Supplementing exogenous enzymes is a safe biological method to promote animal performance and the production of enzymes is becoming cheaper and more efficient (Zilio et al., Reference Zilio, Del Valle, Ghizzi, Takiya, Dias, Nunes, Silva and Rennó2019). Cellulose and xylanase are the most frequently investigated fibrolytic enzymes in dairy cattle (Zilio et al., Reference Zilio, Del Valle, Ghizzi, Takiya, Dias, Nunes, Silva and Rennó2019) and show different effects on nutrient utilization (Yang et al., Reference Yang, Beauchemin and Rode2000) and production performance (Murad and Azzaz, Reference Murad and Azzaz2010). Two meta-analyses (Arriola et al., Reference Arriola, Oliveira, Ma, Lean, Giurcanu and Adesogan2017; Tirado-González et al., Reference Tirado-González, Miranda-Romero, Ruíz-Flores, Medina-Cuéllar, Ramírez-Valverde and Tirado-Estrada2018) demonstrated positive overall effects for fiber digestion and milk production (Adesogan et al., Reference Adesogan, Arriola, Jiang, Oyebade, Paula, Pech-Cervantes, Romero, Ferraretto and Vyas2019). Dietary supplementation of amylolytic enzymes to cows promoted rumen fermentation (Noziere et al., Reference Noziere, Steinberg, Silberberg and Morgavi2014), milk yield (Tricarico et al., Reference Tricarico, Johnston, Dawson, Hanson, Mcleod and Harmon2005) and feed efficiency (Andreazzi et al., Reference Andreazzi, Pereira, Reis, Pereira, Júnior, Acedo, Hermes and Cortinhas2018) without leading to acidosis. However, different results also exist (Andreazzi et al., Reference Andreazzi, Pereira, Reis, Pereira, Júnior, Acedo, Hermes and Cortinhas2018) so it is fair to say that consensus has not been reached.
In theory, exogenous fibrolytic and amylolytic enzymes are considered to perform synergistic effects when supplementing in combination (Tricarico et al., Reference Tricarico, Johnston and Dawson2008). Nevertheless, as far as we know, only two studies reported on this (Hristov et al., Reference Hristov, Basel, Melgar, Foley, Ropp, Hunt and Tricarico2008; Zilio et al., Reference Zilio, Del Valle, Ghizzi, Takiya, Dias, Nunes, Silva and Rennó2019). No effect was found on nutrient ingestion and digestion, rumen fermentation, or milk performance. This might be attributed to the differences in delivery method (Beauchemin et al., Reference Beauchemin, Colombatto, Morgavi and Yang2003; Hristov et al., Reference Hristov, Basel, Melgar, Foley, Ropp, Hunt and Tricarico2008), the time of delivery and the proportion of the diet delivered at each time (Adesogan, Reference Adesogan2005; Zilio et al., Reference Zilio, Del Valle, Ghizzi, Takiya, Dias, Nunes, Silva and Rennó2019). To control the variation, we added the enzyme preparation at the moment of total mixed ration (TMR) production and offered twice per day (Adesogan, Reference Adesogan2005; Adesogan et al., Reference Adesogan, Arriola, Jiang, Oyebade, Paula, Pech-Cervantes, Romero, Ferraretto and Vyas2019). Moreover, the variation in results probably is attributed to the lactation stage (Beauchemin et al., Reference Beauchemin, Colombatto, Morgavi and Yang2003) and parity (Wathes et al., Reference Wathes, Cheng, Bourne, Taylor, Coffey and Brotherstone2007) of cows, which was different between Hristov et al. (Reference Hristov, Basel, Melgar, Foley, Ropp, Hunt and Tricarico2008: late-lactation multiparous) and Zilio et al. (Reference Zilio, Del Valle, Ghizzi, Takiya, Dias, Nunes, Silva and Rennó2019: mid-lactation multiparous). We used early-lactation primiparous cows since they seem to benefit more from the enzyme supplementation (Bachmann et al., Reference Bachmann, Bochnia, Mielenz, Spilke, Souffrant, Azem, Schliffka and Zeyner2018).
Given these various observations, we hypothesized that dietary supplementation with a compound enzyme preparation of fibrolytic (cellulase, xylanase, and β-glucanase) and amylolytic (amylase) enzymes to primiparous early-lactation dairy cows would boost nutrient intake, promote rumen fermentation as well as energy metabolism and enhance milk production in cows.
