MATERIALS AND METHODS

This study was conducted over the period from January 2006 to March 2007 at University of Ondokuz Mayis, Faculty of Agriculture, Department of Animal Science in Samsun Province of the Republic of Turkiye.

Canola Seeds
In this study, canola (Brassica napus) seeds from 4 different winter variety hybrids (Bristol, Eurol, Capitol and Licrown) obtained from the Institute of Karadeniz Agricultural Research in Samsun, Turkiye were used. Canola seed grains were milled in a hammer mill to pass through a 1 mm sieve for subsequent analysis.

Chemical Analysis
Dry Matter (DM) was determined by drying samples at 105°C overnight. Organic Matter (OM) content was determined by ashing in a muffle furnace at 550°C for 8 h. Nitrogen (N) content was determined using Kjeldahl method (AOAC, 1990). Crude protein was calculated as Nx6.25. Crude Fiber (CF) and EE were determined by the methods described by AOAC (1990) and Nitrogen Free Extract (NFE) was determined by difference [100 - (CP + EE + CF +ash)]. Neutral Detergent Fiber (NDF), Acid Detergent Fiber (ADF) and Acid ketergent Lignin (ADL) contents were determined by the methods of Van Soest (1991). Total phenolic matter was determined according to the method proposed by Gurses and Artik (1987). Volatile fatty acids and NH₃-N contents in the rumen fluid were determined using Markham Steam Distillation procedure (Markham, 1942). All chemical analyses were carried out in triplicate.

In vitro Gas Production
Approximately, 200 mg dry weight of samples was weighted in triplicate into 100 mL calibrated glass syringes following the procedures of Menke and Steingass (1988). The syringes were pre-warmed at 39°C before the injection of 30 mL rumen fluid-buffer mixture consisting of 10 mL strained rumen fluid and 20 mL buffer solution into each syringe followed by an incubation in a water bath at 39°C. Rumen fluid from three fistulated Sakýz x Karayaka rams was collected before the morning feeding and strained through two layers of muslin. Sheep were fed twice daily (08.30-16.30) with a diet of grass hay (60%) and concentrate (40%). Gas volume was recorded at 0, 3, 6, 9, 12, 24, 48, 72 and 96 h of incubation. Total gas volumes were corrected for blank incubations. Cumulative gas production data were fitted to the model of Orskov and McDonald (1979) by NEWAY computer package program.

where, a is the gas production from the immediately soluble fraction (mL), b is the gas production from the insoluble fraction (mL), c is the gas production rate constant for the insoluble fraction (mL h^–1), a+b is potential gas production (mL), t is incubation time (h), y is gas produced at time t.

Organic matter digestibility (Menke et al., 1979) and ME (Close and Menke, 1986) contents of canola seeds were estimated using equations given below:

where, GP is 24 h net Gas Production (mL/200mg DM), CP is crude protein (%), EE is Ether Extract (%)

Cellulase Method (In vitro Enzyme Technique)
In vitro digestibility of DM and OM were determined according to Alcicek and Wagener (1995) as follows: ONUZUKA cellulase enzyme was used in this study.

where, S₁ is sample amount as (DM), T₀ is weight of crucible (105°C, 48 h), T₁ is dry sample (105°C, 24 h)+T₀, T₂ is ashed sample (550°C, 4 h)+T₀, A₁ is crude ash amount of sample, (g). Metabolisable Energy (ME) was estimated using equations given below (Jarrige, 1989; Malossini et al., 1993). Calculated values were converted to MJ kg^–1 DM:

In situ Nylon Bag Technique
The in situ degradability characteristics of samples were measured using the nylon bag technique of Orskov and McDonald (1979). Two rumen fistulated SakýzxKarayaka rams were used in in situ study. Three bags for each feed in each of the rams were incubated for 48 h. Triplicate bags containing about 5 g DM were placed into the rumen and incubated for 48 h. After incubation, bags were rinsed in running tap water to remove adhering digesta and then washed twice in a pool of water (30°C) for 5 min to remove rumen fluid. They were dried at 65°C for 72 h. In a forced-drought oven, allowed to air equilibrate and weighed. After incubation, DM, OM and CP degradability (DMD, OMD and CPD) for each bag, for each incubation period and for each ram were calculated separately with formulas suggested by Susmel et al. (1990). Metabolizable Energy (ME) contents of canola seeds were estimated using equations given below (Bhargava and Orskov, 1987):

where, DMD is rumen dry matter degradability for 48 h.

