Introduction
One of the main conditions for the realization of the genetic potential of animals is the organization of their adequate nutrition, taking into account the level of productivity while maintaining a normal physiological state. Proteins directly influence the increase in milk production of animals by supplying them with all the necessary amino acids, increasing the amount of available energy and changing the efficiency of utilizing the nutrients contained in the main diet (Brun-Lafleur et al., 2010; Patton et al., 2014; Lazzarini et al., 2016). The organism of highly productive cows needs to receive a large amount of energy in the first weeks of lactation, as there are significant losses of nutrients along with the milk (Leroy et al., 2008, Amanlou et al., 2017). Therefore, the urgent task of modern intensive livestock breeding, is to provide the organism of lactating cows with the necessary amount of protein.
The rational use of feedstuffs containing a large amount of proteins should not lead to a significant increase in the cost of the basic diet. One of the possible solutions for this problem is the use of protein concentrates obtained during the processing of secondary raw materials of animal husbandry, since it is a widely available product and contains a complete protein. (Jayathilakan et al., 2011; Potapov et al., 2017; Popov et al., 2019). According to the number of studies, parts of the subcutaneous epithelium of farm animals, which remain in tanneries in large quantities after processing the hides, are a source of large amounts of protein in a form easily digestible for animals (Pati et al., 2014). This residual organic material can be used to produce protein feed supplements. In addition, the problem of processing and rational use of secondary raw materials in leather production to prevent environmental pollution in Russia, Ukraine, Bangladesh, China, the United States and some countries of South America and Europe, remains urgent (Jiang et al., 2016; Rigueto et al., 2020). Therefore, the production of a high-protein feed supplement from the subcutaneous epithelium of farm animals could represent an economically feasible way to obtain the required product, as well as reduce the environmental damage caused by solid waste from leather production. Currently, various technological approaches have been developed to obtain safe feed from such waste (Kantarli and Yanik, 2009; Jiang et al., 2016). Despite the fact that such supplements have been produced for a long time, most studies are devoted to the effect of these products on certain physiological indicators of farm animals (Lee et al., 2015; Awawdeh, 2016; Amanlou et al., 2017; Leondro et al., 2019). Comprehensive studies on the effect of protein supplement from minced subcutaneous epithelium of farm animals on indicators of natural resistance, metabolism and milk productivity have not been performed. All of the studied parameters clearly demonstrate the physiological state of highly yielding cows in the early stages of lactation when fed a protein supplement from leather making wastes.
Therefore, the aim of our research was to study the effect of protein supplement made from minced subcutaneous epithelium of farm animals, on hematological, biochemical parameters of metabolism and milk productivity of highly yielding cows during the early lactation.
Materials and methods
To conduct research using the analogue pair method in the first months of lactation, a control (I) and two experimental (II, III) groups of Ayrshire cows, 6 animals each, were formed. The cows were kept fastened in the winter-stall period (Table 1).
Table 1. Characteristics of animals participating in the experiment (mean ± standard deviations)
Animals of the control and experimental groups received a daily ration, which included (kg/animal/day): corn silage - 22, cereal-legume silage - 17, mixed feed (40% soybean meal) - 9.5, grain mixture - 2, crimped grain - 3, fodder molasses - 1.2, barley straw - 0.5, premix - 0.15, glycerin - 0.3, fodder chalk - 0.13, disodium phosphate - 0.15, soda - 0,15, table salt - 0.1, urea - 0.05, fodder sulfur - 0.035. The nutritional value of the main diet was 258.7 MJ. The diets were consistent with the norm in this age period. In addition to the main diet, the cows of the experimental groups were fed protein fodder meal made from minces subcutaneous epithelium of farm animals for 60 days. The aftereffect of the protein feed supplement on the organism of the animals of the experimental groups was studied during the next 30 days (Table 2).
