**1. Introduction**

The wine industry produces annually millions of tons of grape by-products, which are valuable resources of biologically active substances that have many potential uses, also in animal nutrition [1]. Grape pomace (GP) is a by-product from the wine industry and represents about 15–20% of the weight of the grape bunch [2]. The GP is a suitable feed additive for animal nutrition [3–8]. The product can be fed fresh, dried, or ensiled [9]. The nutritive value of grape pomace is variable depending on the grape-growing region, cultivar, technology of winemaking, and the proportion of seeds and pulp [10–12]. The GP is a source of health benefits: flavonoids with antioxidant and anti-inflammatory activity [13–15] that can improve rumen fermentation [16] and delay gas production [17]. Digestibility of crude protein, organic matter, and NDF (neutral detergent fiber) was increased in sheep receiving GP [18,19]. Many studies have focused on the biochemical profile of small ruminant's

**Citation:** Juráˇcek, M.; Vašeková, P.; Massányi, P.; Kováˇcik, A.; Bíro, D.; Šimko, M.; Gálik, B.; Rolinec, M.; Hanušovský, O.; Kolláthová, R.; et al. The Effect of Dried Grape Pomace Feeding on Nutrients Digestibility and Serum Biochemical Profile of Wethers. *Agriculture* **2021**, *11*, 1194. https://doi.org/10.3390/ agriculture11121194

Academic Editors: Lubomira Gresakova and Emilio Sabia

Received: 19 October 2021 Accepted: 25 November 2021 Published: 26 November 2021

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blood with impact on the effect of breed, age, gender, location, and season [20–24]. The effect of different dosages of GP on biochemical parameters of ruminants' blood in different experiments was realized in dairy cows [25], in calves [26], or in sheep [27]. Our previous studies have analyzed the effects of various natural substances obtained as by-products of agricultural production on animal nutrient digestibility, health status, or reproductive efficiency [28–34]. These studies indicate the grea<sup>t</sup> potential of these products for use in animal nutrition, however, the GP addition in animal feeding has to be further examined. The hypothesis is that GP addition to the ruminants' daily diet will increase the nutrients digestibility without the negative effect on the animals' health. Based on the above, the aim of this study was to describe the effect of dried GP feeding on the nutrients digestibility coefficients and blood serum biochemical parameters of wethers.

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

#### *2.1. The Materials Animals and Housing*

Experiments were conducted at the Experimental Center of Livestock at the Department of Animal Husbandry (Slovak University of Agriculture in Nitra). The wethers were of Ile de France breed, obtained from the University farm in Kolinany (Slovak University of Agriculture in Nitra) with an average weight of 34.05 ± 1.97 kg and age of 4 months. The study consisted of 3 groups: control—C, 1% grape pomace—GP1, and 2% grape pomace—GP2 (Table 1). During the preparatory time period, wethers were free housed in group without bedding in pens. Then, the wethers were housed in balance cages individually to monitor proper individual daily diet intake and feces collection in the balance period. The experiment complied with animal health care standards. The animals were under veterinary control and cared for by experienced animal caretakers during the whole experiment. The routine manipulation with animals during the experiment did not cause disproportionate and excessive stress. The conditions of animal care, manipulations, and use corresponded with the instructions of the Ethics Committee of the Slovak University of Agriculture in Nitra, Protocol No. 48/2013.


**Table 1.** Experiment scheme.

C—control group, GP 1—grape pomace 1% from daily dry matter intake, GP 2—grape pomace 2% from daily dry matter intake.

#### *2.2. Feeding and Experimental Design*

The composition of experimental and control daily diets are listed in Table 2. Grape pomace of the Pinot Gris variety (*Vitis vinifera* L.) was obtained from the academic vinery (Slovak University of Agriculture in Nitra). The nutrient content of feed components is shown in Table 3. During the whole experiment, animals were fed two times per day. Half of the daily diet was fed during the morning and another 50% was fed during the afternoon. Water, mineral and vitamin lick was accessible ad libitum. The concentration of biologically active substances (total polyphenols: 27.38 ± 1.38 mg GAE/g—equivalent of gallic acid) was determined in a previous study [35]. The control (C) daily diet from meadow hay, ground wheat, soybean meal, and mineral and vitamin lick was formed. The preparatory period before C diet feeding was 14 days (Table 1). Following this, the experimental balance period lasted 5 days. Daily diet GP1 and GP2 consisted of meadow hay, ground wheat, soybean meal, mineral and vitamin lick, and dried GP (1 and 2% of daily dry matter intake, respectively). The preparatory period before experimental variant GP1 and GP2 lasted 7 days and the balance period 5 days. The difference between the experimental variants was only in the concentrations of dried GP in the diet.


