Abstract: Two experiments were designated to examine the effects of different levels of fish oil and canola oil on in vitro and in vivo nutrient digestibility. Experiment 1 was performed to assess the effects of unsaturated oils (fish oil, canola oil, their combination) in three levels (2, 4 and 6% on DM basis) on In vitro Dry Matter (IVDMD) and Organic Matter (IVOMD) digestibility of alfalfa hay and corn silage. For both forages, oil supplementation decreased IVDMD and IVOMD significantly (p<0.01) and increasing oil levels significantly (p<0.05) decreased IVDMD and IVOMD of both forages but alfalfa hay was more susceptible to increasing oil levels than corn silage. In experiment 2, eight multiparous early lactation Holstein cows (42±12 DIM, 40±6 kg daily milk yield) were fed a total mixed ration supplemented with either 0% oil (Control), 2% Fish Oil (FO), 1% Canola Oil + 1% Fish Oil (COFO) or 2% canola oil according to a double 4x4 Latin square design to examine the effects of unsaturated oil on milk yield, DMI and nutrient digestibility. Each period lasted 3 weeks experimental analyses were restricted to the last week of each period. Diets consist of 20% alfalfa, 20% corn silage and 60% concentrate. Cows were housed in tie stalls and fed the TMR two times a day to allow 5-10% orts (as-fed basis). Experimental diets had no significant (p>0.05) effect on milk production but DMI decreased significantly (p<0.05) in FO diet. Digestibility of OM and NDF were significantly (p<0.05) decreased in FO diet in comparison with three other diets, ADF digestibility was decreased significantly (p<0.05) in all oil containing diet in comparison with control but fat digestibility was not affected by treatments (p>0.05). Results of the current experiments shows that combination of fish oil and canola oil in 2% of dry matter had less negative effects on nutrient digestibility of forages and total mixture rations.

INTRODUCTION

The energy requirement of the modern cow, which has been bred for enhanced milk yield is difficult to meet during early lactation. Due to the high energy value of fat, supplemental fat is frequently added to ruminant diets to increase their energy density ( Oldick and Firkins, 2000; Juchem et al., 2008). Although, dietary fat increase energy density of diets, its influence on nutrient supply to the animal depend on the digestibility of the fat sources and effects of supplemented fat on intake, rumen fermentation and the other diet component digestibility (Khorasani and Kennelly, 1998). Ruminal fermentation and fiber digestion often decrease by addition of fats or oils to the diet; however degree of inhibition varies with amount and type of supplemented fats (Jenkins and Jenny, 1992). In comparison with saturated fatty acids, unsaturated fatty acids, which is more presented in plant oils than animal fat sources had more inhibitory affects on ruminal ecosystem (Jenkins, 1993; Pantoja et al., 1994) decreased dry matter intake (Harvatine and Allen, 2006) and fiber digestibility especially in high concentrate diets (Ueda et al., 2003). Consequently, the proportion of unsaturated and the ratio of unsaturated fatty acids to saturated ones appear to exert the most influence on ruminal fermentation (Oldick and Firkins, 2000). Various in vivo and in vitro methods have been used to measure the extend of nutrient digestion in ruminant animals. The in vitro Tilley and Terry (1963) procedure has been extensively used to measure Dry Matter Digestibility (IVDMD) and Organic Matter Digestibility (IVOMD) of feedstuffs and has been the most accurate and practical laboratory method available for prediction of digestibility data for ruminants (Mabjeesh et al., 2000). This method has been modified and adapted for starch feeds analysis and various researchers have improved its accuracy of prediction.

Objectives of these experiments were to determine the effects of different levels of fish oil and canola oil and their combination on in vitro digestion of alfalfa hay and corn silage as basal forages and to evaluate the effects of supplementing dairy diets in early lactation with fish oil, canola oil and their combination on total tract digestibility of organic matter, ADF, NDF.