Table 2. Experiment design
Blood from the jugular vein was taken for biochemical parameters’ determination on the 1 st, 60 th and 90 th day, before morning feeding in the control and experimental groups. The activity of alkaline phosphatase, the amount of urea, magnesium, phosphorus, total protein, cholesterol, albumin, the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), the level of glucose in the blood serum was determined on a biochemical analyzer Stat Fax 3300 (Awareness Technology, USA) with reagent kits "Diacon" (Russia) and "Vector-Best" (Russia). Serum calcium was determined using a complexometric method with an indicator fluorexon according to Vichev, Karakashev, reserve alkalinity in blood serum by a diffuse method using double flasks, carotene in blood serum according to Karp and Preuss in Yudkin's modification, the total number of ketone bodies in the blood by iodometric method (Kondrahin, 2004), protein index by calculation.
The number of leukocytes and erythrocytes was counted in a Goryaev chamber under a MIKMED-6 microscope (LOMO, Russia). To count leukocytes, 0.02 ml of blood was stained with 0.4 mL of a 3 % solution of acetic acid and methylene blue; to count erythrocytes, 0.02 ml of blood was mixed with 8 mL of 0.9 % sodium chloride solution. The composition and ratio of leukocytes was studied on blood smears, which were fixed with ethyl alcohol. To determine the ratio of leukocytes, peripheral blood smears were stained with azure-eosin according to Romanovsky (1 drop of the basic solution per 1 cm 3 of distilled water with a pH of 6.8-7.0). Under the MIKMED-6 microscope, 200 cells were counted on each blood smear, isolating lymphocytes, monocytes, stab and segmented neutrophils, eosinophils, and basophils. Leukocyte identification was carried out according to Nikitin (1949).
The registration and assessment of milk productivity in animals in all groups was carried out by calculating the daily milk yield according to the monthly control milkings carried out during the experiment. Physicochemical properties of milk: dry matter, content of fat, protein, urea and the number of somatic cells were determined on the analyser "BentleyCombi 150" (Bentley Instruments Inc., USA).
Chemical analysis of protein supplement made from subcutaneous epithelium of farm animals aimed at determining: total moisture - by a two-stage method; crude protein according to the Kjeldahl method with a VELP UDK139 (Velp Scientifica, Italy) semi-automatic steam still (Association of Official Analytical Chemists, 1990); rumen degradable protein by extraction in borate-phosphate buffer (Åkerlind et al., 2011).
Statistical processing of the obtained results was carried out in Statistica program (Stat Soft Inc., USA). For the evaluation of the surveyed parameters we used statistical methods. We calculated the mean arithmetic value and the standard error. The differences between the values in the control and experimental groups were determined using ANOVA, where the differences were considered significant at P<0.05. The results in the tables are presented as mean ± standard error (x ± SE).
Results and discussion
According to the results of the chemical analysis of the composition of protein supplement, it was found to contain the following: 10.3 % moisture and 89.7 % dry matter. It was found that the studied protein supplement contains rumen undegradable protein. The dry matter of the supplement contains 81.9 % crude protein, of which 42.6 % is rumen undegradable protein (RUP). The safety of the additive used is confirmed by the Declaration of Conformity dated November 23, 2018 (Test Report No. 5059 dated November 23, 2018, Test Report No. 5286-1 dated December 07, 2018).
Indicators of protein metabolism in lactating cows of the control and experimental groups were generally within the physiological norm (Table 3).
Table 3. Indicators of protein metabolism
a, b - values within a row with different letters differ at P<0.05 (where "a" is a higher value)
On the 60 th day, there was a 4.5 % increase in the total protein in the blood serum of the animals, which received 300 g of the feed protein supplement in addition to the main diet. This increase remained on the 90 th day of the experiment and was 9.1 % compared to that of animals in the control group. The increase in the total protein content in cows of the II experimental group occurred as a result of albumin content increase on the 60 th and 90 th days by 18.5 % (P<0.05) and 11.8 %, respectively, compared with animals that did not receive a protein supplement. As a result, on the 60 th day, the protein index in animals of the second experimental group exceeded this indicator in cows of the control by 20.4 %.
