Relationship Between Milk pH and Somatic Cell Count in Dairy Cows Treated with Essential Oil-based Teat Spray

Introduction

Subclinical mastitis is one of the most prevalent and economically important diseases affecting dairy cattle worldwide. Unlike clinical mastitis, it does not exhibit visible signs of inflammation, making its detection and management challenging. The condition is characterized by an elevated somatic cell count (SCC), alterations in milk composition, increased milk pH, and gradual reduction in milk yield, resulting in substantial economic losses to dairy farmers. Somatic cell count is considered a reliable indicator of udder health and intramammary infection, while milk pH reflects the physiological status of the mammary gland (Mendake et al., 2026) [11] . During mastitis, damage to mammary epithelial cells and increased permeability of blood capillaries lead to the movement of alkaline ions into milk, causing an increase in milk pH. Therefore, both SCC and milk pH are important parameters for assessing the severity of subclinical mastitis and monitoring the effectiveness of therapeutic interventions. In recent years, the use of herbal and plant-derived products has gained considerable attention as an alternative approach for mastitis control due to concerns regarding antimicrobial resistance and antibiotic residues in milk. Essential oils such as eucalyptus, lavender, peppermint, and tea tree oil possess antimicrobial, anti-inflammatory, antioxidant, and immunomodulatory properties, which may help improve udder health and reduce microbial load on the teat surface. Post-milking application of essential oil-based teat sprays and teat dips can provide a protective barrier against mastitis-causing pathogens and aid in the restoration of normal mammary gland function. Improvement in udder health is expected to reduce SCC, normalize milk pH, and subsequently enhance milk production (Mendake et al., 2025) [12] . The relationship between SCC and milk pH is closely associated with the health status of the udder. An increase in SCC due to infection is often accompanied by an increase in milk pH as a result of inflammatory changes in the mammary gland. Therefore, a reduction in SCC along with normalisation of milk pH serves as a strong indicator of recovery from subclinical mastitis. Evaluating these parameters together provides a comprehensive assessment of the efficacy of essential oilbased teat hygiene practices. Hence, the present study was undertaken to investigate the effect of different concentrations and methods of application of essential oil-based teat disinfectants on milk pH and somatic cell count in dairy cows affected with subclinical mastitis, with the objective of determining their role in improving udder health and milk quality.

Materials and Methods

Milk pH

The milk pH of each milk sample was recorded with a digital pH meter on days 0, 15, 30, 45, 60, 75, and 90. The milk samples from the individual animal were taken in a sterilised vial, and the digital pH meter was inserted into the sample to measure the pH.

Somatic Cell Count Method

The SCC was determined by manual counting as described by (Rana et al., 2023) [17] .

Technique

Sample preparation for SCC, For the preparation of the sample, 20 ml of milk and 10 ml of Phosphate buffer solution (PBS) were taken in a Centrifuge tube which was then subjected to a Centrifuge machine at 4000 rpm for 15 minutes at 25ºC. After centrifugation, the supernatant was removed by pasture pipette, and in the remaining pallet, 10 ml PBS was added to remove fats and again centrifuge at 8000 rpm for 5 minutes. After the process of centrifugation, the supernatant was removed and 2 ml PBS was added again in a fatless pallet and left undisturbed for 5 minutes. 50 µl of methylene blue dye was added to 50 µl of fatless mixture in Eppendorf serum tubes and left undisturbed for 10 to 15 minutes. With the help of a micropipette inject 50 µl of sample in a Neubauer's chamber and observe the sample under a microscope (Rana et al., 2023) [17] .

