased pH. Metabolic changes were associated with variations of markers of oxidative stress at 50 days. Total glutathione (GSx) decreased in the CTR and HST groups, while increased in HFA. Superoxide dismutase (SOD) activity in erythrocytes was higher only for the ewes fed HFA diet. Present data indicate that rapid modification of diet composition affects metabolic and oxidative homeostasis in lactating sheep. © 2007 Elsevier B.V. All rights reserved. Keywords: Ewe; Lactation; Nutrient; Metabolic status; Oxidative status

1. Introduction The imbalance between oxidants and antioxidants in favour of the oxidants is defined “oxidative stress”, a term that describes a metabolic condition of cells, organs, or the entire organism characterized by an oxidative overload. Depending on the pathway of generation of the major reactive oxygen species (ROS) formed, the phenomenon of oxidative stress can be subspecified,

∗ Corresponding author. Tel.: +39 0432 558580; fax: +39 0432 558585. E-mail address: [email protected] (S. Sgorlon).

0921-4488/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2007.04.004

e.g. metabolic oxidative stress, environmental oxidative stress, photooxidative stress, drug-dependent oxidative stress, or nitrosative stress (Sies et al., 2005). The metabolic oxidative stress is related to the mitochondrial activity, because O2 consumption is not perfectly coupled to ATP production (Lopez-Lluch et al., 2006). There is a large body of evidence reporting that caloric restriction reduces ROS production, health risks and delays the onset of most age related diseases by decreasing mitochondrial proton leak (Beckman and Ames, 1998; Weindruch et al., 2001). In high yielding dairy cows, metabolic drive for milk is associated with an increased production of free radicals and ROS (Stefanon et al., 2005; Castillo et al., 2005),

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that can be exacerbated by pathological (Zecconi et al., 2003; Kankofer, 2002) or unfavourable ambient conditions (Gabai et al., 2004; Andres et al., 1999). Moreover, to satisfy the increase of nutrient requirements for the onset of lactation, diet is modified by feeding energydense or fat-supplemented rations. The inclusion of fat in the diet is also a feeding strategy for lactating ewes to increase net energy intake and to modify milk fat composition (Mele et al., 2005). The hypothesis we tested was that energy substrates can modulate metabolic status and antioxidant defences of animal. More specifically, the objective of the present experiment was to study the effect of a substitution of starch and fat contents in the diet, without varying energy intake, on metabolic homeostasis and oxidative balance in the ewe during the lactation period. 2. Materials and methods 2.1. Animals and diets The experimental protocol was approved by state, and local laws and regulations. Twenty-four pluriparous Bergamasca sheep were selected from a flock based on age and live weights and mated in late summer within one week. Until 140 days of pregnancy, sheep were fed a hay and concentrate diet according to the requirements (Van Soest and Fox, 1992) and from 141 days until lambing they received 2 kg/d head (as-fed basis) of a control (CTR) complete pelletted diet (Table 1), which was formulated to cover the nutrient requirements for lactation. At lambing, ewes with single lamb were selected and experimental groups were formed to pair live weight and dates of lambing, which occurred in an interval of 12 days. The final number of ewes used for the experiment was 15, 12 of them with male and 3 with female lambs. Animals were allotted to three experimental groups and kept in three separated boxes (five sheep for group, four with male and one with female lamb). The amount of complete diet offered was increased to 3.25 kg/d head (as-fed basis) at 10 days of lactation and remained constant until 32 days after lambing. At day 33, the amount of pellets administered increased to 3.5 kg/d head (as-fed basis), and kept constant until the end of the trial. A group of ewes (CTR, 22% starch and 3.0% fat) continued to receive the same pellet, while the other two groups received either a diet with high starch (HST group; 28% starch and 2.6% fat) or a diet with high fat (HFA group; 16% starch and 5.4% fat) content, the diets were formulated to be isocaloric (Table 1). The change from CTR to HST or HFA diets was accomplished in 2 days (days 31 and 32); from day 33 until day 50 after lambing, ewes continued to receive the HST and HFA diets. The distribution of the ration was restricted and controlled to avoid differences in individual intakes within and between groups. During the whole experimental period, pellets were distributed at 9.00 and 16.00 h every day and ani-