There was also an increase in total protein on the 90 th day of the experiment by 6.3 % compared to the control in animals that received 500 g of the feed additive in addition to the main diet. The increase in the albumin fraction of the blood serum in the animals of the III experimental group, which was 11 % in comparison with the control on the 60 th day of the experiment led to an increase in the protein index during this period of the experiment by 17.2 %. An increase in whey proteins was found in animals that received a protein supplement containing RUP, which indicates the absence of a violation of their metabolism (Szterk et al., 2017). An increase in the protein index or albumin-globulin ratio in cows from the experimental groups was shown, which was noted on the 60th day of the experiment, probably indicating a more intensive course of protein metabolism in the organism of cows (Gorlov et al., 2015). The increase in the total protein content in the blood of animals receiving the protein supplement may be associated with the enzymatic activity of rumen microorganisms, which leads to a slight increase in the amount of ammonia in the rumen fluid (Hassan and Saeed, 2012; Özsoy et al., 2013).
The level of urea in the blood serum of animals between the groups differed insignificantly, some increase was noted during the experiment, however, it did not exceed the normal values. According to some researchers, a significant increase in the content of urea in the blood and milk of animals may be the result of degradation of protein in the rumen due to its excessive intake along with the feed. Excess protein, which is not converted into milk as a result of metabolism, is converted into ammonia during catabolism, which in turn is converted into urea. Thus, an excess of urea appears as a significant increase in its content in blood and milk (Broderick and Reynal, 2009; Guliński et al., 2016; Zhang et al., 2019; Walkenhorst et al., 2020). Slight increase of urea in the blood of animals of the experimental groups within the acceptable values with the physiological norm of the remaining biochemical parameters may indicate a good level of absorption of dietary protein (Amanlou, 2017).
The study of mineral metabolism in animals during the experiment did not reveal significant deviations of the studied parameters from the physiological norm (Table 4). It should be noted that the phosphorus content in the blood of animals that received 300 g of feed protein supplement in addition to the main diet, statistically significantly increased on the 90 th day of the experiment by 0.39 mmol/L or 34.5 % (P<0.05) in compare to control values.
There was an increase in the level of phosphorus by 10.6 % and magnesium by 18.1 % in the blood serum of lactating cows of the III experimental group on the 90 th day of the experiment, in comparison with the analogous indicators of animals of the I group. A slight increase in the level of calcium in animals of group II was observed on the 60 th (0.19 mmol/L (8.4 %)) and 90 th (0.36 mmol/L (12.4 %)) day of the experiment. The value of this indicator on the 30 th day after the end of feeding the additive in animals of group III exceeded the control one by 0.25 mmol/L or 8.6 %. Alkaline reserve during the entire experiment in all groups was relatively stable, and there was a 4.0 % increase in this indicator in animals of group III compared to the control on the 90 th day.
Cows have an increased demand for calcium at the early stage of lactation, since a large amount of it is excreted from the animals' body along with milk, which is especially typical of high-yielding cows (Jawor et al., 2012; Goff, 2014). An increase in calcium in the blood of animals from experimental groups while feeding a supplement with RUP helps reducing the risk of hypocalcaemia, which can lead to endometritis in highly productive animals (Priest et al., 2013). In addition, cows from the experimental groups showed an increase in phosphorus content on the 90th day of the experiment, which indicates an increase in the metabolism of this macro-element in the organism (Grünberg et al., 2015).
There were no deviations from the values of the physiological norm of the activity indicators of the studied enzymes in the blood serum of the animals of the experimental and control groups (Table 5).
