**Rumen In Vitro Fermentation and In Situ Degradation Kinetics of Winter Forage Brassicas Crops**

**José Daza 1, Daniel Benavides 2, Rubén Pulido 3, Oscar Balocchi 1, Annick Bertrand <sup>4</sup> and Juan Keim 1,\***


Received: 13 September 2019; Accepted: 25 October 2019; Published: 1 November 2019

**Simple Summary:** Winter brassica crops such as kales and swedes are used to supply feed in times of seasonal shortage. However, to the best of our knowledge, there is little information about the fermentation characteristics of these forages in the rumen. This study assessed the nutrient concentration, in vitro fermentation and in situ rumen degradation characteristics of *Brassica oleracea* (L.) ssp. *acephala* (kales) and *Brassica napus* (L.) ssp. *napobrassica* (swedes). The kales and swedes both showed different nutrient concentrations and fermented fast and extensively in the rumen. However, in vitro fermentation of swedes resulted in lower acetate and greater proportions of butyrate and propionate. Varieties of swedes showed more differences in terms of degradation and fermentation in the rumen compared to kale varieties.

**Abstract:** The aim of the present study was to evaluate the nutritional value, the rumen in vitro fermentation, and the in situ degradation of *Brassica oleracea* (L.) ssp. *acephala* (kales) and *Brassica napus* (L.) ssp. *napobrassica* (swedes) for winter use. Five varieties of each brassica were used in three field replicates and were randomized in a complete block nested design. All forage varieties were harvested at 210 days post-sowing to analyze the chemical composition, in vitro gas production, volatile fatty acid (VFA) production and in situ dry matter (DM) and crude protein (CP) degradability. Kales presented higher DM and neutral detergent fiber (NDF) content (*p* < 0.01), whereas swedes showed higher CP, metabolizable energy (ME), glucose, fructose, total sugars, NFC, and nonstructural carbohydrate (NSC) content (*p* < 0.01). The kale and swede varieties differed in their CP and sugar concentrations, whereas the kale varieties differed in their DM and raffinose content. The rates of gas production were higher for swedes than for kales (*p* < 0.01). No differences between the brassica species (*p* > 0.05) were observed in the total VFA production, whereas kales had a higher proportion of acetate and swedes had higher proportions of butyrate (*p* < 0.05). Only the swede varieties showed differences in VFA production (*p* < 0.05). The soluble fraction "a", potential and effective in situ DM degradability were higher in swedes (*p* < 0.01), but kales presented greater DM and CP degradation rates. Differences were observed between brassica species in the chemical composition, degradation kinetics, and ruminal fermentation products, whereas differences among varieties within species were less frequent but need to be considered.

**Keywords:** kale; swede; volatile fatty acids; degradation rates

#### **1. Introduction**

Brassicas such as kales (*Brassica oleracea* (L.) ssp. *acephala*) and swedes (*Brassica napus* (L.) ssp. *napobrassica*) are used for ruminant feed during winter [1], which is a season with low pasture growth in humid temperate regions [2]. These forages can offer high dry matter (DM) production and nutritional quality in a short time, which is related to high metabolizable energy (ME), water-soluble carbohydrates (WSC), and low neutral detergent fiber (NDF) content [3,4]. Winter brassicas have been used successfully in sheep [4], dry cows [5], and lactating dairy cows [6]. In addition, forage brassicas have an environmental advantage; they reduce the amount of enteric methane (CH4) per unit of DM intake compared to ryegrass pasture [4,7]. Although nutrient concentrations in brassicas have been widely described, the nutritive value of these forages depends on the quantity of nutrients available to the animal, which is determined by fermentation processes [8] and the presence of secondary compounds such as glucosinolates and S-methyl-cysteine sulfoxide that are present in brassicas [9], thus meaning animal responses can be affected.

Complementary evaluation methods, such as the ruminal digestibility of nutrients or products of ruminal fermentation and metabolism, have been suggested to determine the real nutritive value of forages [10,11]. The ruminal in situ incubation technique is considered a reference method to estimate degradation parameters, when adjusted to suitable nonlinear models [12]. These parameters are used by feeding evaluation models to estimate nutritive value, nutrient supply, and animal performance [8]. On the other hand, the in vitro gas production technique (IVGPT) allows the determination of fermentation kinetics [13]; estimates of DM, protein, and fiber degradation; ruminal volatile fatty acid (VFA) content; and microbial protein synthesis [14]. The popularity of in vitro gas production (GP) stems mainly from the ability to exercise experimental control, the capacity to nondestructively screen a large number of substrates, the kinetic information obtained, and relatively low costs [10]. Thus, IVGPT offers a unique tool for researchers to address a wide range of nutritional issues in ruminants [15].

Whereas degradation kinetics and ruminal fermentation of summer brassica species (rape and turnip) and varieties [16] have been reported in the literature, few reports exist on the effect of winter brassica species (kales and swedes) and varieties on the in situ degradation kinetics and fermentation end products. For example, Sun et al. [4] have observed that sheep fed swedes showed modified VFA profiles in their rumen fluid and lowered methane yield in contrast with those fed kales or perennial ryegrass. Keogh et al. [1] have reported no effects on the rumen VFA concentration from increases in the dietary proportion of kales in the diets of dry cows. Valderrama and Anrique [17] have reported DM and crude protein (CP) degradation kinetics of kale leaves; however, to the best of our knowledge, such data have not been reported for swedes. Moreover, the nutritive value of brassicas varies among species and varieties within species [3,16], and, therefore, information is still lacking about rumen fermentation and the kinetics of winter brassica species such as kales and swedes.

Hence, the aim of this study was to determine the nutritive value of forage brassica species (kales and swedes) and varieties for winter use, based on their nutrient concentration, in vitro ruminal fermentation, and in situ rumen degradation kinetics.

#### **2. Materials and Methods**

All animal procedures were performed in accordance with the UK Animals (Scientific Procedures) Act and associated guidelines, and approved by the Animal Ethics Committee of the Austral University of Chile (approval number 144/2013).

#### *2.1. Site and Experimental Design*

This experiment was carried out at the Agricultural Research Station (39◦47 S, 73◦13 W) of the Austral University of Chile on a Typic Hapludand soil with an initial water pH of 5.8, Olsen-P of 19.1 mg/kg, exchangeable potassium of 214 mg/kg, and aluminum saturation of 3.1% (measured for the first 20 cm of the soil profile).

Prior to soil preparation and sowing, the weeds were controlled chemically with glyphosate at a dosage of 2025 g/ha of active ingredient. Two brassica species were evaluated (kales and swede), and five varieties were sown for each species: Caledonian (K1), Elba (K2), Sovereign (K3), Regal (K4), and Coleor (K5) for the kales and Major Plus (S1), Aparima Gold (S2), Highlander (S3), Dominion (S4), and Invitation (S5) for swedes. The plot sizes were 6 m by 4 m, with three replicates for each variety, and plots were arranged in field blocks. The varieties were established in October 2014 at a seed dosage of 4.0 (kales) and 1.5 kg/ha (swedes). Fertilizers were applied at sowing to correct any soil nutrient deficiencies. A fertilizer mixture (7 % N-30 % P2O5 -12 % K2O) at doses of 500 kg/ha (35 kg N/ha, 150 kg P2O5/ha, and 60 kg K2O/ha) and 46 kg/ha boronatrocalcite were applied at sowing. After emergence, weeds were controlled chemically by applying Lontrel 3A (clopyralid 475 g of active ingredient (a.i.)/L) and Tordon 24 k (picloram 240 g a.i./L) at doses of 300 and 200 cc/ha respectively. Five weeks after sowing, when the plants had two or three leaves, 125 kg N/ha (urea) was applied, and in January 2015, applications of 750 cc/ha of Aramo (tepraloxydim 200 g a.i./L) and 200 cc/ha of karate (50 g a.i./L Lambda-cyhalothrin) were made.

During the trial, five cuts were made, with an approximate interval of 30 days between cuts, with the first harvest occurring at 90 days after plant emergence. In each cut, 4 m<sup>2</sup> of each crop was harvested. The kale varieties were cut to 20 cm above the ground level, the swede varieties were collected manually, and soil attached to the roots was removed. Plants were weighed and then separated into the main components (leaf and stem for kales and leaf and bulb for swedes). The samples were then dried in a forced-air oven at 60 ◦C for 48 h for determination of the dry matter (DM).

Samples for nutrient concentrations and in vitro and in situ incubations were harvested at 210 days post-sowing, and the plants were separated into their morphological components (leaf and stems for kale varieties and leaf and bulbs for swede varieties) before being chopped and then being frozen at −20 ◦C. Later, they were lyophilized (Virtis 10-45 MR-BA, Gardiner, New York, NY, USA) and then ground (Wiley mill, 158 Arthur H. Thomas, Philadelphia, Pennsylvania, PA, USA) to 5 mm for in situ incubations and to 1 mm for nutrient concentrations analyses and in vitro gas production. For the in vitro and in situ incubations, samples of brassica species were composed of a leaf to stem ratio of 35:65 for kales and a leaf to bulb ratio 30:70 for swedes. The ratios were the average proportions of organ components at harvest obtained in this study.