Materials and methods
The experiment was conducted at the Modern Farm (Baoji, China) and the Laboratory of Animal Nutrition at Northwest A&F University (Yangling, Shaanxi, China). All experimental procedures were approved by the Northwest A&F University Animal Care and Use Committee.
Twenty primiparous early-lactation (47 ± 2 d in milk) Holstein cows of similar body condition score were randomly divided into control (CON, n = 10) and experimental (EXP, n = 10) groups as a completely randomized single-factor design. The CON was only fed a basal TMR diet and the EXP was dietary supplemented with compound enzyme preparation at 70 g/cow/d. Enzyme preparation was added to TMR during its production twice daily. The experiment lasted 4 weeks, with 3 weeks of adaptation followed by a 1-week experimental period. Cows were fed twice per day (0600 and 1400) with at least 5% residues in the feed trough, given free access to water and were milked three times daily (0000–0100, 0700–0800, and 1400–1500).
The basal TMR diet (online Supplementary Table S1) was prepared twice daily before feeding. The compound enzyme preparation (Guangdong VTR Bio-Tech Co., Ltd.) contained fibrolytic enzymes (cellulase 3500 CU/g, xylanase 2000 XU/g, and β-glucanase 17 500 GU/g) and amylolytic enzyme (amylase 37 000 AU/g) (enzyme activities determination details are shown in the online Supplementary File). Feed intake and milk production were recorded daily, whilst the chemical composition and particle-size distribution of feed samples, as well as the physical and chemical indices and SCC of milk samples, were measured for three consecutive days (the first to third day of experimental period). Due to limited labor availability, the rumen pH and volatile acid profile, as well as the serum parameters were sampled for one day. Further experimental details are provided in the online Supplementary File.
Statistical analysis
The study was performed using a completely randomized single-factor design. The daily averages of feed intake, production and feed analysis data were calculated and used for further statistical analysis. For statistical analyses, SPSS software (Version 22.0, SPSS Inc., Chicago, USA) was used to determine the differences of all measures between control and experimental groups, with supplementation of compound enzyme preparation as the fixed factor and the cow as a random factor. The model employed was:
$$Yij = \mu + {\rm treatment}\;i + {\rm cow}\;j + \varepsilon \,ij$$where Yij = the kth observation of the jth cow in the ith treatment, μ = the overall mean, treatment i = the fixed effect of the ith treatment (i = 0 to 1), cowj = the random effect of the jth cow (j = 1 to 10), εij = the residual error associated with the jth cow in the ith treatment. All results were expressed as the mean and sem Significance was declared at P ≤ 0.05.
Results and discussion
Supplementing compound enzyme preparation increased diet non-fibrous carbohydrate content (NFC) (P < 0.05: online Supplementary Table S2). This could probably be explained by the pre-digestive mechanism of fibrolytic enzymes (Adesogan et al., Reference Adesogan, Arriola, Jiang, Oyebade, Paula, Pech-Cervantes, Romero, Ferraretto and Vyas2019), such that the fibrolytic enzymes could partially solubilize acid and neutral detergent fibers, releasing sugars and free or monomeric hydroxycinnamic acids before feeding (Romero et al., Reference Romero, Ma, Gonzalez and Adesogan2015). Moreover, EXP had improved dry matter intake (DMI) and NFC intake (P < 0.05: Supplementary Table S2). The improvement of DMI is consistent with studies of orally supplied fibrolytic enzymes (Beauchemin et al., Reference Beauchemin, Colombatto, Morgavi and Yang2003; Adesogan, Reference Adesogan2005). This is probably due to the improved palatability of the increased sugars released from the hydrolyzation of fiber (Adesogan, Reference Adesogan2005), and the reduced rumen and gut fill by the improved digestion rate (Beauchemin et al., Reference Beauchemin, Colombatto, Morgavi and Yang2003, Adesogan, Reference Adesogan2005).
