Effect of Dietary Supplementation With Fibrolytic Enzymes on the Productive Performance of Early Lactating Dairy Cows

This study was conducted to investigate the impacts of exogenous fibrolytic enzymes (Fibrozyme, Alltech inc company, USA) supplementation for 12 weeks on milk production and composition as well as blood metabolites in early lactating dairy cows. Total of 120 multiparous Holstein dairy cows at early lactation (57±4.2 days in milk) were randomly assigned into two groups according to lactation period "stage of lactation" and lactation season. The first group (control, n=60) were fed total mixed ration (TMR) without a supplement of exogenous fibrolytic enzymes. The second group (treatment, n=60) were fed TMR supplemented with a commercial exogenous fibrolytic enzymes at the rate of 15 g/cow/d for 12 weeks. Each group was placed in a shaded pen equipped with free stalls. An exogenous fibrolytic enzymes was added to the TMR at the time of feeding once per day at 10 am. Cows were fed as a group open feed, with free access to water. The results of this experiment on dairy cows showed that there were not significant changes in dry matter intake of lactating dairy cows with or without a supplement of exogenous fibrolytic enzymes at early lactation. Exogenous fibrolytic enzymes supplementd to lactating dairy cows improved (P<0.003) milk yield (41.0 vs. 39.5 kg/cow/d) compared to untreated dairy cows. Also, the fat corrected milk was increased (P<0.025) as a response to exogenous fibrolytic enzymes supplementation to lactating dairy cows compared to un-supplemented dairy cows. In addition, the supplementation of exogenous fibrolytic enzymes enhanced (P<0.006) the energy corrected milk (40.6 vs. 39.4 kg) and feed efficiency in early lactating dairy cows compared to the control group. The results revealed that supplementation of exogenous fibrolytic enzymes had no significant effect on milk fat, protein lactose and solid not fat (SNF) percentage compared to the control group of dairy cows. While, the quantities of milk protein (1.36 vs. 1.30kg), lactose (2.0 vs. 1.92kg) and SNF (3.47 vs. 3.31kg) in supplemented-dairy cows were improved significantly compared to the control group except quantity of milk fat (P<0.096). Serum glucose, albumin, urea and triglycerides were not affected (P>0.05) but total protein, globulin and cholesterol were declined (P<0.05) due to fibrozyme inclusion compared to control group of dairy cows. The supplementation of exogenous fibrolytic enzymes to early lactating dairy cows achieved higher net profit by 0.93 US$ per cow than control group. It is concluded that exogenous fibrolytic enzymes supplementation to early lactating dairy cows improved significantly milk production, SNF and energy corrected milk.


Introduction
The ruminant production systems are dependent worldwide on forage as the main nutritional components (Wilkins, 2000).The digestion of forage occurs through the microbial fermentation as a result of the presence of microorganisms at the reticulo-rumen and its adaptation to digest lignocellulosic components.The microbial mode of digestion allows ruminants to better unlock the unavailable energy in the plant cell wall components than other herbivores (Krause et al., 2003).This gives ruminant animals the ability to convert low nutritive and resistant lignocellulosic biomass to milk, meat, wool and hides (Weimer et al., 2009).However, most forage plants are high in cell walls and low in nitrogen (N) and energy content (Romney & Gill, 2000).Despite the importance of fibrous components in forages for salivation, rumen buffering and efficient production of ruminal end products (Mertens, 1997) only 10 to 35% of energy intake is available as net energy (Varga & Kolver, 1997).
verge of delivering practical benefits to ruminant production systems.In this regard, 2 cellulases and xylanases are respectively amongst the two major enzyme groups that are specified to break ß1-4 linkages joining sugar molecules of cellulose and xylans found in plant cell wall components (Beauchemin et al., 2003).Several studies with EFE have made mention of the increase of microbial activities in the rumen, which resulted in an enhancement of animal performance traits.Despite the increase in feed digestibility and subsequent production traits, the relationship between the improvement in forage utilization and enzymatic activities is yet to be explained in ruminant systems (Eun et al., 2007).In addition, results with EFE addition in ruminant systems are variable and somewhat inconsistent (Beauchemin et al., 2003;Colombatto et al., 2003), making their biological response difficult to predict.Some studies have shown substantial improvement of feed digestibility and animal performance traits (Cruywagen & Goosen, 2004;Bala et al., 2009;Arriola et al., 2011), while others reported either negative effects or none at all (Baloyi, 2008).If the potential intake and/or the density of available nutrients of forages can be increased with EFE as feed additives, then poor quality forages can be economically and successfully converted into meat and milk for human consumption.Moreover, an increase in the input costs in the dairy industry has demonstrated the need for methods to increase production efficiency.One way of increasing efficiency would be to increase the bioavailability of nutrients in a feedstuff, which might be accomplished through the exogenous fibrolytic enzymes supplement.Therefore, the objective of the undertaken experiment was to evaluate the effect of exogenous fibrolytic enzymes "Fibrozyme" supplementation on dry matter intake, feed efficiency, blood metabolites, milk production and milk composition in early lactating dairy cows.