Statistical Analysis
One-way Analysis of Variance (ANOVA) was carried out to compare the chemical composition, gas production kinetics, ME, NE_L and OMD values using General Linear Model (GLM) of SPSS 10.0 package programs. Significance between individual means was identified using the Duncan’s multiple range tests.

RESULTS

Chemical composition and Total Phenolic Matter (TPM) content of whole fat canola seeds were given in Table 1. Rumen pH, ammonia N (NH₃-N) and total Volatile Fatty Acid (VFA) contents determined for rumen liquid using in vitro gas production technique were; 6.18 (5.88-6.45), 321 mg L^–1 (293-438 mg L^–1) and 112 mmol L^–1 (93-128 mmol L^–1), respectively.

All varieties had low gas production levels. Licrown variety had the lowest production level up to 48 h of the incubation, but there were no differences after 24 h of the incubation (p>0.05). There were not significant differences among the hybrids in terms of estimated parameters except for gas production rate (c). The gas production rate of Licrown was significantly (p<0.05) lower than that of Bristol (Table 2).

There were no differences among the hybrids in terms of DMD, OMD and ME values (p>0.05). (Table 3). There were significant differences among the hybrids in terms of DMD₄₈, OMD₄₈, ME (P<0.001) and CPD₄₈ (p<0.01) (Table 4). The DMD₄₈ and ME values of Eurol and Licrown are higher than those of Capitol and Bristol (p<0.001). OMD₄₈ (p<0.001) and CPD₄₈ (p<0.01)values were found different between Eurol and Bristol.

Table 1:	Chemical compositions and TPM contents of whole fat canola seeds, %DM
DM: Dry matter, CP: Crude protein; EE: Ether extract, NDF: Neutral detergent fiber, ADF: Acid detergent fiber, ADL: Acid detergent lignin, TPM: Total phenolic matter, SEM: Standard error of mean, NS: Non significant. Means in the same row with different letter(s) indicate significance. p<0.01

Table 2:	In vitro gas productions, gas production kinetics and estimated parameters of whole fat canola seeds
DM: Dry matter, a: Gas production from the immediately soluble fraction (mL), b: Gas production from the insoluble fraction (mL), c: Gas production rate constant for the insoluble fraction (mL h–1), ME: Metabolisable energy, OMD: Organic matter digestibility, SEM: Standard error of mean, NS: Non significant; Means in the same row with different letters indicate significance. *p<0.05, **p<0.01

Table 3:	DMD, OMD and ME contents of whole fat canola seeds
DMD: Dry matter digestibility, OMD: Organic matter digestibility, ME: Metabolisable energy, SEM: Standard error of mean, NS: Non significant

Table 4:	In situ DMD48, OMD48, CPD48 and ME values of canola seeds
DMD48: Dry matter degradability, OMD48: Organic matter degradability, CPD48: dry matter degradability, ME: Metabolisable energy, SEM: Standard error of mean, NS: Non significan. Means in the same row with different letters indicate significance. **p<0.01, ***p<0.001

DISCUSSION

In vitro gas production, gas production kinetics, estimated parameter values are largely influenced by the differences in the chemical cosmpositions of feedstuffs. The increase in ash contents of the feedstuffs leads to a decrease in the amount of gas produced (Menke and Steingass, 1988). The similarity of varieties in terms of ash content can be one of the reasons for similar total gas production levels.

Feedstuffs with high CP result in low gas production (Chenost et al., 2001). Lower gas production level observed for Licrown variety might be attributed to its higher CP content. Feedstuffs should contain at least 10% CP for optimum microbial activity in the rumen (Norton, 1998). Feedstuffs with below 10% CP can cause a reduction in the microbial activity in the rumen, thus can lead to less gas production. The hybrids used in the present study did not affect microbial activity significantly. Khorasani et al. (1992) reported that supplementation of ruminally protected canola seed, at levels>3% of the diet, decreased concentrations of total ruminal VFA and molar proportions of acetate, propionate and butyrate.

Licrown had numerically the lowest gas volumes up to 48 h of the incubation. This can be attributed to higher TPM content in Licrown compared to other hybrids (Kilic and Saricicek, 2006). Furthermore, gas production rate differs with relation to the amount and availability of rumen microorganisms (Mauricio et al., 2001). Low gas production rate in Licrown might be caused by higher TPM content which had influence on amount of rumen microorganisms. However, there were no differences among the varieties in terms of gas production levels. Low gas production levels in canola varieties can be attributed to their higher crude fat contents due to the fact that oils decrease VFA concentration and hence gas production in the rumen (Wettstein et al., 2000).