#### *2.2. In Situ Incubations*

Three dry Holstein-Friesian cows (one for each block) fitted with ruminal cannulas (4 Pliable Rumen Cannula w/Stopper and U Bolt, Ankom Technologies, Macedon, New York, NY, USA) were used. At the time of the experiment, the ruminal pH (6.55 ± 0.32) of each cow was measured. Cows were offered grass silage (7.5 kg DM), summer turnips (4.5 kg DM) and commercial concentrate (2.0 kg DM). Samples of each variety were incubated in duplicate (~4 g DM) in Dacron bags (10 cm by 20 cm; pore size of 40–60 μm) and sealed. Up to 20 bags were deposited inside a lingerie bag (30 cm by 40 cm in size). Brassica samples from each block were incubated in a different cow and a control sample (commercial concentrate) was incubated in each cow to evaluate cow-to-cow variation.

Prior to incubation in the rumen, the bags were soaked in warm water (40 ◦C) for 20 min. Nine incubation times were considered: 0, 2, 4, 8, 10, 12, 14, 24, and 48 h. The samples corresponding to 0 h were not introduced into the rumen and were used to determine the soluble fraction. After the incubation, the bags containing the residue were removed from the rumen and were washed under running cold water until no further color appeared; then, they were frozen at −20 ◦C for 24 h to stop fermentative activity. Thereafter, the bags were defrosted, thawed in water at 4 ◦C, and washed with a commercial washing machine for 30 min at a "normal" wash setting. Finally, residues were oven-dried at 60 ◦C for 48 h. The residues were weighed, the DM was calculated by placing the samples in an oven at 105 ◦C for 12 h, and the CP concentration was determined to calculate nutrient loss.

A correction for small particle loss was made as follows. The samples of each brassica variety (1.5 g) were weighed in a beaker. Then, 40 mL of tap water was added and the mix was stored at room

temperature (20 ◦C) for 1 h; afterward, the mix was filtered through a nitrogen-free filter paper and washed eight times with 20 mL of water. The residues were oven-dried and analyzed individually. Degradation parameters (a, b, and c), potential degradation (PD) and effective degradability (ED) were corrected according to Hvelplund and Weisbjerg [18].

#### *2.3. In Vitro Incubations*

Duplicates (1 g) of each sample were incubated in 160 mL glass bottles. Each brassica variety, a control (commercial concentrate) sample and two blanks (bottles without substrate) were used. Subsequently, 85 mL of Goering-Van Soest medium and 4 mL of reducing agent (NaOH 2.5 mM and cysteine-HCl 2.5 mM) were added at 39 ◦C under continuous gasification (CO2) to maintain anaerobic conditions and the bottles were covered with rubber stoppers and aluminum seal.

The inoculum was extracted from two dry Holstein-Friesian cows with ruminal cannulas with a live weight (PV) of 560 ± 20 kg and a ruminal pH of 6.6 ± 0.53; at the time of the extraction, the animals were offered the same diet as in the in situ trial. Rumen fluid was obtained before the cows were fed in the morning and was stored in a thermos flask to preserve the temperature until being transferred to the laboratory. Once there, the fluid was filtered through four layers of cheesecloth while being maintained at a temperature of 39 ◦C and under a constant flow of CO2. Rumen fluids from the two donor cows were mixed in equal proportions and then inoculated (10 mL) into the bottles. After inoculation, the bottles were placed in a water bath at 39 ◦C under continuous horizontal movement at 50 rpm.

Once the rumen fluid was inoculated, the initial gas was extracted from the bottles. The gas pressure in the headspace of the bottles above atmospheric pressure was measured manually with a pressure transducer (PCE Instruments, Tobarra, Albacete, Spain) at 2, 3, 4, 5, 6, 8, 10, 12, 18, 24, 36, and 48 h, and the volume of gas produced was measured by extraction using syringes connected through a three-way Luer valve from the bottles until the visual display of the transducer read zero, and once the volume of gas produced was recorded, it was eliminated. Fermentations were stopped after 48 h by placing the bottles on ice. Each field block (five varieties of kales and five varieties of swedes) was incubated at different runs. Thus, the first block was incubated at a first run, those corresponding to block number two were incubated at the second run, and samples from block three were incubated at the third run. A control standard (commercial concentrate) was incubated at each incubation run to control the day-to-day variation.

Once the in vitro incubation was finished, the samples were kept on ice to stop fermentative processes and residue duplicates from each sample were collected and then centrifuged at 15,000× *g* and 4 ◦C. After centrifugation, 0.9 mL of the supernatant was extracted to determine the VFA concentrations with a GG-2010 gas chromatograph (Shimadzu Corporation, Kyoto, Japan).

#### *2.4. Analyses*

The dry matter content was measured by weighing the samples before and after drying with a forced-air oven, initially at 60 ◦C for 48 h and then at 105 ◦C for 12 h. The CP concentration was determined by combustion (Leco Model FP-428, Nitrogen Determinator, Leco Corporation, St Joseph, Minnesota, MI, USA) based on the DUMAS method (nitrogen × 6.25); digestible organic matter on a dry matter basis (DOMD) was measured according to Tilley and Terry [19]; neutral detergent fiber (aNDF) was measured by using a heat stable amylase [20]; and ash and ether extract (EE) were analyzed according [21] (Methods ID 942.05 and ID 920.39 for ash and EE respectively). Sugars (raffinose, sucrose, glucose, and fructose) were analyzed by Waters ACQUITY ultrahigh-performance liquid chromatography (UPLC, Waters, Milford, Massachusetts, MA, USA), and starch quantification was determined by colorimetric detection of non-soluble residues after enzymatic digestion with amyloglucosidase according to Pelletier et al. [22]. The sum of sugars and starch yielded the content of total nonstructural carbohydrates (NSC). An estimation of the combined organic acids plus neutral detergent soluble fiber (OA + NDSF) was calculated according to Hall et al. [23], where OA + NDSF =

NFC − NSC. Non-fibrous carbohydrates were calculated as follows: NFC = 100 − CP − aNDF − EE − ash + neutral detergent insoluble crude protein (NDICP).

#### *2.5. Calculations*

The in situ disappearance of DM and CP was determined using the non-linear model described by Ørskov and McDonald [12] to determine the potential degradation according to the exponential model

$$\text{PD} = a + b \times (1 - e^{-kt})$$

where *a* is the soluble fraction (fraction washed out at *t* = 0; this value resulted from the incubation of 0 h bags corrected for particle loss and fixed into the model), *b* is the insoluble but potentially degradable fraction, *k* is the degradation rate (per hour), and *t* is the time (h).

The effective degradability (ED) was calculated assuming a fractional passage rate (kp) of 2%, 5%, and 8% per hour according to the following equation:

$$\text{ED} = a + b \times c \% (c + \text{kp})$$

These parameters were corrected for the losses of small particles which are degraded in a similar way to the particles remaining in the bag, as reported by Hvelplund and Weisbjerg [18].

After correcting for gas production of the blanks, the obtained GP data were adjusted to the generalized Michaelis-Menten model without a lag phase [11], as seen in the equation

$$\mathbf{GP} = \mathbf{A} \times \left[ T^n / (T^n + K^n) \right]^2$$

where GP is the gas production at time *T*, A is the asymptote of GP (mL), *n* is the determined value of the shape of the curve, and *K* is the time taken to produce half of A.

The following parameters were calculated according to Groot et al. [24] and France et al. [11], i.e.,

fermentation rate at half-life (C) = *n*/(2 × *K*) maximal fermentation rate (MDR) <sup>=</sup> (*<sup>n</sup>* <sup>−</sup> 1)((*<sup>n</sup>*−1)/*n*)/<sup>k</sup> time to ferment *<sup>x</sup>*% of the substrate (*tx*) <sup>=</sup> *<sup>K</sup>* <sup>×</sup> ((X/(1 <sup>−</sup> X))(1/n))

where X = 0.25, 0.75, and 0.90 of A.

#### *2.6. Statistical Analyses*

Parameters of in vitro GP, in situ degradation kinetics, VFA content and nutrient concentration were averaged for analytical replicates and analyzed with the MIXED procedure of SAS (SAS Institute, Cary, North Carolina, NC, USA).

Data were analyzed under a nested design, with three replicates organized in complete randomized blocks. Varieties were nested within species and the random effect of the field replicate was included as a block. When significant differences (*p* < 0.05) were found, the Tukey-Kramer multiple-comparison test was used in the LSMEANS procedure statement in SAS.

