Newborn calves have incompletely developed defence mechanisms, increasing their susceptibility to diseases, particularly those affecting the digestive and respiratory systems (McGuirk, Reference McGuirk2008). Due to the structure of the ruminant syndesmochorial placenta, which blocks the transfer of immunoglobulins from the cow to the foetus (Peter, Reference Peter2013), calves are born with negligible serum immunoglobulin levels (McCoy et al., Reference McCoy, Reneau, Hunter and Williams1970). Consequently, they rely entirely on passive immunity transfer through colostrum, which contains essential components for neonatal protection and immune development (Morein et al., Reference Morein, Abusugra and Blomqvist2002).
Immunological immaturity in early life, particularly under inadequate management conditions, increases calf susceptibility to enteric diseases such as diarrhoea and respiratory conditions like bovine respiratory disease (BRD), leading to a high use of antimicrobials (Callaway et al., Reference Callaway, Lillehoj, Chuanchuen and E Gay2021). These illnesses are among the leading health challenges in dairy systems and are associated with compromised growth (Cramer MC e Ollivett TL, Reference Cramer and Ollivett2019; Abuelo et al., Reference Abuelo, Cullens and E Brester2021), raise the risk of death and culling (Buczinski et al., Reference Buczinski, Achard and Timsit2021) and impair productive performance (Delabouglise et al., Reference Delabouglise, James, Valarcher, Hagglünd, Raboisson and Rushton2017; Adams and Buczinski, Reference Adams and Buczinski2016). Their impact extends beyond individual animal health, affecting herd productivity, increasing rearing costs and decreasing replacement efficiency.
To mitigate these impacts, nutritional strategies involving nutraceuticals have been explored. Supplementation with minerals such as zinc and selenium, and vitamins A and E, has shown potential to improve immune function and antioxidant capacity in calves, helping to prevent enteric and respiratory diseases (Volpato et al., Reference Volpato, da Silva, Crecencio, Tomasi, Fortuoso, Ribeiro, Secco, Pereira, Bottari, Schetinger, Morsch, Baldissera, Stefani and Machado2018). Likewise, probiotics have demonstrated beneficial effects by modulating gut microbiota, reducing diarrhoea incidence (Frizzo et al., Reference Frizzo, Soto, Zbrun, Signorini, Bertozzi, Sequeira, Armesto and Rosmini2011) and improving nutrient absorption, feed intake and growth (Agazzi et al., Reference Agazzi, Tirloni, Stella, Maroccolo, Ripamonti, Bersani, Caputo, Dell'Orto, Rota and Savoini2014).
However, many studies assessing nutraceuticals were conducted under suboptimal management conditions, including inadequate colostrum feeding or lack of transition milk (Volpato et al., Reference Volpato, da Silva, Crecencio, Tomasi, Fortuoso, Ribeiro, Secco, Pereira, Bottari, Schetinger, Morsch, Baldissera, Stefani and Machado2018). These factors may have compromised the immune development of calves, increasing their disease susceptibility and potentially exaggerating the benefits attributed to the additives. Therefore, additional research is needed in well-managed systems to determine the true efficacy of nutraceuticals. It is also essential to evaluate the performance of commercial products under real-world farm conditions, where environmental and management variables can influence their effectiveness.
The objective of the present study was to evaluate the effect of using a commercial product containing nutraceutical additives – composed of vitamins, minerals and probiotics – during the pre-weaning phase of dairy calves on weight gain and disease occurrence. We hypothesised that calves receiving the nutraceutical would exhibit greater weight gain and improved health.
Material and methods
This study was conducted on a commercial dairy farm (Castro, Paraná, Brazil) and was approved by the Animal Use Ethics Committee of the Pontifical Catholic University of Paraná (02293/2022).
The calves in the study were housed individually (1.2 m wide × 1.5 m long per calf; sand mixed with straw bedding) from the first day of life until 40 days of age. After that, they were moved to group pens (10 calves per pen, 4 m2 per calf; straw bedding) until weaning at 80 days of age. Colostrum was administered using high-quality colostrum (BRIX > 25%) at a volume equivalent to 10% of body weight during the first feeding. To verify transfer of passive immunity, blood serum was collected 48 hours after colostrum ingestion and evaluated using a digital refractometer (Sper Scientific, Scottsdale, AZ, USA). Transition milk and milk was provided twice daily (08:00 and 16:00). Transition milk was offered during the first 3 days of life, and from day 4 onwards, the calves received 6 L of milk replacer (Sprayfo, Trouw Nutrition, Deventer, Netherlands), prepared to achieve 14% total solids, until 60 days of age. Milk was offered via bottle until 40 days of age and subsequently via Milkbar® feeders until weaning. After 60 days of age, calves were fed 4 L per day and weaning occurred at 78 ± 3 days of age. Throughout the entire experiment, water and starter were available ad libitum to the calves from the first day of life; additionally, hay was provided starting from 60 days of age.