MATERIALS AND METHODS

Experiment 1: Using a completely randomized design with 3x3 factorial arrangement of treatments, the effects of three levels (2, 4 and 6% of DM) of three oil sources (fish oil, canola oil and combination of fish oil and canola oil in 50:50 ratios) in an in vitro batch fermentation (Tilley and Terry, 1963) were studied. Treatments were assigned once to alfalfa hay and once to corn silage as forage basic which were dried in a force-air oven at 60°C and milled to pass a 2 mm screen using a wiley mill (Arthur H. Thomas, Philadelphia, PA) and were analyzed for CP, ADF and NDF. The alfalfa hay had the following chemical composition (DM basis): 16% CP, 52.5% NDF and 35.5% ADF. The corn silage contained (DM basis): 6.9% CP, 43.2% NDF and 21.5% ADF. For each forage source, three tubes were used as control without oil supplements. Incubation was performed using rumen fluid from two rumen fistulated sheep maintained on a diet of hay. The liquor was taken before morning feeding, strained through two layers of cheesecloth into a flask and kept under CO₂ gas at 38-39°C until used. The incubation was conducted in 90 mL tube containing 1 g (W₁) of milled alfalfa hay or corn silage which were previously supplemented with different levels of oils, 10 mL of rumen fluid and 40 mL of phosphate bicarbonate buffer (McDougall, 1948). Each tube was gassed with CO₂ before sealing with rubber corks with a gas release valve. Then tubes were incubated at 38°C for 48 h with occasional shaking.

At the end of the first incubation period, 6 mL HCL (20%) and then 5 mL pepsin solution (0.5 g pepsin was solved in 100 mL HCL 0.1 N) were added gradually to each tube. The tubes were then incubated at 38°C for 48 h. Finally, the supernatant were discarded using filter papers (No 42), the residues then were transferred to a 50 mL cruse and dried at 55°C until constant weight. The dry weight of the residue were calculated (W₂) to measure IVDMD and for IVOMD measurement, dried samples were heated at 550°C for 3-4 h. After that samples weight was recorded (W₃). Using following formula, IVDMD and IVOMD were calculated:

Experiment 2: Eight multiparous Holstein cows in early lactation (42±12 DIM, 40±6 kg daily milk yield) were use in a 4x4 Latin square split plot with four treatments, four periods and two cows per treatment as the main plot and the four sampling periods as the subplot. Each period lasted 21 days, which included a 14 days diet adjustment period followed by a sampling period. There were four treatments consist of diet with no supplemental oil (control) and diet supplemented with 2% Fish Oil (FO), 1% Fish Oil-1% Canola Oil (FOCO) and 2% Canola Oil (CO). Diets were composed of 60% (DM) concentrate mix, 20% alfalfa hay and 20% corn silage and formulated to meet energy and protein requirements of lactating cows producing 40 kg of milk and consuming 24 kg of DM day^-1 (Table 1). Oils were added at a level of about 2% of dietary DM, resulting in a dietary ether extract content of 4.7%. Kilika fish oil (khazar Co, Babolsar, Iran) and canola oil (Golestan Soybean Co, Gorgan, Iran) were used in this experiments.

Table 1:	Ingredient component and chemical composition of experimental diets
1Control = diet without oil; FO = diet supplemented with Fish Oil (2% DM); FOCO = Diet supplemented with 1% (DM) fish oil and 1% (DM) canola oil and CO = diet supplemented with 2% (DM) Canola Oil, 2khazar Co, Babolsar, Iran, 3Golestan Soybean, Gorgan, Iran, 4NFC = 100-(NDF+ CP + Ash + Ether Extract)

The oils were added first to the part of the bran and soy bean meal to produce a premix, then after well mixed were added to the other parts of the concentrate weekly. Concentrate mixtures and forage sources were mixed in a weighting and mixing unit and offered in the TMR form twice daily to allow 5-10% orts (as-fed basis).

Cows were housed in free stalls with continuous access to water and were milked daily at 0500, 1200 and 2000. Individual cow intake was recorded daily but only the Dry Matter Intake (DMI) during last 5 days of each experimental period was used for statistical analysis. Milk yield were recorded during last seven days of each experimental period. TMR mixture and feace were sampled on first 5 days of each sampling period and were stored in -20. At the end of each period, feed samples were mixed to get the final sample and were stored in -20 up to the end of experiment. Finally, all the feed and feace samples were dried in a forced-air oven at 60°C and stored in sealed plastic containers at room temperature until analyzed. In preparation for analyses, dried feed and feace were ground first through a 2 mm screen (Wiley; Arthur H. Thomas, Philadelphia, PA) and were analyzed for OM, ADF , NDF and Acid-Insoluble Ash (AIA) (Van Soest and Wine, 1967). Using gas chromatography method, fatty acid composition of diets and supplemental oil has b een determined which are listed in Table 1 and 2, respectively. AIA content of feed and feace was used as a natural marker in ruminant to determine apparent digestibility of some nutrient, using following formula:

Table 2:	Fatty acid composition (g/100 g of fatty acids) of supplemental oils
1khazar Co, Babolsar, Iran, 2Golestan Soybean, Gorgan, Iran

RESULTS AND DISCUSSION

Experiment 1: Results of IVDMD and IVOMD of oil supplemented alfalfa hay and corn silage are presented in Table 3 and 4, respectively. In contrast with control treatments of these two forages, including fish oil, canola oil and their combination decrease IVDMD and IVOMD of both forages significantly (p<0.01). IVDMD and IVOMD of alfalfa hay and corn silage decreased significantly in CO treatments in comparison with two other oil sources (p<0.05) and linear increase in oil levels from 2-6% (DM) cause IVDMD and IVOMD decrease in alfalfa hay but in corn silage including 6% oil in comparison with 2% decrease IVDMD and there were no significant differences in dry matter digestibility when oil level rise from 2-4% and from 4-6%. IVOMD of corn silage tend to be decrease when oil level increased from 2-6% (p = 0.06). Oil levels and sources interactions were not significant for both forage type (p>0.05).

Table 3:	The effects of different levels of fish oil and canola oil on in vitro digestibility of alfalfa hay
1IVDMD = In Vitro Dry Matter Digestibility, IVOMD = In Vitro Organic Matter Digestibility, 2FO = Fish Oil, CO = Canola Oil, FOCO= Fish Oil + Canola Oil (50:50)

Table 4:	The effects of different levels of fish oil and canola oil on in vitro digestibility of corn silage
1IVDMD = In Vitro Dry Matter Digestibility, IVOMD = In Vitro Organic Matter Digestibility, 2FO = Fish Oil, CO = Canola Oil, FOCO = Fish Oil + Canola Oil (50:50)

Table 5:	The effect of experimental diets on DMI, milk yield and nutrient digestibility of early lactating Holstein dairy cows
1Control = diet without oil; FO = diet supplemented with Fish Oil (2% DM); FOCO = Diet supplemented with 1% (DM) fish oil and 1% (DM) canola oil and CO = diet supplemented with 2% (DM) canola oil

The in vitro method of evaluating the digestibility of ruminant feeds is used worldwide. The method is easier than in vivo analysis and avoids the need to surgically prepared animals in different locations in the gastrointestinal tract. The TT method has also been proven more accurate than digestibility predictions based on the chemical compositions of feeds (Van Soest and Wine, 1967). Moreover, because of its similarity to in vivo values (Tilley and Terry, 1963), it is considered a reference method for the prediction of ruminant feed digestibility. Fiber degradation in the rumen may be limited by several dietary factors that alter attachment by, or nutrient availability to, fibrolytic bacteria. Cellulolytic bacteria apparently need to be in close proximity or attached to fiber before degrading it. Among several theories discussed by Palmquist and Jenkins (1980), one states that unsaturated dietary fats may coat fiber and interfere with bacterial attachment. As revealed in results, unsaturated oil supplementation significantly declined IVDMD and IVOMD of both forage type which is mostly related to NDF and ADF section of forages. Including unsaturated oil in diets often decrease digestibility of nutrients, spatially in forages (Avila et al., 2000). IVDMD of soybean oil supplemented broomgrass hay decreased significantly and similar to our results, a linear decline was noted for IVDMD of this hay as the percentage of soybean oil in diet increased from 3-6% (DM) (Whitney et al., 2000). However, addition of 10% (DM) of soybean oil or hydrogenated tallow did not affect in vitro NDF digestibility of crystalline cellulose (Firkins et al., 1991) which has been related to the basal diet used. Including canola oil in comparison with fish oil or combination of fish oil and canola oil significantly decreased IVDMD and IVOMD of both forages which was probably because of higher ratio of unsaturated fatty acids to saturated fatty acids (U:S) in canola oil in comparison with fish oil. As reported previously (Avila et al., 2000) oil suplements with higher U:S ratio will have more adverse effect on fiber digestibility. Alfalfa hay and corn silage had shown different responses of digestibility to oil levels. Increasing levels of oil from 2-4 and from 4-6% (DM) significantly decreased IVDMD and IVOMD of alfalfa hay but IVDMD of corn silage was declined significantly when oil level increased from 2-6%. One of the possible reasons of these differences might be chemical composition of alfalfa hay and corn silage. Alfalfa hay had higher percentages of ADF and NDF than corn silage which made it more sensitive to increasing oil levels. Results of this experiments show that unsaturated oils significantly decreased IVDMD and IVOMD of forages and their depends on oil type, oil levels, degree of saturation, basal diet and chemical compositions of forage.