#### **3. Results**

#### *3.1. Nutrient Concentration and Sugar Profile*

Kales had greater concentrations of DM, EE (+4 g/kg), and aNDF (+123 g/kg) than swedes (*p* < 0.01; Table 1). Swedes showed greater CP (+25 g/kg; *p* < 0.01) and total sugar concentrations (+75 g/kg; *p* < 0.01) than kales, as well as individual sugars, such as glucose and fructose, NSC, OA + NDSF and DOMD. Raffinose and sucrose concentrations were greater in kales (*p* < 0.01). The ash and starch concentrations did not vary between the species (*p* > 0.05).

The kale varieties differed in their DM, CP, EE, raffinose, glucose, fructose, sugars, starch, and NSC concentrations (*p* < 0.01). Coleor had greater concentrations of CP (+30 g/kg) than Regal; Elba and Sovereign greater EE than Coleor; Coleor greater raffinose than Coledonian and Sovereign; and Regal and Coledonian greater concentrations of glucose and fructose than Sovereign. Finally, Regal showed greater concentrations of total sugars than Elba and Sovereign; Sovereign and Coleor greater concentrations of starch than Coledonian; and Regal the greatest NSC concentration of all varieties (231 g/kg), whereas Elba and Sovereign had the lowest (190 g/kg and 194 g/kg, respectively).

Furthermore, the swede varieties differed in their CP, glucose, fructose, sugars, starch, and NSC concentrations (*p* < 0.01). The concentrations of ash, aNDF, sucrose, OA + NDSF, and DOMD for varieties of both species were not different (*p* > 0.05). Invitation showed greater concentrations of CP (+30 g/kg) than Aparima Gold. Major Plus and Highlander showed the greater concentration of glucose, fructose, and sugars (280 g/kg and 288 g/kg, respectively, compared to 233 g/kg, 241 g/kg, and 234 g/kg in Aparima Gold, Dominion and Invitation). For starch, Aparima Gold and Dominion had greater concentrations (32 g/kg and 26 g/kg, respectively) than Highlander (7 g/kg). Finally, the concentrations of NSC in Major Plus and Highlander (291 g/kg and 295 g/kg, respectively) were greater than that for Invitation.

#### *3.2. In Situ Degradation Parameters*

Dry matter degradation parameters differed between brassica species (Table 2) except for the insoluble but potentially degradable fraction "b" (*p* > 0.05). Swedes had a higher soluble fraction "a" compared to kales (591 g/kg and 499 g/kg, respectively; *p* < 0.01) but a lower degradation rate "c" (0.25 h−<sup>1</sup> and 0.34 h<sup>−</sup>1, respectively; *p* < 0.01). However, swedes showed greater PD (+90 g/kg) and ED with ruminal passage rates of 2%, 5%, and 8% per hour compared to kales (*p* < 0.01). No significant differences were found in the in situ degradation parameters for the kale varieties (*p* > 0.05). Within the swede varieties, fraction "a" was greater for Major Plus (634 g/kg) compared to Aparima Gold (545 g/kg), Dominion (581 g/kg), and Invitation (586 g/kg; *p* < 0.01).

For the in situ CP degradation parameters, a species effect was observed on the fractional degradation rate "c", with kales having a faster degradation rate compared to swedes (0.48 h−<sup>1</sup> and 0.36 h<sup>−</sup>1, respectively; *p* < 0.01), whereas no brassica species effect was observed on the other in situ CP degradation parameters with average values of 559 g/kg, 380 g/kg, and 939 g/kg for "a", "b", and PD, respectively (*p* > 0.05). Fractions "a" and "b" were affected by the kale varieties: Coleor presented a higher "a" than Regal and Caledonian (+159 g/kg and +139 g/kg, respectively; *p* < 0.01). On the other hand, Caledonian and Regal had a greater "b" fraction compared to Coleor (*p* < 0.05). Effective degradability at 8% per hour of Sovereign (904 g/kg) and Coleor (900 g/kg) were higher than that of Regal (839 g/kg; *p* < 0.05). No effects for the swede varieties were found for any of the CP degradation parameters (*p* > 0.05).


soluble fiber; DOMD, digestible organic matter on DM basis. Kale varieties: K1, Caledonian; K2, Elba; K3, Sovereign; K4, Regal; K5, Coleor. Swede varieties: S1, Major Plus; S2, Aparima;

S3, Highlander; S4, Dominion; S5, Invitation.

*Animals* **2019**, *9*, 904

**Table 1.** Nutrient

concentration

 (g/kg dry matter) of five varieties of kale (from K1 to K5) and swedes (from S1 to S5).


*Animals* **2019** , *9*, 904

#### *3.3. In Vitro Fermentation Products*

The in vitro GP parameters were affected by the brassica species, whereas for varieties within species no effect was observed (Table 3). The GPs at 24 h and 48 h and "A" were higher for swedes compared to kales (256 mL/g, 285 mL/g, and 285 mL/g against 233 mL/g, 260 mL/g, and 261 mL/g DM, respectively; *p* < 0.01). Fermentation rate parameters "C" and "MDR" (h−1) were slightly faster for swedes (0.15 h<sup>−</sup>1) compared to kales (0.14 h−1; *p* < 0.05). However, no differences were observed for the time to fermentation of 25%, 50%, and 75% of the substrate (h), whereas 90% of the substrate was fermented 1.1 h earlier for swedes (*p* < 0.05).

**Table 3.** In vitro gas production kinetics of five varieties of kales (from K1 to K5) and swede (from S1 to S5).


<sup>1</sup> SEM standard error of the mean; 24 h GP (mL/g DM), gas production after 24 h of incubation; 48 h GP (mL/g DM), gas production after 48 h of incubation; A, asymptotic gas production (mL/g DM); K, time to ferment 50% of the substrate (h); C, degradation rate at half-life (per h); MDR, maximal degradation rate (per h); t25, t75, and t90, time to ferment 25%, 75%, and 90% of the substrate, respectively (h).

For the total volatile fatty acids (tVFA) concentrations, the propionate and branched-chain VFA (BCVFA) molar proportions of tVFAs showed no effects for brassica species (*p* > 0.05; Table 4). The acetate molar proportions of tVFAs (+48 mmol/mol) and A:P ratio were greater for kales, whereas the fermentation of swedes increased the molar proportions of butyrate (+33 mmol/mol). Kale varieties showed no differences for tVFA and the relative proportions of the different VFAs, whereas the tVFAs and the relative proportion of acetate and butyrate were affected by swede varieties. Major Plus showed a higher concentration of tVFAs (61.3 mM) compared to Dominion (45.5 mM), and Major Plus, Aparima Gold, and Invitation had higher relative proportions of acetate (555 mmol/mol, 564 mmol/mol, and 572 mmol/mol, respectively) compared to Dominion, which showed a higher relative proportion of butyrate (162 mmol/mol) than Aparima Gold and Invitation (128 mmol/mol and 120 mmol/mol, respectively).


Highlander; S4, Dominion; S5, Invitation.

**Table 4.**In vitro rumen volatile fatty acids (VFA) of five varieties of kale (from K1 to K5) and swede (from S1 to S5).

*Animals* **2019**, *9*, 904

#### **4. Discussion**

#### *4.1. Nutrient Concentration*

The current study reports the nutritive value of five varieties of two winter forage brassica species that are used to complement the feeding base used in grazing systems when pasture availability is not sufficient to fulfill livestock requirements. Forage brassicas have a higher demonstrated DOMD than perennial pastures; Sun et al. [4], for example, have reported that the DOMD of kales and swedes (883 g/kg and 918 g/kg DM) is 25% higher than that in perennial ryegrass pastures (703 g/kg DM). However, DOMD values observed by the current study are lower than those previously reported for kales and swedes (727 g/kg and 831 g/kg DM).

Sun et al. [4] and Westwood and Mulcock [3] have observed that kales contain higher values of DM, EE, and aNDF than swedes, which is similar to the effect observed in the present study. Regarding CP, swedes showed a higher concentration (136 g/kg) compared to kales (111 g/kg). Westwood and Mulcock [3] have reported similar values for swedes (137 g/kg) but lower values for kales (97 g/kg); however, they have observed that CP is highly variable among kale varieties, ranging from 63 g/kg to 138 g/kg, which is in accordance with our study where the CP concentration of the kale varieties ranged from 88 g/kg to 128 g/kg. These differences are mainly associated with the leaf to stem ratio, as forage brassicas concentrate more CP in their leaves [25]. For example, Valderrama and Anrique [17] have reported CP concentrations of 225 g/kg in kale leaves.

The content of sugars was similar to that reported by Sun et al. [4], with swedes having a higher concentration of sugars compared to kales. Winter brassicas have higher sugars compared to grass-based permanent pastures and perennial ryegrass, where values range from 73 g/kg to 118 g/kg when harvested during winter and early spring, the times when winter brassicas are used [26,27].