Table 1 Ingredients, chemical composition and nutritive value of experimental diets Control Ingredients (%) Corn Alfalfa, dehy Sugar beet pulp, dehy Soybean S.E., 44% Corn gluten feed Soya hulls Palm oil Calcium carbonate Urea Min. Vit. supplement Molasses Lignosulfite Total Chemical composition DM (%) OM (% DM) CP (% DM) Fat (% DM) NEL (Mcal/kg DM) NDF (% DM) Starch (% DM)

High starch

High fat

32.81 30.00 5.60 9.60 9.10 5.00 0.31 0.28 0.00 4.10 3.00 0.20

42.90 30.00 1.20 11.67 1.40 4.59 0.00 0.44 0.50 4.10 3.00 0.20

24.27 35.35 8.22 11.21 5.55 5.00 2.64 0.36 0.10 4.10 3.00 0.20

100.00

100.00

100.00

88.50 90.10 13.80 3.00 1.43 27.00 22.00

88.20 90.10 15.00 2.60 1.45 24.00 28.00

89.10 89.50 14.40 5.40 1.46 29.00 16.00

mal were trained to consume the complete diet within 2 h. To avoid feed consumption by the lambs, they were isolated in a separated box for the following 2 h. Live weight of ewes and lambs was recorded at lambing and at the end of the experiment. 2.2. Blood and milk samples collection and preparation Blood and milk samples were collected for three consecutive days just prior to feed distribution at days 20, 21 and 22 (sample 20 days, before the change of diets), 35, 36 and 37 (sample 35 days) and 50, 51 and 52 (sample 50 days) from lambing. Milk samples were manually collected at 7.00 a.m. and, to avoid suckling, lambs were separated the day before sampling from the ewes at 8.00 p.m. Samples were immediately analysed for fat, protein, lactose and solid not fat (SNF). Blood samples were collected by jugular venipuncture into tubes containing both Li-heparin and K3 -EDTA as anticoagulants or without anticoagulant. Blood for gas analysis was collected in a heparinized syringe (Prov-Vent Plus, Milan, Italy), preserved at 4 ◦ C and analysed within 2 h from the collection. Aliquots of whole blood were stored at −80 ◦ C for the determination of glutathione peroxidase (GSH-px) activity and hemoglobin (Hb) concentrations. For superoxide dismutase (SOD) analysis, erythrocytes were separated from plasma and leucocytes buffy-coat by centrifugation at 1500 × g at 4 ◦ C for 10 min. The erythrocytes were then washed

