Additive «PREVAX» application for mastitis prevention and complex treatment in high-producing cows

. This article presents the results of clinical trials of the supplement «PREVAX». The results of the additive tolerability study «PREVAX» are presented in high-producing cows, the results of preventive and therapeutic use of the supplement «PREVAX» in high-producing cows with subclinical and clinical mastitis, and the effect of the supplement on milk parameters and milk production. The application of the additive «PREVAX» for the prevention of mastitis has a positive effect. The administration of the supplement orally through the watering system at a dose of 1 ml per 100 litres of water for 60 days was found to reduce the incidence of mastitis in cows by 70%. Administration of the additive «PREVAX» to cows with the clinical form of mastitis at a dose of 4 ml per 100 litres of water in the background of classical therapy with Mamikur at a dose of 10 ml intra-cisternally is well tolerated by the animals, gives a pronounced positive clinical effect and leads to normalisation of somatic cell count in milk on the sixth day of treatment. At the same time, treatment without the supplement «PREVAX» only led to a similar result after 12 days of treatment.


Introduction
At present, dairy cattle breeding is undergoing intensification and introduction of advanced technologies. Producers and processors of dairy products are interested in obtaining a quality product [1][2][3]. The main income of dairy cattle breeding is the sale of milk. The quality of production, however, depends on the health of the dairy herd. An important and pervasive problem in dairy farms is the incidence of mastitis [4]. It causes significant economic damage from the reduction in milk productivity, from the costs of treatment and prevention, from the reduction in the operating time of productive animals [5][6][7].
The disease occurs as a result of breaches in the maintenance, care, feeding, and milking of cows. The subclinical form of mastitis, according to statistics from the International Dairy Federation, affects up to 30-50% of cows [8][9][10], in the Russian Federation from 10-12 to 70-80% [11]. Mastitis not only negatively affects cow productivity. Milk from such animals can cause allergic reactions and food toxicosis in humans [12,13].
Products on the veterinary market cannot fully cure the problem, and many have a number of limitations in their use [14].
A variety of chemotherapeutic agents and antibiotics are used in dairy farms to treat the pathologies of various systems in cows and calves. At the same time, microorganisms develop and maintain multiple drug resistance to various drugs [15][16][17]. This also applies to antimastitis drugs used on the farm for a long time. Therefore, to counteract resistant strains of microorganisms and improve the therapeutic efficacy of drugs, a regular rotation of drugs once or twice a year, as well as the use of compound agents with high species sensitivity to the microflora of the farm, complex, farm [18][19][20].
The need of the dairy industry to improve the quality of its products in the absence of effective treatment schemes has stimulated the development of OOO «DeltaBioTech» supplements «PREVAX».
The ingredients in the supplement have anti-inflammatory, anti-edema and analgesic effects and help to restore udder secretory function. In terms of effect on the body PREVAX is classified as a low-hazard substance (Hazard Class 4 according to .
Objective of the study is to conduct clinical trials on the additive «PREVAX», developed by OOO «DeltaBioTech» and determine the preventive and therapeutic effectiveness of the supplement «PREVAX» in high-producing cows with subclinical and clinical forms of mastitis, and the effect of the supplement on the quality and quantity of milk.

