The health-safety and nutritional quality of raw milk are influenced by many factors. One of the main factors that affect the health of and milk production by dairy cows is mastitis. Mastitis is an inflammation of the mammary gland characterized by physical, chemical, bacteriological, and cytological changes in the milk. Changes in the quality and quantity of milk, as well as pathological changes in the glandular tissues of the udder have been observed (Pyörälä and Taponen, 2009).

Mastitis is mainly caused by microorganisms. These are usually bacteria, including gram-negative and grampositive bacteria, mycoplasmas, yeasts, and algae (Zadoks et al., 2011).

The majority of mastitis cases are caused by a few common bacterial pathogens involved: Staphylococcus spp. (S. aureus, S. warneri and S. chromogenes), Streptococcus spp. (Str. agalactiae, Str. dysgalactiae, Str. uberis and Str. bovis), coliforms (mainly E. coli and Klebsiella pneumoniae) and Actinomyces pyogenes (Idriss et al., 2013). Although some pathogens from the group of coagulase negative staphylococci (CNS) and Corynebacterium bovis, are historically considered to be of limited importance and are therefore often described as minor pathogens. In the last decade the impact of CNS has increased probably because the prevalence of major pathogens has decreased (Pyörälä and Taponen, 2009).

Mastitis and other diseases are common problems in dairy herds, resulting in increased costs and decreased production. Most diseases in dairy cows occur at or just after calving, which is a period associated with immune suppression, resulting in increased susceptibility to infections. Prepartum immune suppression is multifactorial but is associated with endocrine changes and destabilization of intestinal flora, leading to impaired digestion and utilization of nutrients from animal feed (Xiaowang, Shaohua and Lixia, 2010; Zigo et al., 2014).

During the past few decades the use of organic feed additives to improve health, wellbeing, and production has been investigated in some areas of animal husbandry. Humic substances (HS) are one such additive (Marcinčáková et al., 2015; Semjon et al., 2020).

Humic substances (HS) are geological deposits made of a complex mixture of acids that arise from the natural decomposition of plant and animal material by soil microorganisms occurring in water, soil, carbon and other sources. They are heterogeneous high molecular weight organic substances and their composition differs according to the geographic region (Jaďuttová et al., 2019; Mudroňová et al., 2020).

A yellow to brown-coloured seam (brown seam) may contain high concentrations of fulvic acid, whereas a dark brown to black-coloured seam (black seam) may contain high amounts of humic acid and humin. Humic acids (HA) are considered to be adsorbent, because of various binding sites present in their structure. It has been assumed that humic acids could reduce the absorption and systemic availability of bacterial endotoxins, which could be of great importance in the protection of animal and human health (Trckova et al., 2005; Galip, Polat and Biricik, 2010).

Moreover, many positive effects on the performance and health of animals have been attributed to humic acids. They inhibit the growth of pathogenic bacteria and moulds and decrease the level of mycotoxins and thus may lead to improved gut health (Marcinčáková et al., 2015).

Humic acids stabilize the intestinal flora, and in this way, improve the utilization of nutrients from animal feed, which affects the composition of dairy cows’ and goatsʼ raw milk (Potůčková and Kouřimská, 2017).

Table 2 illustrates the effect of the supplementation of HA on the milk components and SCC in dairy cows. The two groups of animals, control and experimental, were fed with TMR (Table 1) during the 50 days prior to parturition. In addition to this feed, the experimental group was supplemented with HA at a dose of 100 g per cow per day. Changes of MU content (Figure 2) and SCC on the 10^th day after calving in HA supplemented group was reported. No changes in the composition of dry matter, SNF, lactose, fat, protein content, or pH in raw milk were noted during the monitoring period (Table 2).

The observed decreased level of MU in cows fed diet with HA can be explained by lower blood urea nitrogen (BUN) level which resulted from lower ruminal ammonia nitrogen (NH₃-N) concentration indicated by more efficient utilization of dietary crude protein (CP).

The same trend was observed in the study by Van Soest (1994) who supplemented dairy cows with HA during lactation. In HA treated cows, lower blood urea nitrogen (BUN) value was indicated by more efficient utilization of dietary CP for microbial protein synthesis associated with nitrogen-binding capabilities of HA. This low value of BUN is usually associated with a lower ruminal NH₃-N flux to the bloodstream. Thus, a reduction in MU result can be explained with the reduction of BUN supported by lower ruminal NH₃-N concentration.