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three times with 0.9% NaCl and kept frozen until analysis (−80 ◦ C). For total glutathione (GSx) determination, 100 ␮L of whole blood were added to 500 ␮L of 5% sulfosalicylic acid, mixed and centrifuged at 1750 × g at 4 ◦ C for 10 min to eliminate Hb and other proteins; the supernatant was kept at 4 ◦ C and analysed within the next 6 h. Blood samples were centrifuged (1500 × g/10 min) within 1 h from collection, and serum stored at −80 ◦ C until analyses for glucose, insulin, ␤-hydroxybutyrate (BHBA), nonesterified fatty acids (NEFA) and lactate. 2.3. Laboratory analyses Unless otherwise indicated, all chemicals employed for both GSx and SOD analysis were obtained from Sigma–Aldrich (Milan, Italy). Superoxide dismutase activity was determined by a modification of ferricytochrome c reduction assay proposed by Floh´e and Otting (1984), adapted for microplate determination (Genios, Tecan Austria GmbH, Gr¨odig, Austria) with a filter set at 550 nm (10 nm bandwidth). Total glutathione in blood plasma was determined by an enzymatic recycling method originally described by Tietze (1969). Blood concentration of hemoglobin was measured using Drabkin reagent kit 525-A (Sigma–Aldrich, Milan, Italy) by the method described by Drabkin and Austin (1935). GSH-px activity was measured with a commercial kit (RANSEL, Randox Laboratories Ltd., Crumlin, UK), using a microtitre plate reader. The method is based on the oxidation of glutathione catalysed by glutathione peroxidase in the presence of cumene hydroperoxide (Paglia and Valentine, 1967). Serum samples were analysed using a BM/Hitachi 911 analyser (Roche Boehringer Mannheim Gmbh, Mannheim, Germany) for NEFA (NEFA, Randox Laboratories Ltd., Crumlin, UK) glucose (Cat. No. 1448668, Boehringer Mannheim, Germany), lactate (Cat. No. 11112821035, Boehringer Mannheim, Germany) and BHBA (BHBA reagent, procedure no. 310-UV, Sigma–Aldrich, Milan, Italy). The radioimmunoassay technique was employed for analysis of insulin using a commercial kit (Coat-a-Count insulin, Diagnostic Products Corporation, Los Angeles, USA). The detection limit of the assay was 0.95 ± 0.09 ␮U/mL. The coefficients of variation were 7.0% within assay and 8.1% between assays. Blood gas, electrolytes and pH were measured using a blood gas analyser (Synthesis 20 IL, Instrumentation Laboratories, Instru-Med Inc., Atlanta, USA), and the base excess in the extracellular fluid (BE-ECF) was then calculated as the amount of acid (in mEq/L) that would have to be added to the animal’s blood to bring it to normal pH of 7.4. Fat, protein, lactose and SNF were analysed according to a mid infra-red method employing the instrument Milkoscan 134 A/B (Foss Electric, Hillerod, Denmark), that was calibrated with the procedure of AOAC (AOAC, 1990; methods nos. 905.02, 920.105, and 984.15 for fat, protein, and lactose, respectively).

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2.4. Statistical analysis A mixed model analysis was applied to production, blood and milk data according to the model: Yijk = μ + Ti + Pj + Dk + (T × P)ij + εijk where Yijk is the response variable, μ the overall mean of the population, Ti the mean of dietary treatment (i = 1–3), Pj the mean effect of sampling (j = 1–3) with sampling as a repeated factor, Dk the fixed effect for day of sampling (k = 1–3), (T × P)ij the interaction treatment and sampling and εijk is the unexplained residual element assumed to be independent and normally distributed. Data computation was performed using the repeated measure statement of the SPSS (1997), using sheep within treatment as error term and type III sum of square. To test partial interaction of sampling between diets, simple contrasts were used, comparing 50 days and 35 days versus 20 days sampling times.

3. Results During the experimental period animals ingested all the complete diets offered. The daily amount of starch ingested with experimental diets from day 2 to 32 was 0.63 kg/head and from day 33 to 51 was 0.68, 0.87 and 0.50 kg/head, for CTR, HST and HFA groups respectively. Daily fat intakes varied from 0.10 kg/head in the first period (days 2–32) to 0.11, 0.08 and 0.17 for CTR, HST and HFA groups from day 33 to 51 respectively. The NEL intake increased from the first to the second period, but there was no difference between the treatment groups. The mean (±S.E.) live weight of sheep at lambing was 79.7 ± 2.10 for CTR, 77.3 ± 2.48 for HFA and 80.0 ± 3.28 for HST and did not change during the trial (80.7 ± 1.93 for CTR, 79.3 ± 3.12 for HFA and 79.8 ± 3.20 for HST, at day 51). Milk fat was affected (P < 0.01) by the changes of diet and was higher in HFA group (6.25%) compared to the other experimental groups (5.32 and 5.24% for CTR and HST respectively). No differences were shown between groups for protein, lactose and SNF contents. The average daily gain of lambs (Fig. 1) did not show significant differences between treatments. Blood glucose increased at 35 days in all the experimental groups (P < 0.05) and the variation was more pronounced for HST group (Table 2). The test of partial interaction, solved by the application of contrast analysis, confirmed a different trend during the experiment (35 days versus 20 days, P < 0.05) between CTR and the other diets, underlining the effect of the increased content of starch in the diet (P < 0.01). A rise of insulin concentration was observed immediately after the change of basal diet (35 days), while at 50 days the concentrations