Materials and methods
1st step. Study the tolerability of the additive «PREVAX» The research and production experiments were carried out in «OOO Babaevo» Vladimir region, Sobinsky district, Babayevo village.
Addition «PREVAX» was given to each animal in a dose 10 times the therapeutic dose (therapeutic dose 1 ml) twice daily orally, 5 ml pre-diluted in 10 litres of water, for 5 days using a probe. At the beginning of the study and every day for 5 days, the clinical condition of the animals was assessed: temperature, pulse, respiration, behavioural reactions, appetite, defecation and diuresis, motor activity, skin condition, reproductive system condition (character of discharge, uterine tone, etc.), presence of adverse events and complications. General blood tests were performed before the experiment and 5 days after daily supplementation (Fig.2.) 2nd step. Study the preventive effectiveness of the supplement «PREVAX». Two groups of milking cows of 100 cows in each group were formed (Fig. 3). Experimental group one: additive «PREVAX» animals were given through the watering system daily, at a dose of 1ml per 100 litres of water orally for 60 days (Fig.4.). The control group remained without the supplement. To assess the effect of an additive «PREVAX» on the bodies of the experimental animals, and to exclude concomitant diseases, all experimental animals were clinically examined at the beginning and end of the experiment, and peripheral blood was taken for general clinical analysis and serum biochemical examination of the animals; Before, at the end and during the experiment, mastitis was diagnosed by clinical methods, by dimastine test and by determining the number of somatic cells in the milk (milk viscosimetric analyser "Somatos-B2-K").
The test with dimestine was as follows: 1 ml of milk from each quarter of the udder was added to a special plate, then 1 ml of a 5% dimestine solution was added. After stirring with a stick for 15 seconds, the reaction was recorded.
The reaction was accounted for by the viscosity of the jelly.
backlashhomogeneous liquid (-). No mastitis; questionable reaction -traces of jelly formation (±). This is subclinical mastitis; positive reactiona clearly visible clot (from weak to dense) that can be thrown out of the well with a stick (+). Clinical form of mastitis.
The method for determining the number of somatic cells in milk using a viscometer is to determine the conditional viscosity of milk samples mixed with an aqueous solution of mastoprim according to the time of flow through a capillary. The reading range is from 90 to 1500 thousand cells per 1 cm3 of milk.
Cows with a somatic cell count of less than 500,000/1 cm3 no mastitis. Cows with subclinical mastitis have a somatic cell count of 500,000 to 950,000/1 cm3.
Before and after using the supplement «PREVAX», milk parameters (mass fraction of protein, mass fraction of fat, mass fraction of skimmed milk residue, density) (Fig.5). In the experiment, lactating cows with mastitis. Four groups of lactating cows with clinical and subclinical mastitis were formed. The first two groups consisted of animals with a clinical form of serous-catarrhal mastitis. The first group of animals consisted of 10 cows and were treated with Mamikur at a dose of 10 ml intracisternally; The second group of cows received an additional supplement during treatment «PREVAX» orally at a dose (concentration) of 4 ml per 100 litres.
The third and fourth group (10 cows each) consisted of cows with subclinical mastitis treated with mamikur. The fourth group of cows additionally received a supplement in the course of treatment «PREVAX» orally at a dose (concentration) of 4 ml per 100 litres.
A fifth group of cows was to be formed of animals with mastitis on prophylactic supplementation for 60 days «PREVAX» orally at a dose (concentration) of 1 ml per 100 litres of water. Prophylactic dosing «PREVAX» The mastitis group had a positive effect, with only two cows with mastitis out of 100 cows, and they had a subclinical form of mastitis. A fifth group was therefore not formed.
At the start and end of the treatment, a diagnosis was made of the general condition of the animals (activity, feed requirements, feed digestion, local signs of mastitis, etc.).
The therapeutic efficacy of the supplement was assessed clinically, as well as using a dimastine test and by counting somatic cells in milk samples.
The diagnosis of clinical serous-catarrhal mastitis was made on the basis of clinical examination. The most characteristic signs of acute serous-catarrhal mastitis were: enlargement and swelling of the affected quarter of the udder, increased local temperature (hot to the touch), redness, soreness, stony, dense udder tissue on palpation, often enlarged supravesical lymph nodes. There was a decrease in milk production as well as general depression of the animal, decreased appetite, increased body temperature.
The results of treatment were assessed by clinical examination. In the absence of clinical signs of mastitis, to exclude a subclinical course of mastitis, a dimastine test was carried out, and the number of somatic cells in milk was determined (milk viscosimeter "Somatos-B2-K").