Animals' Management and Experimental Design
This experiment was conducted at Alexandria commercial company of agriculture at Alexandria governorate during March to May, 2011 for 12 weeks.Total of 120 multiparous Holstein dairy cows at early lactation (57± 4.2 days in milk) were randomly assigned into two groups.The first group (control, n=60) were fed total mixed ration (TMR) without a supplement of exogenous fibrolytic enzymes.The second group (treatment, n=60) were fed TMR supplemented with a commercial fibrolytic enzymes (Fibrozyme is enzymes blend, which is prepared from fermentation extract and fermentation soluble of Aspergillus Niger and Trichoderma longibarachiatum, and having xylanase activity by minimum 100 XU/g, Alltech inc company, USA) at the rate of 15 g/cow/d according to the guide of the manufacture for 12 weeks.Fibrozyme was added and mixed to the TMR at the time of feeding once per day.Each group was placed in a shaded pen equipped with free stalls.Cows in the two groups fed a total mixed ration (TMR, Table 1), which composed of alfalfa hay, corn silage, green clover, soybean meal, yellow corn, limestone, vitamins, minerals mixture, protected fat (magnapac), sodium bicarbonate and mono calcium phosphate.The proximate analysis and calculated nutritive value of the TMR is given in Table 2. Cows were fed as a group open feed, with free access to water.Amount of TMR delivered was measured with electronic scales on mixer-feeder wagon.The TMR was mixed and fed using Delaval mixer wagon.The diet was formulated using Gavish computer operated cattle feeding system 2008 to cover or exceed NRC recommendations (NRC, 2001) as in Table 2. Cows were milked three times daily at 4 am, 12 pm.and 8 pm in a Dobell 20-parallel milking parlor equipped with automatic cow identification, milk recording system, and automated detacher milker units.
Milk samples were collected biweekly and analyzed immediately for fat, protein, lactose and SNF content using infrared method by Milk Analyzer (Milko tester Instruments Inc, Bulgaria).Average fat and CP yields were calculated by multiplying milk yield by fat and CP content of milk on an individual cow basis.Milk energy (MJ/kg) was calculated on an individual cow basis using the milk fat, CP and lactose content of the milk (Tyrrell & Reid, 1965).
Blood samples were collected from Jugular vein (10 cows per group were randomly selected) prior to morning feeding monthly.Serum were obtained by centrifugation the blood tubes for 20 min, 3000xg and stored at -20 o C until blood metabolites analysis.Concentrations of serum total protein, albumen, urea, glucose, triglycerides and cholesterol were determined using commercial kits manufactured by Stanbio Diagnostic Company, Germany.The concentrations of serum total protein, albumin, urea, glucose, triglycerides and cholesterol were measured calorimetrically according to Henry (1974); Doumas et al. (1971); Patton and Crouch (1977);and Tietz et al. (1995), respectively.The concentration of globulins in each serum sample was obtained by subtracting the value of albumin from the total blood serum protein concentration.The ratio of A: G was calculated.

Economic Efficiency
Economic efficiency expressed as the daily feed and supplement cost and price of milk.The price of one ton of TMR was 400 US$, while the price of fibrozyme was 12.73 US$/kg and milk price (fat=3.5% and SNF=8.5%) was 0.524US$ according to Egyptian prices of year 2011.