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Table 2 Insulin and metabolic variables measured on jugular blood sampled from ewes fed with control diet (CTR), high starch diet (HST) and high fat diet (HFA) before (20 days) and after the change of the rations DIM (days)

Glucose (mg/100 mL)

Insulin (␮U/mL)

BHBA (mg/100 mL)

NEFA (mEq/L)

Lactate (mg/100 mL)

Diet

Error MSa

CTR

HST

HFA

20 35 50

64.20 64.93 b 62.40

68.33 82.53 a 72.07

66.27 69.87 b 63.27

20 35 50

6.19 7.41 B 6.38 B

6.58 10.53 A 8.22 A

20 35 50

0.440 0.515 A 0.480 A

20 35 50 20 35 50

1.050

Diet

Sampling

D × Sb

Contrast diet × sampling 35 days vs. 20 days

50 days vs. 20 days

**

**

*

*

NS

6.77 9.46 A 0.124 7.33 AB

***

***

***

**

**

0.474 0.428 B 0.389 B

0.437 0.433 B 0.405 B

0.004

***

**

***

**

**

0.206 0.213 0.098 a

0.192 0.212 0.045 b

0.190 0.190 0.086 a

0.003

*

***

**

NS

*

4.15 4.26 B 4.66 B

4.27 5.53 A 5.70 A

4.26 2.57 C 3.68 C

0.110

***

**

***

***

***

Means within a row with different letters (a, b, c) differ (P < 0.05). Means within a row with different letters (A, B, C) differ (P < 0.01). *P < 0.05; **P < 0.01; ***P < 0.001. a Error MS, mean square error. b D × S, diet × sampling interaction.

of the hormone in all the experimental groups decreased (P < 0.001). Also in this case, contrast analysis underlined the effect of the interaction between diets and time of sampling, with insulin concentration being higher in HST and HFA groups than in CTR group at 35 days (P < 0.01). The concentration of BHBA linearly decreased for the HST and HFA groups during the trial, while in CTR group a transient increase at 35 days was shown

Fig. 1. Average daily gains of lambs of ewes fed with control diet (CTR), high starch diet (HST) and high fat diet (HFA) during the periods 0–21, 22–35 and 36–50 days from lambing.

(35 days versus 20 days and 50 days versus 20 days, P < 0.01). NEFA were significantly lower at 50 days in all the groups (P < 0.001), but the variation was higher for the HST group, as assessed by the contrast analysis (P < 0.05). Lactate concentration was affected by diet (P < 0.001) and time of sampling (P < 0.01). Sheep receiving the CTR and HST treatments showed an increase of lactate during the experimental period; instead, the change of diet from the basal to HFA leaded to a sharp decrease (contrast P < 0.001). Blood pH was influenced by diet (P < 0.05) and interaction with sampling time (P < 0.01) (Table 3); HST caused a decrease and HFA an increase of pH values both at 35 days (P < 0.05) and 50 days (P < 0.01). BEECF at 35 days remained constant in the CTR group while it increased in the HFA group and decreased in the HST group (P < 0.05). Total dissolved carbon dioxide (TCO2 ) was influenced by diet (P < 0.01) and also changed with time of sampling (P < 0.05). TCO2 at 50 days was lower for HST and higher for HFA (P < 0.01) in comparison to 20 days.