Clinical indicators
Both before, during and at the end of the experiment, no significant changes in body temperature, pulse rate and respiratory movements were observed in the experimental group cows. No general body reaction to the drug administration was detected. No changes in behavioural reactions, decreased appetite, defecation and diuresis, or decreased milk productions were observed.
Temperature, pulse and breathing readings.
Clinical examination results (temperature, pulse and breathing) in experimental cows before and after supplementation «PREVAX» are presented in table 1.  Table 1 shows that temperature, pulse and respiration of the experimental animals remained within the physiological norm before and after the experiment and had no statistically significant differences.

Haematological indicators
Results of haematological examination of cows in the experimental group before and after supplementation «PREVAX» are presented in table 2. As shown in Table 2, haematological parameters before and after the experiment were within the physiological norm and had no statistically significant differences.

The second stage of the work
Study on the preventive effectiveness of the supplement «PREVAX».
Results of haematological tests on experimental cows before and 60 days after oral supplementation «PREVAX» through the watering system daily, at a dose of 1ml per 100 litres of water are shown in Table 3. Table 3. Results of haematological examination of healthy high-producing cows before and 60 days after oral supplementation «PREVAX» through the watering system daily, at a dose of 1 ml per 100 litres of water. As Table 3 shows, the additive «PREVAX» does not lead to a significant change in haematological parameters in cattle. Nevertheless, it can be seen that while the relative values of eosinophils and stab neutrophils were at the beginning of the experiment in the upper limits and even above the physiological norm, by the end of the experiment these indicators returned to their physiological limits. In addition, the relative value of lymphocytes in the experimental animals by the end of the experiment was 7% higher than in the control group, while being within normal limits. This, combined with an increase in serum immunoglobulins, indicates a possible immunomodulatory property of the supplement. Table 4 shows the results of serum biochemistry of the experimental groups.  Table 4. Results of serum biochemistry of healthy, highly productive cows before and 60 days after oral supplementation «PREVAX» through the watering system daily, at a dose of 1 ml per 100 litres of water. Note: * -differences are significant at p≤0.05 (comparison between experimental and control groups)

Indicators
As can be seen from Table 4, if at the beginning of the experiment the indices of the experimental and control groups did not differ significantly, by the end of the experiment we see differences between them. In the experimental group significantly (p≤0.05) higher than in the control group values of total protein, albumin and immunoglobulins. At the same time, the content of Alanine transaminase (ALT) and Aspartate aminotransferase (AST) in the experimental group decreased by the end of the experiment, although the difference is not significant. An increase in serum immunoglobulins combined with an increase in lymphocyte count (Table 3) suggests a possible immunomodulatory property of the supplement. It should be noted that all these fluctuations were within the physiological norm.
Prophylactic effectiveness of the protein-vitamin mixture «PREVAX» is shown in table 5. Table 5. Results of a study on the effectiveness of the protein-vitamin mixture «PREVAX» for the prevention of mastitis in cows 60 days after oral supplementation «PREVAX» through the watering system daily, at a dose of 1ml per 100 litres of water. As shown in Table 5, the additive «PREVAX» in this dosage has a pronounced prophylactic effect. While 10% of cows in the control were affected by mastitis by the end of the experiment, and two out of ten were affected by clinical mastitis, in the experimental group we observed only three animals with subclinical mastitis.

Groups
When analysing the physico-chemical properties of the milk ( Table 6, Figure 6), there was a tendency for somatic cells in the experimental group to decrease by an average of 7.2% compared to the control group. When analysing the fat and protein content of the milk, a slight increase was observed compared to the control and to the initial data before the application of «PREVAX», thus the fat index reached 3.9% (by 0.25%) and the protein index 3.4% (by 0.2%) relative to control. Table 6. Results of a study on the effectiveness of the protein-vitamin mixture «PREVAX» on milk performance of cows 60 days after oral supplementation «PREVAX» through the watering system daily, at a dose of 1ml per 100 litres of water.