The amount of raffinose and sucrose were higher in kales; however, these amounts were not enough to compensate for the higher concentration of fructose and glucose in swedes. Importantly, varieties of both kales and swedes differed in the concentrations of fructose and glucose. In the case of kales, the Regal concentrated almost twofold the amounts of glucose and fructose compared to Sovereign, whereas for swedes, the Major Plus and Highlander concentrated approximately 20 g/kg and 30 g/kg more than the other three varieties. This approach is important, as it considers the type and amount of sugars demonstrated to affect fermentation in the rumen and the end products such as the VFAs [28].

Additionally, the concentration of OA + NDSF was evaluated, and this was higher for swedes (327 g/kg DM) than for kales (291 g/kg DM). This parameter was evaluated in summer brassicas by Keim et al. [16], who found it to be 313 g/kg and 314 g/kg DM for turnip and rape, respectively. Neutral detergent soluble fiber is mainly composed of galactans, β-glucans, soluble hemicelluloses, and pectic substances [23], while organic acids (OA) are mainly carbohydrate derivatives and may contain lactate, citric acid cycle components and secondary plant compounds such as oxalate and shikimate [23]. Previous studies have reported concentrations of pectins between 77 g/kg and 129 g/kg DM for brassicas in general [29]. Likewise, Sun et al. [4] have reported 80 g/kg and 69 g/kg DM for kales and swedes, respectively. In addition to sugars, these components of the NDSF could have an effect on ruminal fermentation and VFA concentrations, and although NDSFs are readily and extensively broken down in the rumen, they do not mimic the pH-lowering effect of starch because they generally produce little or no lactate, and their fermentation ceases at low pH [30]. On the other hand, organic acids do not support microbial growth [23] and therefore have little impact on rumen metabolism.

#### *4.2. In Situ Degradation Parameters*

Brassica forages are characterized by their high concentration of readily fermentable carbohydrates and high digestibility [9], which is in accordance with the high soluble fraction and fast degradation rates observed in our study for both species. To the best of our knowledge, limited data exist from reports on degradation kinetics of kales [17] and no published literature has been found regarding the degradation kinetics of swedes. The greater soluble fraction observed in swedes is in accordance with its greater concentration of sugars and nonstructural carbohydrates compared to kales. Both, kales and swedes showed a fast degradation rate and extensive degradation of DM and CP compared with other forages used for livestock feeding during winter such as grass pastures, silages, and hay [31,32], and they showed a similar potential degradability but faster degradation rate compared to other winter fodder crops, such as fodder beets [33], and summer brassicas, such as turnips and forage rape [16]. The faster degradation rates observed in our study compared to those observed by Keim et al. [16] in summer brassicas and the values reported by Valderrama and Anrique [17] for kales might be explained by the composition of the diet offered to the cannulated cows. In this experiment, cows were offered brassicas in their diet, and, therefore, rumen microbes might have been adapted to degrade these kind of ingredients, which would have increased the fermentation rate [34].

Even though kales showed a faster degradation rate of crude protein than swedes, due to the similar soluble (a) and insoluble but potentially degradable (b) fractions, PD and ED were similar among species. The crude protein ED of kales was similar to the values reported by Valderrama and Anrique [17] when calculated at a passage rate of 2% per hour and was slightly greater with passage rates of 5% and 8% per hour because of the fast degradation rate observed in our study (0.48 h−1). Importantly, the CP fractions "a" and "b" differed among the kale varieties and need to be considered for the ration formulation models. This variation in degradation characteristics associated with genetic differences has been reported previously by Sun et al. [35] with perennial ryegrass. The varieties with greater CP concentration (Coleor) also showed the highest soluble fraction of crude protein.

#### *4.3. In Vitro Fermentation*

Higher 24 h, 48 h, and asymptotic GP in swedes reflects greater digestibility compared to kales, since total GP is an indicator of forage digestibility [36] and has been related to DM degradability [37], which is in accordance with the greater DM potential and effective degradability observed for swedes compared with kales. This difference may be attributed to the chemical composition, as forages with lower aNDF content and higher NSC, such as swedes, present greater GP [38]. As observed for DM potential and effective in situ degradability, the in vitro GP parameters were not affected by varieties within species, as a good correlation between the GP measurements and in situ degradability has been found [39]. Contrary to what was observed with in situ DM degradation rates (faster for kales than swedes), in vitro gas production rates (C and MDR) were faster for swedes. This is because in vitro gas production rates take into account the gas produced from the soluble fraction, whereas the in situ degradation rate comes only from the insoluble but potentially degradable fraction. Nevertheless, C and MDR values of both swedes and kales (0.14 and 0.15, respectively) demonstrated faster fermentation compared to other typical feedstuffs used for ruminant feeding during winter, such as concentrate (0.11 h−<sup>1</sup> for both c and MDR), hay (0.03 and 0.05 h−<sup>1</sup> for c and MDR), silage (0.07 and 0.08 h−<sup>1</sup> for c and MDR) and pasture (0.09 and 0.10 h−<sup>1</sup> for c and MDR) [40].

Fast fermentation of both kales and swedes is reflected by parameters t25, k, t75, and t90, where, for example, 90% of both species ferment in less than 23 h, mainly due to the presence of readily fermentable carbohydrates such as sugars and NDSF. Hall et al. [41] have shown that fermentation of NDSF is faster than fermentation from NDF, indicating that forages high in NDSF tend to exhibit a rapid fermentation.

In contrast to the GP, the total VFA production was similar among species. This finding could be because the digested OM is fermented to VFA and GP or converted to microbial protein and therefore total VFA and GP are not always well correlated [42]. Total VFA production was different across swede varieties and related to the in situ DM soluble fraction; that is, the Major Plus variety presented the greater soluble fraction and tVFA production, whereas Dominion showed the lowest values for both tVFA and DM soluble fraction. The acetate and acetate to propionate ratio were greater for kale, which is related to the high aNDF and lower NSC concentrations in kales compared to swedes, as has been reported previously [27,42]. In addition, the highest proportion of butyrate found in swedes

coincides with the higher sugar concentration, since the fermentation of sugars generally increases the amount of butyrate in the rumen [28]. The greater acetate and lower butyrate proportions of tVFA for kales compared with swedes are in agreement with in vivo studies with sheep, where acetate was reduced from 515 mmol/mol to 412 mmol/mol, while butyrate was increased from 118 mmol/mol to 176 mmol/mol for sheep fed swedes compared with those fed kales [4].

Kale varieties had no effect on VFA production and the relative proportions of VFAs, whereas differences in the acetate and butyrate relative proportions of tVFA were observed for varieties of swedes. The varieties with greater butyrate relative proportions of tVFA were those with greater sucrose and fructose concentrations as well as a higher DM soluble fraction.

#### *4.4. Implications*

Although nutrient concentrations of winter brassicas and variations among varieties have been widely described [3] and their use in sheep [4], dry cows [5] and lactating dairy cows [6] have been reported, to the best of our knowledge few studies have evaluated the rumen fermentation processes of winter brassicas.

The main products of rumen fermentation are VFAs, and among these, propionate is a substrate for gluconeogenesis and is the main source of glucose in the animal, whereas the non-glucogenic acetate and butyrate are sources for long-chain fatty acid synthesis [43]. Glucogenic and lipogenic nutrient supply and VFA profile have been associated with animal energy balance. It has been suggested that in lactating cows increased energy intake is channeled largely through increases in rumen production of butyric and propionic acids and their yield of ATP to the host animal [43,44]. From an environmental point of view, production of acetate and butyrate liberates hydrogen, whereas propionate serves as a net hydrogen sink [45]. Consequently, diets that increase propionate and decrease acetate in the rumen are often associated with a reduction in ruminal CH4 production [46].

Therefore, the results we obtained for in vitro fermentation end products may lead us to infer some animal responses. However, these extrapolations must be done carefully, because the mechanisms governing microbial efficiency and VFA molar proportions in vitro are not necessarily valid in vivo. For example, in the rumen itself, feed and microbial biomass are subject to passage and VFA subject to passage and absorption [10], processes that do not occur under in vitro conditions.

One of our main findings is that swedes increased the relative molar proportion of butyrate at the expense of acetate compared with kales, however there are differences among varieties of swedes that must be considered and depend basically on their sugar concentration and type of sugars. There is generally a positive association of feeding sugars, such as sucrose, and increased milk and milk fat production, which may be due to the greater molar proportion of butyrate produced from those sugars [44,47]. However, to the best of our knowledge no studies have reported milk production responses of dairy cows fed swedes. In vivo results reported by Sun et al. [4] are similar to our study, with total rumen VFA concentrations being similar for sheep fed swedes or kales and greater butyrate and lower relative molar proportions of acetate found for sheep fed swedes. Complementarily, dry matter intake (DMI) was greater when kales were offered but energy intake was greater when feeding swedes resulting in lower methane emissions; however, no animal responses were reported. Conversely, Keogh et al. [6] have observed no differences in body weight and body condition score change between pre-calving dairy cows supplemented with kales or swedes during the dry period, but rumen fermentation data was not shown in their work.