Statistical Analysis
Statistical analysis was completed using a completely randomized design with a 2 factorial arrangement to analyze the data.The MIXED procedure of SAS (Version 9.In addition, the supplementation of exogenous fibrolytic enzymes enhanced (P<0.006) the ECM (39.4 vs. 40.6kg) in dairy cows compared to un-treated group.The significance contrasts in Table 3 showed that there were no interaction between treatment and time, while time had significant effects on milk yield, FCM, ECM and feed efficiency in treated group compared to the control group.
Effects of exogenous fibrolytic enzymes supplement to the diet of lactating dairy cow at early lactation on milk composition are given in Table 4.The results denoted that supplementation of exogenous fibrolytic enzymes had no significant effect on milk fat, protein lactose and solid not fat (SNF) percentage compared to the control group of dairy cows.While, the quantities of milk protein (1.36 vs. 1.30kg), lactose (2.0 vs. 1.92 kg) and SNF (3.47 vs. 3.31 kg) in supplemented-dairy cows were improved significantly compared to the control group of dairy cows except quantity of milk fat (P<0.096).The significance contrasts in Table 4 showed that there were no interaction between treatment and time, while time had significant effects on yield of milk fat, protein, lactose and SNF in treated group compared to the control group.Effects of exogenous fibrolytic enzymes inclusion to lactating dairy cows at early lactation on blood metabolites are presented in Table 5.  * The difference in milk price between two groups was due to higher 0.1% in SNF.
The results revealed that exogenous fibrolytic enzymes supplementation caused significant decline in serum total protein (12.8 vs. 10.4 g/dL), globulin (8.9 vs. 6.3 g/dL), A/G ratio content (0.81 vs. 0.54) and cholesterol (242.0 vs. 193.7 mg/dL) compared to control group of dairy cows.While, there were no significant changes regarding glucose, albumin, urea and triglycerides when exogenous fibrolytic enzymes was supplemented to the diet of lactating dairy cows at early lactation compared to control group.The results of statistical analysis showed no interaction was detected between treatments and months in the blood metabolites, while only values of total protein and blood urea N were highly affected by months.
Economic evaluation of the exogenous fibrozyme enzymes supplementation at early lactation of dairy cows is given in Table 5.The results showed the inclusion of 15 g of exogenous fibrolytic enzymes will cost 0.191 US$ /cow/day, which will increase both milk production and SNF by 1.5 kg and 0.1% per cow/day.The daily profit of individual cow was 10.79 and 11.72 US$in control and treated group of lactating dairy cow at early lactation, respectively excluding the labor, veterinary medicines and other management's costs.Moreover, the net profit for exogenous fibrolytic enzymes inclusion in diet of early lactating dairy cows was higher 0.93 US$ than untreated cows and economic efficiency increased from 2.09 to 2.19 by the treatment.