EXP had increased molar percentage of rumen butyrate and isobutyrate (P < 0.01) as well as decreased molar percentages of propionate and valerate (P < 0.05: Table 1). These results are similar to the research of Tricarico et al. (Reference Tricarico, Johnston, Dawson, Hanson, Mcleod and Harmon2005), who found improved acetate and butyrate as well as reduced propionate molar proportions in steers and lactating dairy cows with dietary addition of α-amylase. In a subsequent study, Tricarico et al. (Reference Tricarico, Johnston and Dawson2008) hypothesized a cross-feeding mechanism that the supplemented α-amylase and fibrolytic enzymes would hydrolyze amylose, cellulose and xylans into oligosaccharides, thereby providing substrate to non-fibrolytic or non-amylolytic bacteria, giving these bacteria a competitive advantage. This hypothesis could probably explain the current study. Due to the ratio between fibrolytic and amylolytic enzymes, or the ratio of forage to starch in diet, or the presence of butyrate producing bacteria (Selenomonas ruminantium GA192) that could use both malto- and xylo-oligosaccharides (Tricarico et al., Reference Tricarico, Johnston and Dawson2008) as substrate, the supplemented enzymes in the current study provided a competitive advantage to butyrate producing bacteria. EXP exhibited an increase in serum alkaline phosphatase (ALP) activity (P < 0.01) and albumin concentration (P < 0.05: Supplementary Table S3). The serum ALP activity and albumin concentration of both CON and EXP were in the normal and healthy range (70–144 U/l and and 27–47 g/l, respectively) as reported by Cozzi et al. (Reference Cozzi, Ravarotto, Gottardo, Stefani, Contiero, Moro, Brscic and Dalvit2011) and Lager and Jordan (Reference Lager and Jordan2012).
Effects of compound enzyme preparation on rumen fermentation (n = 10 cows/group) and production performance in dairy cows (n = 3, milk samples were collected from the 1st to 3rd day of the experimental period)
1 Control (CON) group, without supplementation of compound enzyme preparation.
2 Experimental (EXP) group, with supplementation of compound enzyme preparation.
3 Feed efficiency = FCM yield (kg/d)/DMI (kg/d).
a,bDifferent superscripts within a row indicate a significant difference (P < 0.05). sem, standard error of the mean; FCM, 4% Fat corrected milk yield = Actual milk yield (kg/d) × (0.4 + 15 × Milk fat content (%)); ECM, Energy corrected milk yield = Actual milk yield (kg/d) × (0.3246 + 12.86 × Milk fat content (%) + 7.04 × Milk protein content (%)); SSC, somatic cell count.
EXP exhibited increased yield of milk, fat corrected milk (FCM), energy corrected milk (ECM), milk fat and milk protein (all P < 0.01 or better: Table 1). Improved milk performance was also shown in the studies of supplemented fibrolytic enzymes (Arriola et al., Reference Arriola, Oliveira, Ma, Lean, Giurcanu and Adesogan2017) and amylolytic enzymes (Tricarico et al., Reference Tricarico, Johnston, Dawson, Hanson, Mcleod and Harmon2005; Andreazzi et al., Reference Andreazzi, Pereira, Reis, Pereira, Júnior, Acedo, Hermes and Cortinhas2018; Bachmann et al., Reference Bachmann, Bochnia, Mielenz, Spilke, Souffrant, Azem, Schliffka and Zeyner2018). It was attributed to the improvement of DM and NDF digestibility by Arriola et al. (Reference Arriola, Oliveira, Ma, Lean, Giurcanu and Adesogan2017), and to the promotion of rumen starch fermentation and the propionate absorption for liver gluconeogenesis by Andreazzi et al. (Reference Andreazzi, Pereira, Reis, Pereira, Júnior, Acedo, Hermes and Cortinhas2018). By contrast, in the research of Tricarico et al. (Reference Tricarico, Johnston, Dawson, Hanson, Mcleod and Harmon2005), the nutrient digestibility was not affected but the rumen VFA profile (increased butyrate and decreased propionate proportions) and serum metabolite concentration (higher BHBA, NEFA concentrations and unaffected glucose concentration) were changed, indicating that the supplementation of amylolytic enzyme might improve milk yield by affecting ruminal fermentation and concomitantly changing serum metabolite concentrations. Similarly, the enhanced milk performance in the current study is probably because of the effect on ruminal fermentation. However, it should be remembered that increased butyrate and decreased propionate proportion is normally considered to reduce blood glucose concentration because propionate is gluconeogenic and butyrate is ketogenic, and butyrate has been reported to inhibit the hepatic uptake of propionate. Tricarico et al. (Reference Tricarico, Johnston, Dawson, Hanson, Mcleod and Harmon2005) found that when cows were supplemented with α-amylase with a dose of 240 DU per kg TMR, the increase in rumen butyrate, which was at the expense of propionate, was not large enough to decrease liver gluconeogenesis or blood glucose content. Additionally, the rumen butyrate concentration has been reported to have a strong positive correlation with milk yield (Seymour et al., Reference Seymour, Campbell and Johnson2005).