Table 4. Indicators of mineral metabolism
a, b - values within a row with different letters differ at P<0.05 (where "a" is a higher value)
Table 5. Enzyme activity indicators
AST - aspartat transaminase, ALT - alanine aminotransferase
On the 60 th and 90 th day of the experiment, an excess of ALT activity level was observed in the experimental groups of lactating cows, in comparison with the values of this indicator in the control: in the II experimental group the increase was 4.63 U/L or 20.1 % and 0.44 U/L or 1.3 %, in III experimental group - 4.73 U/L or 20.5 % and 2.92 U/L or 8.9 %, respectively. On the 30 th day after the end of feeding the feed protein supplement in cows of groups II and III, the AST activity indices in comparison with the control were higher by 16.66 U/L or 19.4 % and 7.64 U/L or 8.9 % respectively. Nevertheless, the activity of the studied enzymes in both experimental groups was at the optimal level.
It was found that the studied parameters of carbohydrate, lipid and vitamin metabolism of lactating cows in the experiment did not have significant deviations from the physiological norm (Table 6).
Table 6. Indicators of carbohydrate, lipid and vitamin metabolisms
a, b - values within a row with different letters differ at P<0.05 (where "a" is a higher value)
The level of cholesterol in the blood serum of cows of the II experimental group was 0.32 mmol/L (7.2 %), and in the III experimental group 0.27 mmol/L (6.1 %) higher than that of the control on the 90 th day of the experiment. A slight increase in the total lipid index (by 53 mg% and 39 mg% on the 60 th and 90 th days, respectively) was observed in animals of the II experimental group, compared with the control values; in cows of the III group, this indicator was 34 mg% higher than in the control on the 30 th day after the end of feeding the supplement. The content of vitamin A in the serum of cows of group II exceeded the control indicator by 8.39 μg% in comparison with the control group on the 60 th day of the experiment. An increase in cholesterol along with an increase in blood glucose levels in animals from the experimental groups is possibly associated with an increase in the production of propionate in the rumen, since propionate is the main substrate for gluconeogenesis in ruminants, which for the most part actively proceeds in the liver (Aschenbach et al., 2010). The combination of these processes indicates an improvement in the metabolism of nutrients passing through the liver (Schlegel et al., 2012; Hashemzadeh-Cigari et al., 2014; Kessler et al., 2014). The absence of pathological changes in the liver, as well as the metabolic rate, is to some extent indicated by an increase in the main hepatic enzymes ALT and AST along with increased albumin and cholesterol levels in the blood serum of animals treated with RUP supplement (Mordak et al., 2020).
Hematological indicators of lactating cows of all studied groups at all stages of the experiment were within the physiological norm (Table 7).
Table 7. Blood indicators of animals in the experiment
The increase in the number of leukocytes in the blood of animals of the experimental groups in group II was 2.5 % on the 60th day, in III experimental group by 7.9 % and 8.1 % on the 60 th and 90 th day, respectively. The number of erythrocytes in the cows of the experimental group increased in comparison with the animals of the control group: in the blood of cows of the II experimental group on the 60 th day of the experiment by 7.9 %, and this excess remained on the 30 th day after the end of feeding additives and amounted 3.2 %; in the blood of cows of the III experimental group by 4.0 % on the 60 th day of the experiment. Some increase in erythrocytes within the physiological norm in the blood of cows in the experimental groups may indicate an increase in the rate of redox reactions, which contributes to a better supply of oxygen to tissues and affects an increase in the intensity of metabolic processes. An increase in the number of leukocytes may indicate an increase in the non-specific immunity of the animal organism (Gorlov et al., 2015; Kalyani et al., 2018).
The ratio of individual forms of leukocytes in the control and in the experimental groups of lactating cows was within the physiological norm (Table 8).
Table 8. Leukocyte blood count of cows in the experiment
STN - stab neutrophils; SEN - segmented neutrophils
An increase in the proportion of lymphocytes in the blood of animals of the experimental groups on the 60 th and 90 th day of the experiment in relation to the control values was observed: in the II experimental group by 1.67 % and 2.00 %, in the III experimental group by 1.34 % and 1.20 %, respectively. The content of basophils in the blood of lactating cows of groups II and III on the 30 thday after the end of feeding the additive was reduced by 0.60 % and 0.40 %, respectively, in comparison with animals in the control group. At all stages of the experiment, the number of basophils in the blood of cows of all groups was within the normal range, which indicates the absence of allergic reactions in the body, including the inclusion of a supplement from leather industry wastes (Otter, 2013).