Discussion
Feeding high-producing cows continues to challenge dairy farmers and nutritionists.Also, dairy profit margins vary as milk prices and feed costs shift yearly.Feed additives are a group of feed ingredients that can cause a desired animal response in a non-nutrient role, such as pH shift, metabolic modifier, or performance (Hutjens, 1991).The use of exogenous fiber-degrading enzyme additives for ruminants was first examined in the 1960s, as reviewed by Beauchemin and Rode (1996).Enzyme products for ruminant diets are of fungal (mostly Trichoderma longibrachiatum, Aspergillus niger and A. oryzae) like our tested product (Fibrozyme); bacterial (Bacillus spp., Pendleton, 2000) or rumen bacterial (Gado et al., 2009) origin.According to Sheppy ( 2 0 0 1 ) , there are four main reasons for u s i n g enzymes in animal feed: 1) to break down anti-nutritional factors; 2) to increase the availability of starches, proteins and minerals enclosed within fiber-rich cell walls; 3) to break down specific chemical bounds in raw materials which are not usually broken down by the animals' own enzymes, thus releasing more nutrients, and.4) to supplement the enzymes produced young animals.Beauchemin et al. (1999) used lactating and cannulated Holstein cows to investigate the effects of grain source and fibrolytic enzymes supplementation on ruminal fermentation, nutrients digestion in the rumen and in intestine, and milk production.Two grains (barley and hull-less barley) were combined with and without enzymes.They proposed three ways for the arrival and action of the enzymes into the intestine: enzymes applied to dry feed may enhance the binding of the enzyme to the substrate, which may increase the resistance of the enzymes to proteolysis and prolong their residence time within the rumen; enzymes applied to silage or TMR immediately prior to feeding, may be released into the ruminal fluid and may pass through the rumen quickly before they can be effective, which would provide larger intestinal effects and exogenous enzymes may alter digestion and nutrient absorption in the small intestine.Digestion of plant cell walls, which is carried out by ruminal microorganisms, provides a large amount of energy for ruminants.Fibrolytic enzymes isolated from fungi fermentation cultures have been utilized to improve DMI and forage digestibility; particulate passage rate; and digestibility of DM, NDF, and ADF in beef steers (McAllister et al., 1999).
In consistence with our finding on DMI, Ahn et al. (2003); Bernard et al. (2010) and Arriola et al. (2011) reported that adding fibrolytic enzymes supplementation to dairy cow diet did not enhance DMI and no difference was found between cows fed supplemented diet or un-supplemented diet with fibrolytic enzymes.On the other hand, several researchers recorded an increase in DMI of dairy cows when fibrolytic enzymes was applied to forage before mixing with other ingredients (Lewis et al., 1999) or applied to TMR or concentrate portion of the diet (Bowman et al., 2002;Ware & Zinn, 2005).However, the effects of fibrolytic enzymes on DMI appear to be vary among enzymes products and the method of applying of enzymes (Bowman et al., 2002) A number of studies have examined the effects of fibrolytic exogenous enzymes on digestibility and milk production in dairy cows.In some studies, dietary addition of fibrolytic enzymes either to forages or concentrate portion increased milk production from 5-16% (Lewis et al., 1999;Gado et al., 2009;Holtshausen et al., 2011) as noticed at the current study but no milk response was reported in others (Elwakeel et al., 2007;Bernard et al., 2010).Furthermore, these enhancement in milk yield at the current study are in line with those found by Guerra et al. (2007) who used Fibrozyme in diet containing alfalfa hay and they reported that Fibrozyme supplementation increased milk yield, which may be due to improved utilization of nutrients in digestive tract and in rumen and increased gain of net energy.Differences in enzyme activity, form and application rates, and diets across these studies complicate elucidation of the reasons for these discrepancies and highlight the need for caution when comparing studies involving different enzyme preparations.
The improvement in feed efficiency observed in the current lactation study might be attributable to greater NDF digestibility in the rumen and the similar trend was concluded by Holtshausen et al. (2011).Improvements in feed conversion efficiency were due to lower DMI rather than a change in milk yield.Improved feed efficiency indicates better utilization of nutrients when TMR was treated with enzymes, with the magnitude of improvement being a linear function of enzymes dosage.
There were no responses in the percentages of milk fat, protein, lactose and SNF for supplementing exogenous fibrolytic enzymes to early lactating dairy cows under the conditions of the current study, but the yield of milk protein, lactose and SNF was enhanced significantly by fibrozymes inclusion in the early lactating dairy cows.In agreement with our finding, several studies has been reported that fibrolytic enzymes supplementation to Holstein dairy cows did not affect (P>0.05) on milk composition (Lewis et al., 1999;Knowlton et al., 2002;Reddish & Kung, 2007;Elwakeel et al., 2007;Bernard et al., 2010;Arriola et al., 2011).On the other hand, Yang et al. (1999) and Mansour (2009) found that milk fat increased when adding fibrolytic enzymes.The increase in fat percentage may be due to the increase in available energy and fatty acids for fat synthesis.Gado et al. (2009) concluded that milk protein yield for Brown Swiss cows was (P<0.05)increased (0.57 %) for cows fed ZADO ® supplemented diet compared with 0.45 kg/h/day for cows fed control diet.The variability in responses among studies may be attributed to variety of enzyme products and experimental conditions.
Contents of albumin, glucose, triglycerides and urea were not differing significantly due to fibrozymes supplementation to early lactating dairy cows (Table 5).Broderick et al. (1997) treated alfalfa silage with four levels of fibrolytic enzymes mixture (xylanase and cellulase) and fed dairy cows, they found that the adding of fibrolytic enzymes did not influence blood glucose and urea in all levels.Also Viktor (2006) concluded that there was no significant difference in blood plasma glucose concentrations for Holstein dairy cows fed diet supplemented with fibrolytic enzyme (xylanase).On the other hand, total protein and globulin and cholesterol were declined significantly by fibrozymes supplementation, which is similar to that found by Abd El-Kareim (2004).A/G ratio indicated that the treated animals were in good health since A/G ration was more than 1.00.Exogenous fibrolytic enzymes inclusion in TMR fed to the early lactating dairy cows improved economic efficiency and achieved daily net profit 0.93 US$ per cow over control cows.These results agreed with those obtained by Tozer et al. (2003) who found that although costs per kilogram of milk produced were lowest for pasture-concentrate cows, cows on TMR had the highest net income per cow per day because of higher yields of milk and milk components, but cows on the pasture-concentrate had lower daily net income due to lower yields of milk and milk components.

Conclusion
The results of the current study concluded that feeding a fibrozyme applied to the total mixed ration of dairy cows in early lactation has the potential to increase milk production, SNF and economic efficiency.

Table 4 .
Milk composition of lactating dairy cows at early lactation fed total mixed ration with or without exogenous fibrolytic enzymes (EFE) (± SE) SE: standard error; SNF: solid not fat; W: weeks.

Table 5 .
Least square means (± SE) of blood metabolites of lactating dairy cows at early lactation fed total mixed ration with or without exogenous fibrolytic enzymes (EFE) Different letters (a, b) in the same row indicate significant differences.SE: standard error; M: months.

Table 6 .
Economic evaluation of the exogenous fibrolytic enzymes supplementation at early lactation of dairy cows