S. Sgorlon et al. / Small Ruminant Research 74 (2008) 123–129

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Table 3 Gas analysis and markers of oxidative stress measured on jugular blood sampled from ewes of CTR, HST and HFA groups before (20 days) and after the change of the rations DIM (days)

Diet CTR

pH

BE-ECF (mmol/L)

TCO2 (mmol/L)

SOD (U 37 ◦ C/L)

GSx (␮mol/L)

GSH-px (U/g Hb)

Error MSa HST

Diet

Sampling

D × Sb

HFA

Contrast diet × sampling 35 days vs. 20 days

50 days vs. 2 days

20 35 50

7.452 7.456 ab 7.441 AB

7.441 7.406 b 7.384 B

7.437 7.486 a 7.499 A

0.009

*

NS

**

*

**

20 35 50

5.44 5.46 b 4.34

5.26 3.74 c 1.50

5.18 9.24 a 6.08

0.515

NS

*

NS

*

NS

20 35 50

30.57 31.13 29.59 B

31.77 29.94 25.74 C

30.99 34.65 33.24 A

0.371

**

*

**

NS

**

20 35 50

31.88 32.20 34.36 b

33.28 35.30 37.84 b

34.64 37.85 45.90 a

1.090

NS

***

*

NS

*

20 35 50

896.40 824.40 784.80 B

865.80 745.20 673.20 C

892.80 820.80 918.00 A

16.192

*

**

**

NS

**

20 35 50

177.54 177.12 199.34

176.91 174.09 207.86

182.95 197.88 223.58

NS

***

NS

NS

NS

5.317

Means within a row with different letters (a, b, c) differ (P < 0.05). Means within a row with different letters (A, B, C) differ (P < 0.01). *P < 0.05; **P < 0.01; ***P < 0.001. a Error MS, mean square error. b D × S, diet × sampling interaction.

The activity of SOD in erythrocytes increased with DIM (P < 0.001) and at 50 days the values were higher than in the other periods (P < 0.001). Although SOD was not affected by diet, contrast 50 days versus 20 days evidenced a higher increase in the HFA compared to the other two groups (P < 0.05). GSx concentrations significantly (P < 0.01) decreased with time of sampling, but a different pattern was shown for HFA group at 50 days (P < 0.01). The GSH-px activity increased (P < 0.001) with DIM irrespectively of the diet. 4. Discussion Experimental diets were formulated to be isoenergetic (Table 1). The length of lactation of Bergamasca sheep is about 3 months (Colitti et al., 2000) and the time of dietary change was selected to avoid the stress condition related to peripartum, meanwhile having animals still in full lactation.

The effect of fat-supplemented diet on milk yield is controversial. In goats, high fat diets cause a sharp increase of milk fat percentage and have smaller effect on milk yield (Chilliard et al., 2003). In sheep, a positive (Rossi et al., 1991) and negative (Horton et al., 1989) effect of dietary fat on milk yield is reported, but milk fat content is always improved. Fat inclusion in the diet consisted in a significant increase (P < 0.01) of fat content in the milk, while the growth rate of lambs was not affected (Fig. 1). Body weight and body weight changes of sheep did not significantly differ between groups, as the final weight of lambs (data not reported). These findings do not allow to ascertain if a relevant variation of milk yield would have been caused by the dietary changes. The inclusion of some fats at high dietary concentrations may interfere with rumen microbial fermentation, and may lead to a reduction of VFA production or a variation of acetate to propionate ratio (Micek et