**Table 2.** Feed rations used in the digestibility experiment.

\* 1% from daily dry matter intake, \*\* 2% from daily dry matter intake, mineral and vitamin lick (Jan Valasek, Ludrova, Slovakia) content was as follows: MnO (as Mn) 3100 mg, ZnO (as Zn) 4800 mg, Ca(IO3)2 (as I) 125 mg, Se 31 mg, CoSO4.7H2O (as Co) 42 mg, vit. A 300,000 i.u., vit. D3 125,000 i.u., vit. E 100 mg, ash 95%, Ca 9.9%, P 5.0%, Na 13.7%, Mg 5.1% in 1 kg of dry matter.

**Table 3.** Chemical composition of feed components.


DM: dry matter, CP: crude protein, EE: ether extract, CF: crude fiber, ADF: acid detergent fiber, NDF: neutral detergent fiber, NFE: nitrogen free extract, NFC: nonfiber carbohydrates, OM: organic matter, \* in g/kg of original matter, other nutrients in g/kg of dry matter.

#### *2.3. Blood Sampling and Analyses*

Blood samples were collected from *vena jugularis externa* on the morning of the last day of the nutrition balance experiment in each variant. Sampling and analysis of blood were realized. For biochemical analysis of blood serum blood samples were centrifuged at 1006× *g* for 30 min. Potassium (K), sodium (Na), and chloride (Cl) ions were analyzed by an EasyLite analyzer (Medica, Bedford, MA, USA) with an ion-selective electrode [36,37]. Blood serum concentrations of calcium (Ca), magnesium (Mg), phosphorus (P), triglycerides (TG), cholesterol (CHOL), glucose (GLU), total protein (TP), urea, albumin (ALB), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma glutamyl transferase (GGT), were determined using DiaSys (Diagnostic Systems GmbH, Holzheim, Germany) kits on the Randox RX Monza analyzer (Randox Laboratories, Crumlin, UK) [37,38]. Globulin (GLB) was calculated mathematically by subtracting the serum levels of albumins from serum total proteins [32].

#### *2.4. Feed and Feces Collection, Analysis and Determination of Digestibility*

During the balance period once daily in the morning the rests and samples of feeds, daily diets, and feces were collected. The content of organic and inorganic nutrients was analyzed in the rests and samples of feeds and in pooled samples of feces for each animal for 5 days. Dry matter content (DM) was analyzed by gravimetric method at 103 ◦C, crude protein (CP) by Kjeldahl method, ether extract (EE) by gravimetric method according to the Soxhlet principle, crude fiber (CF) by gravimetric method as a residue insoluble in

acid and alkaline media after deduction of ash (Fibertec System, Tecator), acid detergent fiber (ADF) by gravimetric method as a residue after hydrolysis in acid detergent solution (Fibertec System, Tecator), neutral detergent fiber (NDF) by gravimetric method as a residue after hydrolysis in neutral detergent solution (Fibertec System, Tecator) and ash (A) by gravimetric method at 550 ◦C (muffle furnace) were determined. The content of organic matter (OM), nitrogen free extract (NFE), and nonfiber carbohydrates (NFC) were calculated according to formulas:

$$\text{OM} = \text{DM} - \text{A (g/kg)} \tag{1}$$

$$\text{NFE} = \text{DM} - \left(\text{CP} + \text{EE} + \text{CF} + \text{A}\right) \left(\text{g}/\text{kg}\right) \tag{2}$$

$$\text{NFC} = \text{DM} - \left(\text{CP} + \text{EE} + \text{NDF} + \text{A}\right) \left(\text{g}/\text{kg}\right) \tag{3}$$

The content of Ca, Mg, Na, K was determined by High Resolution Continuum Source Atomic Absorption Spectrometer contrAA 700 (ANALYTIC JENA, Jena, Germany) and content of P by 6400 Spectrophotometer (JENWAY, Montreal, QC, Canada). In vivo apparent digestibility coefficients of CP, EE, CF, NFE, NFC, OM, ADF, and NDF in the diets (in %) were calculated as:

In vivo digestibility coefficient = [(nutrient intake − nutrient excreted)/nutrient intake] × 100 (%) (4)