Finally, the in situ ruminal degradation parameters of kales and swedes generated in this study can be used by researchers and nutritionists in feeding evaluation models to estimate the nutritive value, nutrient supply, and animal performance of livestock fed with winter brassicas.

#### **5. Conclusions**

Relative to winter forage brassica crops, our study demonstrated high digestibility for ruminant feeding in times when pasture availability is low. Differences between the two species were observed in terms of chemical composition, gas production, and dry matter degradation parameters, with swedes exhibiting a faster and more extensive degradation due to their greater concentrations of readily fermentable carbohydrates and lower NDF, which resulted in a lower acetate to propionate ratio and greater butyrate concentrations in the rumen. Additionally, differences among varieties within species were observed and must be considered when selecting certain varieties for use. For example, varieties of swedes showed differences regarding their in situ DM soluble fraction and in vitro tVFA and acetate and butyrate relative proportions of tVFA, whereas kale varieties differed in their in situ soluble CP and insoluble but potentially degradable CP fractions. Continuing with studies under in vivo conditions when feeding winter brassicas, especially to lactating dairy cows, is important because less information is available.

**Author Contributions:** Conceptualization, J.K., O.B., and R.P.; data curation, D.B., J.D., A.B., and J.K.; funding acquisition, J.K., O.B., and R.P.; investigation, A.B., D.B., J.K., and J.D.; methodology, J.K., O.B., and R.P.; project administration, J.K.; resources, J.K., O.B., and R.P.; supervision, J.K.; validation, J.K., O.B., and R.P.; writing—original draft, D.B., J.D., and J.K.; writing—review and editing, A.B., O.B., and R.P.

**Funding:** This research was funded by Dirección de Investigación y Desarrollo, Universidad Austral de Chile, Valdivia, Chile (DID-S2014-17).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

### **Comparative Grain Yield, Straw Yield, Chemical Composition, Carbohydrate and Protein Fractions, In Vitro Digestibility and Rumen Degradability of Four Common Vetch Varieties Grown on the Qinghai-Tibetan Plateau**

### **Yafeng Huang, Fangfang Zhou and Zhibiao Nan \***

State Key Laboratory of Grassland Agro-ecosystems, Key Laboratory of Grassland Livestock Industry Innovation of Ministry of Agriculture and Rural Affairs, College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020, China

**\*** Correspondence: zhibiao@lzu.edu.cn; Tel.: +86-931-8912-582

Received: 6 July 2019; Accepted: 23 July 2019; Published: 31 July 2019

**Simple Summary:** The common vetch (*Vicia sativa* L.) is an important legume crop of mixed crop-livestock systems that provides high-quality grains used as food/feed and straw used as ruminant feed. The objective of this study was to determine the variability in grain yield, straw yield, straw chemical composition, carbohydrate and protein fractions, in vitro gas production, and in situ ruminal degradability of four different varieties of common vetch grown on the Qinghai-Tibetan Plateau. The results showed that grain yield, straw yield, and straw nutrient value varied significantly among the four varieties. Overall, the findings indicated that in terms of straw yield and nutritive quality, variety Lanjian No. 1 has the greatest potential as a crop for supplementing ruminant diets in the smallholder mixed crop–livestock systems on the Qinghai-Tibetan Plateau.

**Abstract:** Four varieties of common vetch, including three improved varieties (Lanjian No. 1, Lanjian No. 2, and Lanjian No. 3) and one local variety (333A), were evaluated for varietal variations in grain yield, straw yield and straw quality attributes on the Qinghai-Tibetan Plateau. Crops were harvested at pod maturity to determine grain yield, straw yield, harvest index, and potential utility index (PUI). Straw quality was determined by measuring chemical composition, carbohydrate and protein fractions, in vitro gas production and in situ ruminal degradability. Results showed a significant effect (*p* < 0.01) of variety on the grain yield [875.2–1255 kg dry matter (DM)/ha], straw yield (3154–5556 kg DM/ha), harvest index (15.6–28.7%) and PUI (53.3–63.2%). Variety also had a significant effect on chemical composition, carbohydrate and protein fractions (*p* < 0.05) except non-structural carbohydrates and rapidly degradable sugars. Significant differences (*p* < 0.05) were observed among the varieties in potential gas production [188–234 mL/g DM], in vitro organic matter (OM) digestibility (43.7–54.2% of OM), and metabolizable energy (6.40–7.92 MJ/kg DM) of straw. Significant differences (*p* < 0.001) were also observed among the varieties in rapidly degradable DM fraction and effective DM degradability of straw; however, no difference was observed in other DM degradation parameters and neutral detergent fiber degradation parameters. In conclusion, based on straw yield and quality, Lanjian No. 1 has the greatest potential among the tested varieties as a crop for supplementing ruminant diets for smallholder farmers on the Qinghai-Tibetan plateau.

**Keywords:** common vetch; straw; nutritive value; varietal effect; ruminants

#### **1. Introduction**

Ruminants provides the majority of food worldwide, contributing approximately 25.9% of global meat production and nearly 100% of global milk production [1,2]. The demand for cattle and sheep meat has been growing at a global growth rate of 2.0% per year from 2016 and is predicted to continue till 2026 [3]. The Qinghai-Tibetan plateau supports approximately 41 million sheep and over 14.3 million cattle, one of the largest livestock systems in Asia [4,5]. The plateau is also the headwater region of most of Asia's five major rivers, including Yangtze, Yellow, Mekong, Ganges, and Indus rivers [4]. Thus, sustainable management of the Plateau is important for the livelihood of over 9.8 million nomadic populations and for protecting these crucial river systems.

Unfortunately, harsh climatic conditions (e.g., low temperatures) and short growing season on the Qinghai-Tibetan plateau limit crop production [4–6]. Combined with grassland degradation, the annual fodder gap in this region is expected to reach 2 million tonnes, which implies that each year 2.7 million sheep units with insufficient feedstuffs will result in 20–30% liveweight loss during winter and early spring [5,7]. An effective remedy to stabilize feed supply is to incorporate nitrogen-fixing annual cool-season feed legumes such as common vetch (*Vicia sativa* L.) into fallow lands that will improve soil properties and lead to increased grain and straw production [8].

The use of common vetch in the mixed crop-livestock systems has expanded greatly in the last decade [4,9–11]. Several studies showed high generation of crop residues after harvesting common vetch that has substantial potential to be used for feeding ruminant livestock [9,12]. Previous studies also reported that the nutritive quality of common vetch straw is relatively high, containing an average of 9.41% crude protein (CP), 55.2% in vitro organic matter digestibility (IVOMD) and 7.3 MJ/kg metabolizable energy (ME) [9,13]. This makes common vetch straw a good feed supplement for ruminants, which are offered low-quality cereal straw-based diets in many small-holder crop-livestock systems [9,14]. In addition, the fatty acid composition of grains has increased the use of common vetch as a feed for ruminants [15]. Although common vetch is important as a feed for ruminants, limited information is available on the varietal variations in straw composition (carbohydrate and protein fractions), in vitro gas production, and in situ ruminal degradability, because most of the earlier studies focused on grain yield, straw yield, and straw quality including only chemical composition, IVOMD and ME [9–13]. Russel et al. [16] suggested that carbohydrate and protein fractions can be used as a reliable indicator to accurately predict biological value and performance of feed in ruminants. Marcos et al. [17], and Blümmel and Ørskov [18] demonstrated that the chemical composition in combination with in vitro digestibility and in situ ruminal degradability can be used as crucial parameters to evaluate the nutritive value of feed.

Accordingly, the objective of this study was to evaluate grain and straw yields, as well as chemical composition, carbohydrate and protein fractions, in vitro gas production and in situ ruminal degradability of straw of four different common vetch varieties grown on the Qinghai-Tibetan Plateau.

#### **2. Materials and Methods**

#### *2.1. Location, Experimental Design, and Sampling*

A detailed description of the location, experimental design and sampling is in the companion paper on the nutritive value of common vetch grain [4] and are summarized here.

This experiment was conducted during the cropping season of 2015 at the Xiahe Experimental Station of Lanzhou University, Gansu, China (35◦45 N, 102◦34 E; altitude 2880 m). The region under this study is situated at the eastern margin of the Qinghai-Tibetan Plateau. The location received an annual average precipitation of 452 mm (80% in May and September) and recorded a mean annual air temperature of 3.5 ◦C (1984–2014, 31 years). The previous crop was rape (*Brassica campestris* L.).