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al., 2004). However, this effect has not always been reported, depending upon the quality and amount of lipids included (Zervas et al., 1998). If plasma lactate is used as an indicator of rumen fermentation of non-structural carbohydrates (Kaneko et al., 1997), our data confirmed that HFA caused a reduction while HST leaded to an enhancement of the rumen activity of amilolytic microflora. The higher availability of starch in the HST group leaded to a significant increase of plasma glucose and insulin (Table 2; P < 0.01; P < 0.001), but it is likely that milk yield was not improved, because of the genetic constraint of Bergamasca sheep. High starch content in ruminant diets can be associated with rumen acidosis and the gas analysis data (Table 3) indicated a significant reduction of pH and TCO2 at 35 and 50 days in HST diet. On the contrary, fat addition was associated with an increase of these parameters. Taken together, gas test data would indicate that HST animals were close to metabolic acidosis, while HFA animals were directed to a condition of metabolic alkalosis (Kaneko et al., 1997), consistent with the variation of BE-ECF at 35 days (P < 0.05). High starch content in the diet can lead to an increase of blood glucose availability directly through its digestion or indirectly from the propionate glucogenic pathway. The enhancement of glucose concentration and utilisation can modify the acid–base balance, as a consequence of the increase of the reducing power (H+ ions) from glycolysis and pentose phosphate shunt. This condition of metabolic acidosis can be emphasized by a shift of glucose aerobic pathway to the anaerobic glycolysis with production of lactic acid (Table 2). High dietary fat is known to induce lipoperoxidation (Djuric et al., 2001) and to enhance cytochrome P450 activity (Chen et al., 2003), which is involved in fatty acid oxidation, in turn stimulating the production of endogenous reactive oxygen species. To face with the fat-induced oxidative stress, HFA animals enhanced antioxidant defences, increasing GSx concentrations and SOD activity. Higher plasma GSx in human (Piolot et al., 2003) and increase of plasma GSx and erythrocytes SOD activity in rats (Ruiz-Guti´errez et al., 1999) after lipid inclusion in the diets have already been reported. A similar trend was observed also for GSH-px activity, which showed higher values in HFA group than in other experimental groups, even if differences were not statistically significant (Table 3). Administration of HST had less evident effects on oxidative stress markers (Table 3), also because the reducing power achieved from glycolysis and pentose phosphate shunt could per se have counteracted the

enhancement of reactive oxygen species derived from the catabolism of glucose (Osawa and Kato, 2005). 5. Conclusion The study of the environmental and nutritional conditions that affect the redox status is a fascinating area of research and there is growing body of evidence underpinning the patho-physiological consequences of oxidative stress also in farm animals. Data obtained in the experiment indicated that rapid modification of diet composition affected the biological response of lactating sheep. Fat inclusion modified milk composition and had a moderate effect on redox homeostatsis, thus stimulating the biological response to dietary-induced stress, while the increase of starch mainly affected metabolism. The mid-term and long-term consequences of repeated dietary-induced stress on lactating ewes, as in the case of grazing animals in adverse climate conditions, requires to be further evaluated. Acknowledgments This work was founded by the Italian National Research Council (CNR, 2000). The authors thank feed company Martini for the co-operation. References Andres, S., Ma˜ne, M.C., Sanchez, J., Barrera, R., Jimenez, A., 1999. Temporal variations in blood glutathione peroxidase (GSHPx) activity in sheep at pasture in a Mediterranean area. Vet. J. 157, 186–188. Association of Official Analytical Chemists, International, 1990. Official Methods of Analysis, 15th ed. AOAC, Arlington, VA. Beckman, K.B., Ames, B.N., 1998. The free radical theory of aging matures. Physiol. Rev. 78, 547–581. Castillo, C., Hernandez, J., Bravo, A., Lopez-Alonso, M., Pereira, V., Benedito, J.L., 2005. Oxidative status during late pregnancy and early lactation in dairy cows. Vet. J. 169, 286–292. Chen, H.W., Tsai, C.W., Yang, J.J., Liu, C.T., Kuo, W.W., Lii, C.K., 2003. The combined effects of garlic oil and fish oil on the hepatic antioxidant and drug-metabolizing enzymes of rats. Br. J. Nutr. 89, 189–200. Chilliard, Y., Ferlay, A., Rouel, J., Lamberet, G., 2003. A review of nutritional and physiological factors affecting goat milk lipid synthesis and lipolysis. J. Dairy Sci. 86, 1751–1770. Colitti, M., Stradaioli, G., Stefanon, B., 2000. Effect of ␣-tocopherol deprivation on the involution of mammary gland in sheep. J. Dairy Sci. 83, 345–350. Djuric, Z., Lewis, S.M., Lu, M.H., Mayhugh, M., Tang, N., Hart, R.W., 2001. Effect of varying dietary fat levels on rat growth and oxidative DNA damage. Nutr. Cancer 39, 214–219. Drabkin, D.L., Austin, J.H., 1935. Spectrophotometric studies. 1. Spectrophotometric constants for common hemoglobin derivates in human, dog and rabbit blood. J. Biol. Chem. 98, 538–543.

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