Stage three of the work
Study on the therapeutic effectiveness of the supplement «PREVAX» in the complex treatment of mastitis in high-producing cows.
The results of the clinical study are presented in Table 7. At the beginning of the experiment in animals with clinical form (groups 1 and 2) of mastitis the following clinical signs were observed: increase and swelling of affected quarter of udder, local temperature increase (hot to the touch), reddening, painfulness, at palpation udder tissue could be stony, dense, often noted increased supramental lymph nodes, reduced milk productivity. There was no increase in pulse and respiration, general depression of the animal, decreased appetite and increased body temperature.
After treatment, the clinical signs described above were not observed in animals in all experimental groups. Symptoms of mastitis in the second group of sick animals disappeared by the fifth day of treatment, whereas in the first group, without «PREVAX» complete disappearance of clinical mastitis signs was observed by day 9 of treatment.
During the course of the treatment, we carried out additional tests with dimestine, as well as somatic cell counts in the milk.
The results of the study are presented in Table 8. As shown in Table 8, the disappearance of clinical signs of mastitis in the first group of animals was observed by day 7 of treatment in five animals. The disease had become subclinical, as confirmed by dimestine tests and somatic cell counts, which averaged 792.4±57.5 thous/ cm 3 with a normal rate of up to 500. By day 9 of treatment, the clinical signs had disappeared in all animals, but the test for dimestine and somatic cells was positive (subclinical form). By day 12, the animals in the whole group had completely recovered, as confirmed by the negative dimatin test and the somatic cell count, which was 456.0±27. 6, In the second group using the additive «PREVAX» By day 3 of treatment, four cows (40%) no longer showed clinical signs of mastitis and the disease had become subclinical. By the fifth day of treatment, all 10 cows were clinically cured, and by the 6th day of treatment, the animals had fully recovered. By this date, all cows were negative for dimastin and somatic cell counts were within physiological limits. Therapeutic efficacy «PREVAX» In subclinical mastitis (groups 3 and 4), mastitis was evaluated with a dimastine test and by counting somatic cells in milk samples.
The results of the dimestine test were negative at the end of the treatment in all experimental groups.
Viscometric method. The results of somatic cell counts in milk samples are shown in Table 9. Table 9. Results of somatic cell counts in milk samples from cows with subclinical mastitis before and after treatment.   Table 9 showed that before therapy, the number of somatic cells in the third and fourth experimental groups exceeded the norm and indicated the presence of subclinical mastitis in cows. After therapy, the number of somatic cells in all experimental groups corresponded to the norm and had no significant differences. On completion of therapy, the number of somatic cells significantly decreased in both the third and fourth experimental groups compared with the beginning of the experiment and became consistent with normal values. The treatment period was three days everywhere, so there was no advantage in one or the other group in terms of recovery time.

Conclusions
1. The results of clinical and laboratory tests show that the protein-vitamin mixture «PREVAX» is well tolerated by lactating cows. Administration of the drug orally in a dose of 10 ml prediluted in 10 litres of water daily for 5 days did not cause changes in clinical parameters (temperature, pulse, respiration), behavioural reactions, appetite, defecation and diuresis, motor activity, skin condition, reproductive system condition (character of discharge, uterine tone, etc.), did not cause side effects and complications, did not reduce milk productions.
The analysis of the hematological parameters at the end of the experiment also did not reveal any negative changes in the blood parameters; they were within the physiological norm and had no noticeable and statistically significant differences with the parameters obtained before the experiment.
2. The protein and vitamin mix «PREVAX» has a pronounced prophylactic effect on the incidence of mastitis. It is established that the administration of the additive orally through the watering system at a dose of 1 ml per 100 litres of water for 60 days reduces the incidence of mastitis in cows by 70%. Its positive effect on the physicochemical properties of milk was also noted.
3. The protein and vitamin mix «PREVAX», used in the complex treatment of clinical serous catarrhal mastitis in cows at a dose of 4 ml per 100 litres of water, not only is it well tolerated by animals, but also gives a pronounced positive clinical effect and leads to normalisation of the somatic cell count in milk, that is, complete recovery of all animals in the experimental group on the sixth day of treatment. At the same time, treatment without the «PREVAX» supplement only led to a similar result on day 12.