Experiment 2: The result of milk production and DMI are shown in Table 5. Milk yield was not affected by oil supplemented diets (p>0.05). Supplementing diets with oils rich in Polyunsaturated Fatty Acids (PUFA) often results in a reduction in nutrient intake and milk production (Chilliard et al., 2001; Lock and Shingfield, 2004). However, there are several previous reports which have reported no significant effects of supplemented oil on milk yield (AbuGhazaleh and Holmes, 2007; Juchem et al., 2008). Donovan et al. (2000) also reported no significant decline in milk production until cows were consuming 3% (DM) FO; Wheres, in Whitock et al. (2002) study milk production appeared to be lower numerically in cows consuming 2% FO diet in comparison with three other diets. Including 2% (DM) fish oil in diets decreased DMI significantly (p<0.05) in comparison with control and other supplemented diets. Intake is regulated by the type and temporal pattern of available fuels (Allen, 2000), the interaction of available fuels and metabolic state, the potential negative effect of feeding supplemental oil on food palatability and ruminal fiber digestion. Such an effect can be expected to be most important with oils or ground seeds because of direct contact between lipids and rumen microorganisms (Bayourthe et al., 2000). In the current experiment DMI was decreased just in FO diet, which is probably because of the lower palatability of FO diet. Digestibility of OM and NDF were significantly decreased in FO diet in comparison with three other diets, ADF digestibility was decreased in all oil containing diet (Table 5). The digestibility of structural carbohydrates often decreases in oil containing diets, especially when unsaturated oil sources are included in diets. However, data on the interaction between basal diet and lipid supplementation for total-tract digestibility are inconsistent and depend on different parameters such as levels of oil in diets, saturation of oil supplements and the basal diet especially forage to concentrate ration as reported by Ueda et al. (2003), who reported that ruminal NDF digestibility increased with linseed oil supplementation to the forage-rich diet, whereas it decreased with supplementation to concentrate-rich diet. Moreover, the method of feeding, ad libitum or restricted, (Kucuk et al., 2004) and the number of feeding a day (Oldick and Firkins, 2000) also affect the nutrient digestibility when oil are included in diets. Different levels of soybean oil from 0-9.4% (DM) by limit feeding (Kucuk et al., 2004) and feeding fat supplemented diets twelve time a day (Oldick and Firkins, 2000) did not affected digestibilities of OM and NDF. Strong negative effects of linseed oil (Ikwuegbu and Sutton, 1982) and rapeseed oil (Ben Salem et al., 1993) on ruminal fiber digestibility with high proportion of concentrate in diet were reported previously. In contrast, in corn silage-based diet (Doreau and Chilliard, 1997) with around 35% concentrate ration in diet, a positive effect of n-3 fatty acids from fish oil, on ruminal fiber digestibility was reported. In the present study, the ratio of concentrate to forage in diet was 60:40 which group diets in high concentrate diets and as reveals in Table 5, total tract digestibility of fiber fraction of diet (NDF and ADF) decreased in oil containing diets with high concentrate ratio. However, Cottyn et al. (1971) showed that total-tract crude fiber digestibility in sheep was decreased by 5% linseed oil supplementation to diets with both high (75:25) and low (25:75) forage:concentrate ratios. In contrast with an in vitro digestibility results, fish oil had more negative effect on total tract digestibility of OM than canola oil which might probably related to lower DMI intake in FO diet. According to lower total tract digestibility of some diet nutrients, the benefits of increased energy density associated with fat supplementation maybe lost with increasing the levels of oil in diets.

CONCLUSION

This study shows that oil levels, basal diets and degree of oil saturation are the most important factors affecting nutrient digestibility of forages and total mixed rations of Holstein dairy cows. In vitro results revealed that canola oil had more negative effects of alfalfa hay and corn silage and digestibility of alfalfa hay was more decreased than corn silage when oil levels were increased. Total tract digestibility results illustrated that diet with 2% (DM) fish oil had lower OM and NDF digestibility of all treatments. In conclusion, combination of canola oil and fish oil had less negative effect on fiber digestibility that separately feeding them.