Similar results were observed by Degirmencioglu (2014) after supplementation of HA to ruminants for 90 days of lactation, with no improvements in milk composition (nonfat dry matter, lactose, fat and protein content). The effect on milk yield was inconsistent.

Other studies have reported positive effects of HA on milk production, milk fat (Thomassen and Faust, 2000) and milk protein (Potůčková and Kouřimská, 2017) in dairy cows. Another study showed that the use of HA as an animal feed supplement leads to increased milk production and increased butterfat percentage in dairy cows (Islam, Schumacher and Gropp, 2005).

However, it is difficult to compare the effects of HS across studies due to the different sources and preparations of HA used, as well as because animals reared in various regions of the world are exposed to different climates and environmental conditions.

Milk SCC is a useful tool for measuring milk quality, the health status of the mammary gland and changes in milk composition. In the European Union the legal limit for cows is 400 000 cells.mL^-1 (Zajác et al., 2012) and in the USA the legal limit established by the Food and Drug Administration for cows is 750 000 cells.mL^-1 (Paape et al., 2007).

In our study, the control group shoved increased SCC value above the legal limit on the 10^th day after calving (Figure 2). However, the HA supplemented group had lower SCC values on the 10^th day after calving (Table 2) and positive quarters according to the CMT (Table 3).

A score of 1 to 3 in the CMT indicates an increased SCC over the legal limit, most commonly caused by the presence of pathogenic bacteria. The penetration of pathogenic bacteria into the teat canal irritates the delicate mammary tissue causing an inflammatory response and changes in milk quality and composition (Sharma, Singh and Bhadwal, 2011).

In particular, differences were found by comparing the positive quarters in both groups with CMT score 1 and 3. According to our results the number of positive quarters detected by the CMT on the 10^th day after calving was reduced by 20.0% in the group of cows supplemented with HA (Table 3).

Similar to our study, Thomassen and Faust (2000) reported that HA supplementation significantly decreased the SCC in milk. They reported that a diet containing 3 g.HA.kg^-1 decreased the SCC level in milk dairy by about 50%.

Similarly, Xiaowang, Shaohua and Lixia (2010) reported that a lower (by 40.1%) SCC was observed in their humate-supplemented group compared to the control.

Intramammary bacterial invasion occurs immediately after calving and leads to glandular damage in parenchymatous tissue. The glandular tissue damage leads to increased SCC and reduced milk production. The cellular presence in milk is one of the important protective mechanisms of the mammary gland (Sharma, Singh and Bhadwal, 2011).

The addition of HA at a dose 100 g per cow per day in our study had a positive nutraceutical effect that stimulated neutrophil activity, which may protect against bacterial pathogens and reduce mortality during acute bacterial infection. The results show that in addition to reducing SCC the number of positive quarters infected with CNS was reduced by 12.5% during the first 30 days of lactation (Figure 3). On the 10^th day of lactation, 15 and 9 quarters were infected in the control and experimental group, respectively. The same trend was observed on the 30th day of lactation. A large proportion of CNS (40%) was noted in the control group from infected quarters.

In recent years CNS have become increasingly important in udder infections. They are normal inhabitants of the skin and teat canal and are frequently isolated from milk samples (Taponen et al., 2006). S. chromogenes, S. haemoyticus and S. warneri were pathologically important in intaramammary infection with increased SCC. The addition of humates to the feed reduced the incidence of staphylococcal infections by 5%.

According to Dabovich et al. (2003) the use of HA increases the body's defenses by stimulating neutrophil activity in response to the onset of inflammation. Testing of milk during field trials often indicates an increase in the number of microbes in the milk, an indication to the dairyman of impending mastitis.

As a result of feeding humates, mastitis cases within the milking herd dropped from an average of 3 to 4 cases daily to 4 cases in a month (Islam, Schumacher and Gropp, 2005).

According to our results dietary supplementation with HA significantly reduced the MU content, SCC and the number of positive quarters detected by the CMT on the 10^th day after calving but did not affect the others milk parameters. Although the mechanism by which HA supplementation affects milk synthesis and mastitis reduction has not been fully described, its indirect beneficial effects could improve the immunity of the mammary gland. Based on the information above, further research into the use of humates for the prevention of mastitis during the peripartum period and early lactation will be needed.