Milk pH

The results of the milk pH of the dairy cows under different treatment groups are presented in Table 4.10 and graphically depicted in Fig. 4.10. There was a non-significant (p<0.05) difference in the pH value of the milk at the initiation of the experiment. The pH value of the milk sample of the cows under different groups on initiation of the experiment, i.e., 0 th days, was T 1 (7.61±0.08), T 2 (7.65±0.07), and T 3 (7.55±0.10). The values reveal that the pH of the milk at the initial stage was slightly alkaline for all the treatment groups. However, all three groups have shown a decrease in milk pH over the experimental period. The pH value of the milk sample recorded at the end of the experimentation, i.e., on the 90 th day, was 6.68±0.05, 6.58±0.04, and 6.75±0.05 for Group T 1 , Group T 2 , and Group T 3 , respectively. This indicated a gradual normalisation of the milk pH at the end of the experiment. All treatments show a progressive decline over time in milk pH. Non-significant difference (p<0.05) observed in group T 1 on day 0 th to day 30 th, and a significant drop in pH was observed on day 45 th up to the end of experimentation. Group T 1 and group T 2 had sharper declines after Day 45 th , and group T 2 had the lowest value at Day 90 th observed. Group T 3 had shown a significant improvement from day 60 th of the experiment. The values for day 0, day 15, day 30, day 45, day 60, day 75 and day 90 for Group T 1 were 7.61±0.08, 7.58±0.08, 7.46±0.09, 7.35±0.09, 7.13±0.07, 6.87±0.04, and 6.68±0.05, respectively, for Group T 2 were 7.65±0.07, 7.65±0.07, 7.56±0.05, 7.36±0.04, 7.02±0.04, 6.80±0.02, and 6.58±0.04, respectively and for Group T 3 were 7.55±0.10, 7.51±0.09, 7.48±0.09, 7.38±0.07, 7.12±0.07, 6.86±0.04, and 6.75±0.05, respectively. The present study findings were in line with Deshmukh (2023), who observed that the pH value of the T 2 (2% EObased teat dip) and T 1 (1% iodine-based teat dip) was improved from the 30 th day of the application of the teat dip and restored to normal on 60 th days. The decrease in milk pH can be attributed to the antimicrobial and anti-inflammatory activities of the essential oils ( [,, 3] which has led to the improvement in sub-clinical mastitis condition. Similarly, Rasool et al., (2021) [19] , found that post-milking essential oilbased teat dips containing eucalyptus and lavender oils significantly reduced the pH of milk in dairy cows suspected of having subclinical mastitis on the 4 th week of treatment. Likewise, Tomar et al., (2018) [26] reported a notable drop in pH throughout the trial of the experiment in goats. On the 100 th day of using post-milking aloe vera-based teat dips in goats with subclinical mastitis, the pH of the milk returned to normal. Similarly, Waghmare et al., (2013) [28] found that post-milking teat dip consisting of Barberis lyceum, Curcuma longa, and Eucalyptus globus significantly decreased the pH of milk in subclinical mastitis in cows on day 30 th respectively. Milk components that maintain pH (casein citrate, phosphate, dissolved CO 2, and bicarbonate), are regulated by udder cells' permeability to blood capillaries. Higher pH levels in the milk are caused by mastitis because of the gland's increased permeability to blood components, including bicarbonate ions (Kolte et al., 2008) [8] . The movement of bicarbonate ions increases in milk resulting from a drop in lactose production and the addition of blood-derived alkaline salt, which has a pH above 7.0 (Bagri et al., 2018) [2] . Increasing in pH is due to higher levels of Clˉ ions (Chen et al., 2018) [4] . The decrease in the pH in present study might be due to improvement in udder condition resulting from application of essential oils in all treatment groups.

Somatic Cell Count (SCC)