Recall that the two previous studies using combined fibrolytic and amylolytic enzymes (Hristov et al., Reference Hristov, Basel, Melgar, Foley, Ropp, Hunt and Tricarico2008; Zilio et al., Reference Zilio, Del Valle, Ghizzi, Takiya, Dias, Nunes, Silva and Rennó2019) did not find any effect on milk yield. One explanation for Hristov et al. (Reference Hristov, Basel, Melgar, Foley, Ropp, Hunt and Tricarico2008) is insufficient enzyme dose. Another explanation is the delivery method of direct supplementation into rumen (Beauchemin et al., Reference Beauchemin, Colombatto, Morgavi and Yang2003). Because the pre-ingestive effects of enzymes on feed were lacking, and the homogeneity between feed and enzymes was decreased, the lack of effect on milk yield for Zilio et al. (Reference Zilio, Del Valle, Ghizzi, Takiya, Dias, Nunes, Silva and Rennó2019) could probably be attributed to the differences in time and diet portion of adding enzyme. In the research of Adesogan (Reference Adesogan2005), the enzymes were supplemented once a week into concentrate during its preparation. This delay may have been too long, resulting in reduced enzyme activity. These workers did show positive responses on production when utilizing high concentrate to forage ratio (62:38) in the diet, but not when a lower concentrate to forage ratios (45:55 to 40:60) was used Adesogan, Reference Adesogan2005). Hence, a proper diet composition for enzyme addition should consider the concentrate to forage ratio. The concentrate to forage ratio of Zilio et al. (Reference Zilio, Del Valle, Ghizzi, Takiya, Dias, Nunes, Silva and Rennó2019) was about 52:48, which is closer to the range of 45:55 to 40:60. Therefore, adding enzymes into the concentrate may not be effective.
Lactation stage (Beauchemin et al., Reference Beauchemin, Colombatto, Morgavi and Yang2003) and parity (Wathes et al., Reference Wathes, Cheng, Bourne, Taylor, Coffey and Brotherstone2007) of cows might result in variable responses. As indicated by Bachmann et al. (Reference Bachmann, Bochnia, Mielenz, Spilke, Souffrant, Azem, Schliffka and Zeyner2018), the supplementation of exogenous amylase only promoted the milk yield of high-producing (≥32 kg milk/day) early lactation primiparous cows, whilst late-lactation lower producing primiparous cows as well as early- and late-lactation multiparous cows were not affected. Late- and mid-lactation multiparous cows were used in the research of Hristov et al. (Reference Hristov, Basel, Melgar, Foley, Ropp, Hunt and Tricarico2008) and Zilio et al. (Reference Zilio, Del Valle, Ghizzi, Takiya, Dias, Nunes, Silva and Rennó2019). Early-lactation cows are more responsive to exogenous enzyme addition due to higher energy requirements caused by calving and higher milk yields (Beauchemin et al., Reference Beauchemin, Colombatto, Morgavi and Yang2003), and primiparous cows have larger magnitude of response than multiparous, because they have not previously experienced calving and lactation, therefore they are more sensitive to energy in the early lactation period (Wathes et al., Reference Wathes, Cheng, Bourne, Taylor, Coffey and Brotherstone2007).
In conclusion, the dietary supplementation of a fibrolytic (cellulase, xylanase, and β-glucanase) and amylolytic (amylase) compound enzyme preparation increased diet NFC content, DMI and intake of NFC, affected rumen fermentation by increasing butyrate proportion at the expense of propionate, and enhanced milk performance in primiparous early-lactation dairy cows.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0022029924000475
Acknowledgements
The authors thank Guangdong VTR Bio-Tech Co., Ltd. for providing enzyme preparation product and thank Modern Farm (Baoji, China) for assistance to carry out this work. Besides, the authors are thankful to Kai Dong and Xiaoting Sun for their help on experiments. The study was supported by the National Natural Science Foundation of China (grant no. 31972592 and 32072761), the Science and Technological Project of Shaanxi Province (grant no. 2021NY-019) and the Science and Technological Project of Yulin (grant no. CYX-2020-075).