In this way, the stimulating effect of a feed supplement containing RUP on the erythro- and leukopoiesis of lactating cows, as well as a shift in the leukocyte formula towards an increase in the relative number of individual forms of leukocytes, was established.
It is known that high-yielding cows often experience liver dysfunction due to inflammatory processes as a result of postpartum stress, unbalanced nutrition, metabolic disorders in the first months of lactation (Trevisi et al., 2012). Inflammatory processes in the liver negatively affect the synthesis of many proteins, including albumin (Osorio et al., 2014). An increase in the amount of albumin in the blood of animals of the experimental groups, together with haematological parameters, which are within the physiological norm, indicates the absence of pathological changes in the liver.
It was found that on the 90 th day of the experiment there was an increase in the mass fraction of fat in the milk of animals of all groups, however, in cows of group II, the value of this indicator was higher by 0.14 %, in cows of group III by 0,72 % compared to the control group (Table 9). On the 30 th day of the experiment there was an almost twofold decrease in urea in the milk of cows compared to the beginning of the experiment, while this indicator in the milk of animals of groups II and III statistically significantly exceeded the control value by 2.00 mg % and 2.32 mg % respectively. On the 60 th day, an increase in the amount of urea in the milk of cows of all groups was found, and in the milk of animals of the II group, there was 1.86 mg% more urea than in the milk of cows in the control.
Table 9. Indicators of milk production
a, b - values within a row with different letters differ at P<0.05 (where "a" is a higher value); SCC - somatic cells count
The somatic cells count (SCC) in the milk of animals of all groups at the beginning of the experiment did not exceed the permissible standard values. There was a significant increase in this indicator in the milk of cows of the I control group on the 30 th day of the experiment, while the value of this indicator remained within the normal range in the milk of cows of the II and III experimental groups. On the 60 th and 90 th days of the study, the SCC in the milk of cows receiving the protein supplement also did not have significant fluctuations from the earlier established values in these experimental groups. The value of this indicator was lower than the control in groups II and III by 44 10 3 cells and 39 10 3 cells on the 60 th day and 210 10 3 cells and 121 10 3 cells in 30 days after the end of feeding the supplement, respectively.
On the 30 th day, the experimental groups showed an increase in the daily milk yield with 4% fat content; group II exceeded the control value by 31.4 % (P<0.05) and group III by 13.1 %. On the 60 th day, cows of all groups showed a slight decrease in the average daily milk yield, however, the value of this indicator was higher than in the animals of the control groups, and when converted to 4 % milk, the difference in the average daily milk yields of animals in groups II and III with control was 33.9 % and 21.6 % respectively. This trend continued on the 30 th day after the end of feeding the protein supplement containing the RUP. The productivity in terms of 4 % fat content milk in the cows of the II group was higher by 27.2 %, in the III group - by 19.5 % compared to the control.
The study showed that protein supplement from minced subcutaneous epithelium of farm animals containing RUP helped to supply high-yielding cows with the necessary amount of protein, which is especially deficient in animals during the early lactation. This had a stimulating effect on increasing the milk productivity of cows and the biosynthesis of milk constituents. According to the data of milk control, an increase in milk productivity as well as quality indicators of milk was established along with a decrease in the number of somatic cells in the milk of cows in the experimental groups. It should be noted that during the entire period of the experiment, mastitis was not detected in the cows of the first experimental group. An increase in the number of SCC in cows at the beginning of lactation may be the result of the accumulation of macrophages in the secretion prior to milking, which is not the cause of udder disease. The activation of defence mechanisms caused by exogenous and endogenous factors is a likely cause. In addition, the value of this indicator depends on the technological methods of milking, udder hygiene, the presence of stress in the animal (Auldist et al., 1995; Græsbøll et al., 2016; Alhussien and Dang, 2018).