There is a strong relationship between the OMD of feedstuffs and the rate of gas production (Chenost et al., 2001). In the present study, Licrown variety with lowest c value had numerically the lowest OMD value. This finding is consistent with the results of Chenost et al. (2001). However, Kilic and Saricicek (2006) suggested that feeds with lowest c value do not always have lowest OMD value. Lower c value of Licrown compared to Bristol explains why the Licrown had lower gas production level up to 48 h of the incubation. As expected, the feeds with lower c values had lower gas productions at the beginning of the incubation. Thus, the lack of difference with the progression of incubation explains this case.

Data from cattle fed with whole canola suggest that the seeds are relatively resistant to digestion in the rumen and intestine unless processed (Khorasani et al., 1992; Leupp et al., 2006). Gralak et al. (1997) reported 71.90 and 74.98% values for effective DM degradability and CP degradability of whole canola seeds. This finding is consistent with present findings. Micronized whole canola seeds had higher gas productions and lower DM and CP degradability compared to unprocessed seeds (Wang et al., 1997). DM and CP degradability found in this study are similar to present findings.

At 5%/h flow rate, effective CP degradability of WCS was 86.7.2%. Extrusion did not affect WCS and rumen CP degradability. Without some form of protection whole canola seed CP is obviously, highly degradable (Deacon et al., 1986, 1988). These results are in agreement with our findings. The highest degradability values were found for CP.

If whole canola seed makes up more than 12-14% of the ration it may lead to depressed rumen function reduced feed intake and digestibility of nutrients. Conversely, fed dairy cows whole canola seeds, raw or extruded, at 14% of the diet without effecting Crude Fibre digestibility (Ellwood, 2004). Murphy et al. (1987) reported reduction in rumen digestibilities of DM, NDF and cellulose, however hindgut fermentation compensated for the reduction at 1 kg/day Whole Canola Seed supplementation, but not at 2 kg day^–1.

Although, there were no differences between the canola hybrids in terms of dry matter digestibility, organic matter digestibility and in vitro ME values in enzyme technique, Bristol, which had the lowest CP value, also had the lowest CPD₄₈ value in in vitro gas production technique. Variability in feeds and also their production locations is one of the most important factors affecting the results of nylon bag technique (Kilic and Saricicek, 2004). Yilmaz (1997a, b) found wide variations for SunFlower Meal (SFM) samples obtained from 12 different locations and for Alfalfa Hay (AH) samples obtained from 15 different locations in terms of degradability characteristics. The author reported a value, from which DMD was calculated, as 3.90 -60-72% for SFM and 0.00- 41.75% for AH. This explains why the large variations were observed among the canola varieties used in present study.

ME values found in in vitro gas production technique and in in vitro enzyme technique were not different, but there were differences among the hybrids in in situ bag technique. This can be attributed to the fact that the varieties have significant effects on the results of nylon bag technique (Yilmaz, 1997a, b; Kilic and Saricicek, 2004). ME values found in in vitro gas production technique were lower than those found in in vitro enzyme technique and in situ nylon bag technique due to the lower gas productions of the canola seeds. This difference might be due to the fact that high oil levels found in all the canola varieties prevent gas production.

Leupp et al. (2006) reported that supplementation with canola seed at 8% of dietary DM did not affect intake or fiber digestion in low-quality forage diets. Canola supplementation increased apparent and true ruminal CP degradation but decreased small intestinal CP digestion. Canola supplementation decreased ruminal pH and the molar proportion of acetate. Decrease in acetate content explains the lower gas productions in canola seeds. The researchers explanained that their study results suggest that ground canola included at 8% of the diet can alter ruminal VFA concentrations and increase in situ degradation of canola seed when offered as a supplement for cattle fed low-quality forage diets.

CONCLUSION

The results of the present study indicate that feed value of different canola seed varieties is similar. Furthermore, they have lower in vitro gas production values due to their higher oil content. While there were no differences among the hybrids in in vitro enzyme technique, there were significant differences in in situ technique (p<0.01). If whole canola seed makes up more than 12-14% of the ration it may lead to a depressed rumen function, reduced feed intake and digestibility of the nutrients. However, canola seeds should be used up to 20% of the total ration dry matter or the oil supplied from canola should not exceed the 6% of the total oil content of the ration.It can be said that canola seeds incorporated into the ration can decrease the feed energy waste in rumen due to lower gas production levels.