Three improved varieties (Lanjian No. 1, Lanjian No. 2, Lanjian No. 3) and one local variety (333A) were utilized in the field study. These improved varieties, developed by the Common Vetch Breeding Program of Lanzhou University, are well-adapted and extensively grown by smallholder farmers in the

region surrounding the research site. Agronomic characteristics of these tested varieties are shown in Table 1. All four varieties were planted under the same agronomic conditions following a completely randomized design with four replicates. The individual plot area was 40 m<sup>2</sup> (8 m <sup>×</sup> 5 m) with a row spacing of 20 cm, and grains were sown by hand at a depth of 3–5 cm at a density of 150 viable grains m-2. Grains were sown (6 May 2015) before inoculation with rhizobium (CCBAU01069, China Agricultural University, Beijing, China), which was recommended based on the symbiont performance of these varieties [19]. Irrigation and fertilizers were not applied after sowing, and weeds in each plot were adequately controlled manually.


**Table 1.** Agronomic characteristics of the common vetch varieties utilized in this study.

Source: Ministry of Agriculture, Beijing, China.

Harvesting of the common vetch plants was done at the pod maturity (6, 15, 15, and 26 September for Lanjian No. 3, 333A, Lanjian No. 2 and Lanjian No. 1, respectively). The crops were manually harvested from two representative subplots (1 m × 1 m) of each plot for each variety and threshed to obtain grain and straw samples. Harvest index was calculated as follows: Harvest index (%) = Grain yield × 100 / (Grain yield + Straw yield). All samples were oven-dried at 65 ◦C for 48 h and ground to pass through a 2-mm sieve for analysis of in situ ruminal incubation and to pass through a 1-mm sieve for chemical analysis and in vitro gas production measurement. All procedures involved animals were approved by the Animal Ethics Committee of Lanzhou University (protocol AEC-LZU-2016-01).

#### *2.2. In Vitro Gas Production*

Four adult Dorper rams (approximately 33-month-old; 58.4 ± 1.24 kg body weight) fitted with flexible rumen cannulas were used as donors of ruminal fluid. The rams were kept in individual stalls and had free access to fresh water and mineral/vitamin licks. The rams were daily fed 1.2 kg of 550 g/kg DM sheepgrass [*Leymus chinensis*, (Trin.) Tzvel], 140 g/kg DM soybean meal, 294 g/kg DM maize (*Zea mays* L.) seed, 8.6 g/kg DM calcium hydrophosphate, 5.0 g/kg DM salt and 2.4 g/kg DM mineral-vitamin mix at maintenance energy level in equal portions at 08:00 and 16:30 hours. In vitro gas production (GP) was measured as described by Blümmel and Ørskov [18]. Rumen fluids were obtained before morning feeding and strained through four layers of cheesecloth into a preheated, insulated bottle. Briefly, approximately 200 mg DM of each sample (in duplicate) was weighed into calibrated glass syringes (100 mL). Each syringe was preheated at 39 ◦C before injecting 30 mL rumen fluid/buffer mixture [20]. Then, the syringes were placed vertically in a water bath at 39 ◦C with three syringes without a sample used as blank. The volume of GP was manually recorded after 0, 4, 6, 8, 12, 24, 36, 48 and 72 h of incubation and was blank corrected. The incubation run was repeated three times. All operations involving rumen fluid were conducted under a continuous flush of CO2 to ensure anaerobic conditions.

The GP data were fitted with time using the following equation [17], as follows: *y* = *b* × [1 – e−*<sup>c</sup>* (*<sup>t</sup>* - *lag*) ], where *y* = the volume of GP at time *t*; *b* = the potential GP (mL/g DM); *c* (h<sup>−</sup>1) = the fractional rate of GP (h−1); *lag* = the initial delay in the onset of gas production (h); *t* = incubation time (h). Parameters *b*, c and *lag* were determined by an iterative least square method using the NLIN procedure of SAS 9.2 (SAS Inst. Inc., Cary, NC). The IVOMD [% organic matter (OM)] and ME (MJ/kg DM) were estimated using the equations of Menke and Steingass [21] as: IVOMD = 14.88 + 0.889 GP + 0.45 CP + 0.0651 ASH; ME = 2.20 + 0.136 GP + 0.057 CP + 0.0029 EE2, where GP = the net gas production after

24 h of incubation (mL/200mg DM), ASH = the ash content (% DM), CP = the crude protein content (% DM), and EE = the ether extract content (% DM).

#### *2.3. In Situ Ruminal Incubation*

In situ ruminal degradability of DM and neutral detergent fiber (NDF) of straw samples was determined using the nylon bag technique described by Nandra et al. [22]. Briefly, approximately 5.0 g of each sample (in duplicate) was weighted into nylon bags (9 × 5 cm; 50-μm pore size) and incubated in the ventral sacs of the rumen of the same four rams used for the production of ruminal fluid in Section 2.2. The bags were inserted into the rumen for 0 (control), 4, 8, 12, 24, 48 and 72 h. Following removal, the bags were briefly washed under cold running water and frozen (–20◦C) until further analysis. All bags were defrosted, manually washed in cold tap water until the water was clear, oven-dried at 60°C for 48 h, and weighed. The dried undigested residues of replicates per same time within sheep were pooled to measure DM and NDF. Straw DM and NDF disappearance rates were estimated from the difference in straw weight before and after incubation. The kinetic parameters of DM and NDF degradation were determined using the exponential equation described by Ørskov and McDonald [23]. The effective degradability of DM (EDDM) and NDF (EDNDF) were calculated as ED= A + [(B × C)/(C + k)], where A = the soluble fraction, B = the potentially degradable fraction, C = the rate of degradation of fraction B, k = the rumen outflow rate (0.031 h<sup>−</sup>1) [4].

#### *2.4. Laboratory Analysis*

Determination of DM (ID 930.15), nitrogen (N; ID 988.05), ether extract (EE; ID 920.85), ash (ID 938.08), acid detergent fiber (ADF; ID 973.18) and acid detergent lignin (ADL; ID 973.18) were analyzed following the methods of the Association of Official Agricultural Chemists [24]. The CP content was calculated by multiplying the nitrogen value by 6.25. The NDF content was determined following the method by Van Soest et al. [25] using heat-stable α-amylase and sodium sulfite. Contents of NDF and ADF were expressed inclusive of residual ash. Acid detergent insoluble protein (ADIP), neutral detergent insoluble protein (NDIP) were measured by Kjeldahl analysis of the ADF and NDF bag residues, respectively, using the procedure described by Licitra et al. [26].

Carbohydrate fractions of straw samples from the four common vetch varieties were determined as proposed by the Cornell Net Carbohydrate and Protein system (CNCPS) [27]. The system divides carbohydrate into four fractions in terms of their degradation rate as follow: CA, rapidly degradable sugars; CB1, intermediately degradable pectin and starch; CB2, slowly degradable cell wall; and CC, undegradable/lignin-bound cell wall. Total carbohydrates (TCHO) content was calculated as TCHO = 100 – (CP + EE + Ash) [28]. Non-structural carbohydrates (NSC) and structural carbohydrates (SC) were estimated using the equations given by Caballero et al. [29] as: NSC = TCHO – SC and SC = NDF – NDIP. Starch content was determined by enzymatic hydrolysis of α-linked glucose polymers [30].

The CP of straw samples was fractionated into five different fractions according to CNCPS as described by Licitra et al. [26] and Sniffen et al. [27]. These fractions include: fraction PA, non-protein nitrogen (NPN), calculated as the difference between total N and true protein N, analyzed using sulphuric acid (0.5 M) and sodium tungstate (0.30 M); fraction PB1, buffer-soluble protein, estimated by subtracting buffer-insoluble protein precipitated with freshly prepared (1 g/10 mL) sodium azide and borate-phosphate buffer (pH 6.7–6.8) solution from true protein; fraction PB2, neutral detergent- soluble protein, calculated by subtracting NDIP from buffer-insoluble protein; fraction PB3, acid detergent-soluble protein, calculated by subtracting ADIP from NDIP; and fraction PC, ADIP, is indigestible protein. All measurements were performed in duplicate and appropriate chemical standards were included in each analytical run.

#### *2.5. Calculations and Statistical Analyses*

Potential utility index (PUI) was estimated from the amount of utilizable portion of the total biomass yield for grain and straw regardless of the economic value as described by Alkhtib et al. [31]:

PUI (%) = 100 × [grain yield + 0.01 × IVOMD (%) × straw yield]/ total biomass yield

Data collected were subjected to one-way ANOVA using SPSS software (Version 21.0. IBM Corporation, Armonk, NY, USA). The fermentation parameters were subjected to separate analysis of variance with varieties as fixed effect and the incubation run as random effect. Differences between means were compared using the Duncan significant difference test at *p* < 0.05.