The stimulating effect of the protected protein on the body of high-yielding cows has been convincingly proven by many researchers (Dijkstra et al., 2013; Yatsko et al., 2013; Matyaev and Andin, 2015; Zhou et al., 2016). Its presence in the main diet is one of the key points that have a beneficial effect on the synthesis of milk and milk components (Batistel et al., 2017; Wang et al., 2017). Obtained results indicate that the effect of the proposed protein supplement containing RUP is consistent with the effect of similar feed products (Lee et al., 2015; Awawdeh, 2016; Leondro et al., 2019).
This study demonstrates that the use of a feed supplement containing protein from subcutaneous epithelium of farm animals, in the amount of 300 g and 500 g per animal per day for 60 days, in feeding cows for milk production, promotes the activation of metabolic processes in the organism of cows.
It should be noted that within 30 days after the end of feeding the protein supplement from leather industry wastes, the animals of the experimental group experienced a prolonged effect of the supplement, which consists in higher values of blood biochemical parameters, an increase in milk productivity compared to same indicators of the control.
Conclusions
It was found that the use of a protein supplement from minced subcutaneous epithelium of farm animals, in an amount of 300 g and 500 g per animal per day in feeding lactating cows for 60 days, provides a more balanced protein nutrition of animals at the beginning of lactation, which has a beneficial effect on their productive performance. According to the obtained data, it can be assumed that the use of RUP as part of an additive in feeding high-yielding cows in the first months of lactation improved the physiological state of animals due to an increase in the indicators of all types of metabolism within the physiological norm. However, further research is needed in order to recommend this protein supplement from leather industry wastes, containing RUP, as means to balance the protein nutrition of high-yielding cows at the beginning of lactation and to accurately analyse its effect on metabolism, liver health, digestion processes and milk production of animals.
Funding
This study was supported within the framework of the program of fundamental scientific research of the state academies of sciences for 2013-2020 in the direction of research X 10.7. Zootechnics 157. Theoretical foundations of molecular genetic methods of control of the breeding process in order to create new genotypes of animals, birds, fish and insects with economically valuable traits, their maintenance and feeding systems "To improve methods of genetic control and management of the breeding process, technology of feeding and keeping cattle and sheep in order to increase the productivity and profitability of livestock production" (No. 0597-2019-0016-C-01).
Učinak dodatka proteina iz otpada kože na hematološke i biokemijske pokazatelje metabolizma i mliječnost visokoproduktivnih krava
Sažetak
Cilj je ovog istraživanja utvrditi učinak dodatka bjelančevina iz mljevenog potkožnog epitela domaćih životinja, na hematološke i biokemijske parametre metabolizma te proizvodnost visokoproduktivnih krava tijekom rane laktacije. Za to su prema principu analognih parova formirane tri skupine krava, svaka po šest životinja. Kontrolna skupina hranjena je osnovnim obrokom, a druga i treća eksperimentalna skupina svakodnevno su uz osnovnu hranidbu hranjene s 300 g i 500 g proteinskog dodatka tijekom 60 dana. Učinak dodatka proučavan je tijekom sljedećih 30 dana. U svih životinja proučavani su hematološki i biokemijski parametri krvi, te kvalitativni i kvantitativni sastav mlijeka. Životinje pokusnih skupina pokazale su porast metaboličkih i redoks procesa tijekom pokusa. Utvrđen je porast metabolizma hranjivih sastojaka u jetri. Zbog dovoljnog unosa bjelančevina u organizam životinja eksperimentalnih skupina došlo je do povećanja pokazatelja mliječne proizvodnosti i pokazatelja kvalitete mlijeka.
Ključne riječi: proteinski dodatak; otpad od kožne industrije, krave s visokom proizvodnjom, metabolizam, mliječna proizvodnost