The results of the somatic cell count of the cows under experimental trial are presented in Table 4.1 and the same was represented graphically in Fig. 4.1. The results show a nonsignificant (p<0.05) value of SCC on days 0 th was observed for the Groups T 1 , T 2 , and T 3 . The values for Groups T 1 , T 2 , and T 3 were 4.00±0.15, 4.12±0.16, and 4.35±0.13 respectively, which reveals that all the experimental animals exhibit sub-clinical mastitis. With the progress of the treatment, there was a decrease in the somatic cell count every fortnight i.e. on the 15 th , 30 th , 45 th , 60 th , 75 th, and 90 th day in all the groups. All treatment groups T 1 , T 2 , and T 3 , showed statistically significant (p<0.05) reductions in somatic cell count from day 0 th to day 90 th within the rows. The values of group T 1 for day 0, 15, 30, 45, 60, 75, and 90 days being 4.00±0.15, 3.62± 0.18, 3.40± 0.18, 2.77±0.12, 2.10±0.1, 2.05±0.05, and 1.97±0.04, respectively. Group T 1 had shown non-significant (p<0.05) difference within the treatment group till day 15 th . however, a significant difference within group T 1 was observed between day 30 th and day 45 th . Also, a significant difference (p<0.05) was observed between day 45 th and day 60 th . However, no significant difference (p<0.05) was observed between day 60 th and day 75 th for group T 1 . Further, a significantly lower SCC (p<0.05) was also observed for group T 1 between day 75 th and day 90 th . For group T 2 , a non-significant (p<0.05) difference within the treatment group was observed between day 0 th and day 15 th . However, a significant difference (p<0.05) within group T 2 was observed between day 15 th and day 30 th . Also, a significant difference (p<0.05) was observed between day 30 th and day 45 th . Similarly, a significant difference (p<0.05) was also observed for group T 2 between day 75 th and day 90 th . However, no significant (p<0.05) difference in SCC in group T 2 was observed between day 45 th and day 60 th ; further, no significant (p<0.05) difference in SCC in group T 2 was observed between day 75 th and day 90 th . The values for Group T 2 for day 0, 15, 30, 45, 60, 75 and 90 being 4.12±0.16, 4.12±0.16, 3.65±0.15, 2.90±0.16, 2.47±0.1, 2.22±0.08 and 1.95±0.07, respectively. For group T 3 a non-significant (p<0.05) difference within the treatment group was observed between day 0 and day 15. However, significantly lower SCC values (p<0.05) within group T 2 were observed between day 15 and day 30. Also, significantly lower values for SCC (p<0.05) were observed between day 30 and day 45. Further, with the advancement of the experiment, there is a reduction in the SCC on days 60, 75, and 90. However, no significant (p<0.05) difference in SCC in group T 3 was observed between day 45, day 60, day and day 90. The values for Group T 3 for day 0, 15, 30, 45, 60, 75, and 90 days being 4.35±0.13, 4.17±0.12, 3.85±0.10, 3.45±0.09, 3.02±0.05, 2.55±0.07, and 2.25±0.07, respectively. Significant difference (p<0.05) in Group T 1 (2.77±0.12) and T 2 (2.9±0.16) and Group T 1 (2.77±0.12) and T 3 (3.45±0.09) was observed from day 45 th . A significant difference (p<0.05) in Group T 1 and T 2 and Group T 1 and T 3 continues up to day 90 th with values as 1.97±0.04, 1.95±0.07, and 1.97±0.04, 2.25±0.07 in Group T 1 and T 2 and Group T 1 and T 3 respectively, whereas, the non-significant difference was observed in values of Group T 1 (1.97±0.04) and T 2 (1.95±0.07). The present findings were in line with the findings of Deshmukh (2023) who reported a decrease in SCC in groups treated with Essential Oil (EO)-based teat dip and Iodine-based teat dip. The readings of SCC in Groups T 1 and T 2 show that the animals have recovered from subclinical mastitis on the 90 th day of experimentation. The results of in line with the findings of Rasool et al., (2021) [19] who reported a decrease in SCC of the treatment group in dairy cows on the application of EO based post milking teat dips (Eucalyptus oil and lavender oil with dimethyl sulphoxide). Rathaur et al., (2020) [20] reported a significant decrease in SCC on the 10 th day of treatment with the application of herbal paste (turmeric, castor oil, lemon, and aloe vera) after milking in cows with subclinical mastitis. Similarly, Waghmare et al., (2013) [28] , and Vala et al., (2013) [27] also reported a significant reduction in the SCC in cows on the application of Mastidip (Berberis lyceum, Curcuma longa, Eucalyptus globulus) post-milking teat dip. Biradar et al., (2022) also reported a significant decrease in somatic cell count on the 60 th day with the application of Aloe vera-based teat dip in HF crossbred cows positive for subclinical mastitis. The somatic cells in milk include Macrophages, Neutrophils, Epithelial cells, and Mononuclear cells whereas Neutrophils and PMN Leukocytes act as the second line of defense in the mammary gland and they act as both morphological and chemical barriers for pathogens. Macrophages are found abundant (66-88%) in the mammary gland, they identify the pathogens and signal the leukocytes and immune cells resulting in the accumulation of leucocytes and immune cells in mammary glands and thereby increasing the count of somatic cells Alhussien & Dang (2018) [1] , Orlandini & Bijgaart, (2011), and Harmon, (1994) [7] . On the 90 th day, all the groups showed a decrease in SCC, however, the lowest values were observed in Group T 2 (1.95±0.04) followed by Group T 1 (1.97±0.04) and Group T 3 (2.25±0.07) indicating better efficacy of 2% EO-based Teat spray over 2% EO-based teat dip and 1% EO-based Teat spray.

Conclusion

The present study demonstrated that the application of essential oil-based teat spray and teat dip formulations effectively improved udder health in dairy cows affected with subclinical mastitis, as evidenced by a significant reduction in somatic cell count (SCC) and normalization of milk pH. All treatment groups showed a progressive decline in SCC and milk pH throughout the 90-day experimental period, indicating recovery from subclinical mastitis. The reduction in SCC may be attributed to the antimicrobial and antiinflammatory properties of the essential oils, which helped control intramammary infections and reduce inflammation of the mammary gland. Simultaneously, the decrease in milk pH towards the normal physiological range reflected restoration of mammary gland integrity and reduced leakage of alkaline blood constituents into milk. Among the treatments evaluated, the 2% essential oil-based teat spray (T 2 ) showed the greatest efficacy, recording the lowest SCC and milk pH values at the end of the experiment, closely followed by the 2% essential oil-based teat dip (T 1 ). The findings indicate a strong relationship between decreasing SCC and normalisation of milk pH, confirming that both parameters can serve as reliable indicators of udder health improvement. Therefore, the use of essential oil-based teat spray, particularly at a 2% concentration, can be recommended as an effective natural strategy for the management of subclinical mastitis and for improving milk quality in dairy cows.

Acknowledgement

AcknowledgementThe authors are grateful to the Department of Livestock Production and Management, Nagpur Veterinary College, Nagpur, under Maharashtra Animal and Fishery Sciences University (MAFSU), for providing the necessary facilities and support for this research. Sincere thanks are also extended to the Dean, Gorakshan Sabha Gaushala, Dhantoli, Nagpur, and all faculty members, laboratory staff, and colleagues for their cooperation, guidance, and assistance throughout the study. The authors also acknowledge everyone who directly or indirectly contributed to the successful completion of this work.

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