#### **3. Results**

#### *3.1. Grain Yield, Straw Yield, and PUI*

Table 2 shows a significant effect of variety on grain yield and straw yield (*p* < 0.01). The grain yield ranged from 875.2 to 1255 kg DM/ha with an average value of 1088 kg DM/ha. The grain yield of 333A and Lanjian No. 1 was significantly less than that of other improved varieties. The straw yield varied from 3154 to 5556 kg DM/ha. The least straw yield was observed for Lanjian No. 3 and the greatest for Lanjian No. 1.

Variety had a significant (*p* < 0.001) effect on harvest index and PUI. Harvest index varied from 15.6 to 28.7%. Harvest index of Lanjian No. 2 was less than that of Lanjian No. 3, but greater than that of the other varieties (*p* < 0.01). The PUI varied from 53.3 to 63.2%. The PUI of the local variety was less than the improved varieties, which had similar PUI (average 62.1%).

**Table 2.** Influence of variety on grain yield, straw yield, harvest index, and potential utility index (PUI) of four common vetch varieties.


a,b Within a raw, different letters represent the significant differences at *p*-value < 0.05. <sup>1</sup> DM, dry matter; SEM, standard error of the mean.

#### *3.2. Chemical Composition*

Table 3 shows a significant effect of variety on DM, ash, CP and EE contents of the straw samples (*p* < 0.01). The DM content among varieties varied from 90.1 to 90.5% and was the greatest in Lanjian No. 2. The ash content varied from 10.2 to 13.5% DM. The least ash content was for 333A and the greatest for Lanjian No. 1. The CP content varied from 9.76 to 13.8% DM. The local variety had significantly less CP content compared to the improved varieties. There were significant differences in CP content among the improved varieties, which was greater for Lanjian No. 1. The EE content varied from 0.459 to 1.11% DM. The EE content of 333A was similar to that of Lanjian No. 3 but was significantly greater than that of Lanjian No. 1 and Lanjian No. 2.

Significant differences (*p* < 0.05) were observed in the cell wall contents of different varieties. The NDF, ADF, and ADL contents varied from 45.0 to 54.1% DM, 27.4 to 33.2% DM, and 6.08 to 9.56% DM, respectively. The NDF, ADF and ADL contents were the greatest in 333A and the least in Lanjian No. 1. Hemicellulose and cellulose contents varied from 17.6 to 21.4% DM and 21.4 to 23.7% DM, respectively. The hemicellulose and cellulose contents of variety 333A were higher than those of Lanjian No. 1, but similar to those of other varieties. Phosphorus content varied from 0.185 to 0.296% DM. The phosphorus content was considerably greater (*p* < 0.05) in Lanjian No. 3 than in Lanjian No. 2

and 333A, with Lanjian No. 1 being intermediate. Calcium content was also significantly influenced by variety (*p* < 0.001). It varied from 1.00 to 1.54% DM and was the greatest in Lanjian No. 1.

**Table 3.** Influence of variety on chemical composition (% dry matter unless stated otherwise) of straw in four common vetch varieties.


a,b Within a raw, different letters represent the significant differences at *p*-value < 0.05. <sup>1</sup> SEM, standard error of the mean.

#### *3.3. Carbohydrate and Protein Fractions*

As shown in Table 4, the TCHO content was significantly different (*p* < 0.001) among varieties and varied from 72.2 to 79.0% DM. The greatest TCHO content was observed in 333A and the least in Lanjian No. 1. The NSC content varied from 29.3 to 31.2% DM with no difference (*p* > 0.05) among varieties. The SC content varied from 41.1 to 49.7% DM. The variation in SC content was the greatest in 333A and the least in Lanjian No. 1. Significant differences (*p* < 0.05) were observed in CHO fractions except CA fraction (Table 4). The CB1 fraction was greatest (*p* < 0.001) in Lanjian No. 1 (26.0% CHO), intermediate in Lanjian No. 2 (22.9% CHO), and least in Lanjian No. 3 (20.1% CHO) and 333A (17.7% CHO). The CB2 fraction varied from 33.9 to 39.5% CHO. Variety 333A had significantly less (*p* < 0.05) CB2 fraction than Lanjian No. 2 and Lanjian No. 3 but similar to Lanjian No. 1. The Cc fraction varied from 20.2 to 29.0% CHO. The CC fraction of variety 333A was significantly greater (*p* < 0.001) compared to the improved varieties, which had similar CC fraction.

**Table 4.** Influence of variety on carbohydrate and protein fractions of straw in four common vetch varieties.


a,b Within a raw, different letters represent the significant differences at *p*-value < 0.05. <sup>1</sup> CA, rapidly degradable sugars; CB1, intermediately degradable pectin and starch; CB2, slowly degradable cell wall; CC, unavailable/lignin bound cell wall; CP, crude protein; TCHO, total carbohydrates; NSC, non-structural carbohydrates; PA, non-protein nitrogen; PB1, buffer soluble protein; PB2, neutral detergent soluble protein; PB3, acid detergent soluble protein; PC, indigestible protein; SC, structural carbohydrates; SEM, standard error of the mean.

Variety had a significant influence on the protein fractions of straw (*p* < 0.01; Table 4). The PA fraction varied from 7.16 to 11.5% CP, and the least value was recorded in 333A and the greatest in Lanjian No. 1. The PB1 fraction varied from 25.8 to 37.3% CP; the greatest value was recorded in 333A. The PB2 fraction varied from 10.8 to 34.0% CP. The variation in PB2 fraction was the least in 333A and the greatest in Lanjian No. 1. The PB3 fraction varied from 13.3 to 23.6% CP. The PB3 fraction was significantly greater for 333A than for Lanjian No. 3, and significantly greater for Lanjian No. 3 than the other varieties. The PC fraction varied from 15.4 to 21.1% CP. The Pc fraction was greatest in 333A and least in Lanjian No. 1.

#### *3.4. In Vitro Gas Production*

Table 5 shows the gas production parameters, IVOMD and ME of straw in four common vetch varieties. The potential gas production differed considerably (*p* < 0.001) between the varieties and varied from 188 to 234 mL/g DM. The potential gas production of Lanjian No. 3 was greater than that of 333A; however, it was less than that of the other varieties. The fractional rate of GP was not affected by variety (*p* > 0.05), averaging 0.0631 h<sup>−</sup>1. The *lag* value was similar (*p* > 0.05) for the four varieties (average 0.633 h).

Variety had a significant influence on the IVOMD content of straw (*p* < 0.001; Table 5). Straw IVOMD varied from 43.7 to 54.2% OM, and the varieties were ranked in order Lanjian No. 1 > Lanjian No. 2 > Lanjian No. 3 > 333A. Significant difference in the ME content was also observed among the different varieties (*p* < 0.001), and it varied from 6.40 to 7.92 MJ/kg DM. The least value was recorded for 333A and the greatest for Lanjian No. 1 and Lanjian No. 2.

**Table 5.** Influence of variety on gas production parameters, in vitro organic matter digestibility (IVOMD) and metabolizable energy (ME) of straw in four common vetch varieties.


a,b Within a raw, different letters represent the significant differences at *p*-value < 0.05. <sup>1</sup> *b*, potential gas production; *c*, fractional rate of gas production; DM, dry matter; *lag*, initial delay in the onset of gas production; OM, organic matter; SEM, standard error of the mean.

#### *3.5. In Situ Ruminal Degradability*

The soluble DM fraction was significantly different (*p* < 0.001) among varieties and varied from 22.8 to 28.4% DM (Table 6). The soluble DM fraction of 333A was similar to that of Lanjian No. 3, but less than that of Lanjian No. 1 and Lanjian No. 2. The potentially degradable DM fraction and rate of DM degradation were similar (*p* > 0.05) for the four varieties and recorded an average of 43.2% DM and 0.0442 h<sup>−</sup>1, respectively. The EDDM value was significantly different (*p* < 0.001) among the four varieties. Average EDDM value was 51.0% DM and ranged from 46.7 to 55.2% DM. Variety 333A had significantly less EDDM value compared to Lanjian No. 1 and Lanjian No. 2, but it was similar to Lanjian No. 3.

The NDF degradation profiles in situ are given in Table 5. Soluble NDF fraction, potentially degradable NDF fraction, and rate of NDF degradation were not influenced (*p* > 0.05) by variety and recorded an average of 11.5% NDF, 46.7% NDF and 0.0374 h−1, respectively. No difference was observed in EDNDF value among varieties, but there was a trend toward greater EDNDF for Lanjian No. 1 (*p* = 0.078).


**Table 6.** Influence of variety on in situ ruminal degradation kinetics of dry matter (DM) and neutral detergent fiber (NDF) of straw in four common vetch varieties.

a,b Within a raw, different letters represent the significant differences at *p*-value < 0.05. <sup>1</sup> *A*, soluble fraction; *B*, potentially degradable fraction; *C*, rate of degradation of fraction B; EDDM, effective dry matter degradability; EDNDF, effective neutral detergent fiber degradability; SEM, standard error of the mean.

#### **4. Discussion**

#### *4.1. Grain Yield, Straw Yield, and PUI*

In smallholder crop-livestock systems, improvement in crop straw yield implies increase in milk and meat production from ruminants [6,32]. Substantial variability differences among varieties were found for grain and straw yields, partly due to differences in days to pod maturity and harvest index as suggested by Abd El-Moneim [8], Larbi et al. [9], and Kafilzadeh and Maleki [32]. Our findings are consistent with the earlier reports on common vetch [9] as well as faba bean (*Vicia faba* L.) [31], lentil (*Lens culinaris*) [32] and chickpea (*Cicer arietinum*) [33]. The grain yield recorded in this study (875.2–1255 kg DM/ha) is within the reported range (287–1783 kg DM/ha) [9], but less than ranges reported value of 1340–2240 kg DM/ha [12]. Meanwhile, the straw yield (3154–5556 kg DM/ha) is greater than that reported by Larbi et al. [9] (629–2226 kg DM/ha), but slightly less than that reported by Albayrak et al. [12] (4620–7320 kg DM/ha). The yields vary between studies [9,12] as consequence of the differences in the varieties, agronomic practices and growing conditions (e.g., soil type and climate) [8,12,34]. The grain yield of the improved varieties was significantly greater than that of the local variety in this study. Similar results were reported for other leguminous crops such as faba bean [31] and lentil [35]. The local variety demonstrated inferior PUI compared to the improved varieties. This is consistent with the findings in faba bean [31]; however, contrary to the findings of Tolera et al. [34] who observed less PUI for the improved varieties compared with the local varieties.

#### *4.2. Chemical Composition*

Higher CP and lower cell wall contents (NDF, ADF, cellulose and ADL) can be used as indicators of good feed quality [28]. Large varietal differences in straw chemical composition observed in our study is in agreement with the earlier findings in common vetch [9,11,13], as well as faba bean [31], lentil [32] and chickpea [33]. The range of CP content observed in this study (9.76–13.8% DM) was more than the threshold content (8.0% DM CP) required for optimum activity of rumen microorganisms in ruminants [36]. The NDF content among the varieties varied from 45.0 to 54.1% DM with a mean value of 49.9% DM. Van Soest [37] indicated that NDF content over 65% leads to effects on voluntary intake and production by ruminants. This makes common vetch straw a good source of CP supplements for ruminants in smallholder crop-livestock/agro-pastoral systems. Common vetch straw NDF and ADF contents of 52.2 and 36.1% DM reported by Makkar et al. [13] are consistent with our results; however, the CP contents (6.2% DM) is less than our results. Phosphorus and calcium contents observed in this study are similar to those reported by Abreu and Bruno-Soares [38]. The differences in chemical composition of straw between studies may be due to varietal variability, differences in growing condition (e.g., soil type and climate), or differences in harvesting and postharvest handling practices.

#### *4.3. Carbohydrate and Protein Fractions*

The significant varietal differences in carbohydrate fractions of common vetch straw are in agreement with reported for sorghum (*Sorghum bicolor* [L.] Moench) [28], timothy (*Phleum pratense* L.) [39] and wheat (*Triticum aestivum* L.) [40]. The values for TCHO, NSC and SC obtained in this study are comparable with the previous studies on other legume crops such as *Trifolium alaxendrinum* [41]. Among the CHO fractions, CB2 fraction was the highest in the straw of common vetch varieties analyzed. Others have reported similar results in sorghum, berseem (*Trifolium alexandrium*) and cowpea (*Vigna sinensis*) [28,41]. However, there is limited data available on the CHO fractions of common vetch straw. The pattern of CHO fractions observed in our study for common vetch straw is comparable with the earlier reports on other forage crops [41–43].

Wide range of protein fractions observed in the varieties is consistent with the findings in sorghum [28] and wheat [40]. Swarna et al. [44] indicated that PB2 and PB3 fractions represent a bypass protein of forage, while Pc fraction represents the non-degraded fraction. Compared to other varieties, Lanjian No. 1 had lower Pc fraction and higher PB2 + PB3 fractions. These observations on protein fractions suggest that straw of variety Lanjian No. 1 could be used as the better nitrogen source for ruminants. There is limited data available on the contents of protein fractions of common vetch. The pattern of protein fractions revealed here is similar to the reports on lucerne (*Medicago sativa* L.) [43] and black gram [*Vigna mungo* (L.) Hepper] [44].

#### *4.4. In Vitro Gas Production*

The gas produced by in vitro fermentation reflects the degree of feed fermentation and digestibility [34]. In this study, we observed significant differences among varieties in potential gas production, but not in gas production rate and *lag* time. Our results are partially in line with previous studies on other crops such as spineless cacti (*Opuntia* spp) [20] and chickpea [33]. Studies on gas production from common vetch straws are scarce, with the exception of the Spain studies [45]. López et al. [45] reported greater potential gas production, and less gas production rate and *lag* time in comparison with those observed in the current study. Cone and van Gelder [46] indicated that despite high degradability, feed with high CP typically produce less gas during fermentation as protein fermentation produces ammonia, which affects the carbonate buffer balance by neutralizing H<sup>+</sup> ions from volatile fatty acids without releasing carbon dioxide. The differences in gas production parameters between studies may be attributed to the differences in straw CP content, varieties, season, and location.

The IVOMD and ME contents of straw varied among the varieties, which is partly due to varietal variations in straw ADF and NDF and may be due to the proportions of straw morphological fractions, which were not measured in this study. In this study, varieties Lanjian No. 1 and Lanjian No. 2 recorded straw CP and IVOMD as high as 8.0% DM and 50.0% OM, respectively, which suggests that the straw of these varieties may be effectively used as a CP supplement to ruminants fed low-quality cereal straw-based diets [9]. The straw IVOMD range in this study is comparable to the range reported earlier in other vetch varieties [9], while the ME range is slightly less than those reported for vetches and other legume straws [13]. The straw quality varied between studies possibly due to differences in varieties, cell wall lignification, leaf to stem ratio, and the stage at which the straw was harvested. Larbi et al. [9] and Makkar et al. [13] earlier reported that straw IVOMD and ME contents of common vetch are influenced by varieties, growing season, and stage of straw harvest.

#### *4.5. In Situ Ruminal Degradability*

Higher ruminal degradability of high-fiber forages is satisfying because it implies improved the nutrient availability to rumen microbes [47]. The observed differences in DM degradation profiles of the straw varieties may be related to their varietal traits reflected as substantial differences in morphological and chemical composition. Our results are consistent with those reported for other crops such as maize [47]. For DM degradation parameters, the value of *A* fraction observed in this study for common vetch straw is greater and the values of *B* and *C* fractions are similar to those reported by Bruno-Soares et al. [48]. In this study, the value of *A* fraction of Lanjian No. 1 was significantly greater than other varieties, while the values for *B* and *C* fractions were similar for the four varieties analyzed. The differences in EDDM between the varieties can be mainly attributed to the differences in *A* fraction and not to *B* and *C* fractions, which is consistent with the literature [47]. The EDDM reported for common vetch straw in our study are similar to that reported for chickpea straw (51.8% DM) [49], but greater than that reported for fenugreek (*Trigonella foenum-graecum*) straw (32.2% DM) [50]. The degradation profiles of NDF were not influenced by variety in our study, which is in agreement with the earlier reports on maize and chickpea [47,49]. Bruno-Soares et al. [48] reported less rapidly degradable NDF fraction and rate of NDF degradation, and similar potentially degradable NDF fraction in comparison with those observed in this study. The EDNDF of straw in this study was less than reported by Abbeddou et al. [51] for lentil straw (45.9% NDF), but greater than that reported by Mustafa et al. [50] for fenugreek straw (20.3% NDF). Different studies recorded different ruminal degradation kinetics due to differences in varieties/species, straw composition, and animal species [4,49–51].

#### **5. Conclusions**

The results of this study showed varietal differences in grain and straw yields, and straw nutrient value in common vetch. Evaluation of common vetch varieties showed that Lanjian No. 1 had straw yield, straw CP, non-protein nitrogen, neutral detergent soluble protein, IVOMD, ME, and EDDM greater than other varieties, despite its less grain yield. Variety Lanjian No. 3 demonstrated early maturity and greater grain yield; however, straw yield and quality were less than Lanjian No. 1. Variety Lanjian No. 2 had greater grain yield and moderate straw CP; however, straw IVOMD, ME, EDDM and EDNDF contents were comparable with Lanjian No. 1. Based on these results, variety Lanjian No. 1 is the best option among varieties examined for smallholder farmers on the Qinghai-Tibetan plateau.

**Author Contributions:** Y.H. and Z.N. conceived and designed the experiment, Y.H. and F.Z. performed the experiments, Y.H. analyzed the data, and Y.H. and Z.N. wrote the manuscript.

**Funding:** This research was funded by the National Basic Research Program of China (No. 2014CB138706).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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