Study design
In total, 126 Holstein female calves with an average birth weight of 38.37 ± 3.06 kg were enrolled in the study between September 2022 and February 2023. All calves were randomly grouped according to birth date; every 10 calves born formed a group, alternating between the control (CON) group and the treatment (TRT) group. Thus, 63 calves were included in the CON group, managed according to the farm’s routine practices. The TRT group consisted of 63 calves, managed in the same way as the CON group, with the supplementation of the commercial additive (Table 1). Once allocated to their respective pens, each calf had a corresponding monitoring sheet where data on the mother, birth condition, volume and amount of colostrum ingested and other present conditions were recorded. The physical examination of the calves in both groups to investigate the presence of pulmonary and enteric diseases was conducted weekly by the research team, with details provided in the following sections.
Nutritional composition of the nutraceutical additive used for calf feeding in the treatment group (100 g/calf/day)
Treatment delivery
The product was given in the milk at every feeding from birth until weaning, at 78 ± 3 days. The product was weighed and dissolved in the milk replacer at the time of its preparation by the research team. To facilitate dilution, we used an industrial mixer with an amount of 50 g of the product per calf per feeding (100 g/calf/day). For easier identification, the bottles used for feeding the TRT group were marked with red tape. The pens of the calves belonging to the TRT group, as well as an identification collar at birth, were also marked with red tape for easy identification.
Health assessment
The physical examination was performed weekly by the research group, consisting of veterinarians and veterinary students, following the calf health assessment protocol described in the Wisconsin manual (McGuirk, Reference McGuirk2008). The evaluation is based on scores for rectal temperature, nasal discharge, cough, ocular discharge and ear position. In the Wisconsin score, each sign is rated on a scale from 0 to 3, where 0 indicates normal, 1 represents a mild change, 2 a moderate change and 3 a severe change. Calves with a total score of ≥5 were considered positive for BRD (McGuirk, Reference McGuirk2008).
To enhance respiratory health assessment, lung ultrasonography (Croxxvet, Montes Claros, Brazil; linear probe, frequency of 6.5 MHz) was performed to improve ultrasound image visualisation, using 70% liquid alcohol as the transducer medium in the 3rd to the 11th intercostal space. Ultrasonographic evaluations were conducted at 7, 30 and 75 days of life, and pulmonary consolidation scores were categorised from 0 to 5, based on Ollivett and Buczinski (Reference Ollivett and Buczinski2016). Where score 0 represents only reverberation artifacts, few comet tails and consolidations <1 cm; score 1, diffuse comet tails occupying one or more lung lobes; score 2, pulmonary consolidations >1 cm but not occupying the entire lobe; score 3, complete consolidation of one lung lobe; score 4, consolidation of 2 lung lobes; and score 5, when 3 or more lung lobes were consolidated (Ollivett and Buczinski, Reference Ollivett and Buczinski2016; Cramer et al., Reference Cramer, Ollivett and Stanton2016). Calves with pulmonary consolidations above score 3 were considered positive for BRD in the study.
The diagnosis of diarrhoea was performed weekly by the research team using a visual scoring system according to faecal consistency and the rectal temperature (°C) using a digital thermometer (Joytech, Hangzhou, China); scores ranged from 1 to 3, with scores 2 and 3 considered diarrhoea. Score 1 was considered normal faeces, with preserved consistency (McGuirk, Reference McGuirk2008). The severity of diarrhoea was measured according to the maximum stool score of each calf (score 3).
Neither serum Brix (TRT: 9.55 ± 0.97; CON: 9.38 ± 0.86; P = 0.90) nor the failure in transfer of passive immunity (cut off = 8.1% Brix) differed between groups (P = 0.44).
The evaluation of each calf's navel was conducted using scores from 0 to 2, where score 0 represents a normal, dry navel without deformities; score 1 represents an oedematous navel, diameter larger than a thumb; score 2 represents an infected navel with or without the presence of myiasis (Gomes et al., Reference Gomes, Madureira, Borges, Pinheiro, Martin, Baccili, Santos, Yasuoka, Decaris and Bombardelli2021).
Calves were monitored daily by trained farm staff, and clinical diagnosis of BRD and diarrhoea was based on the Wisconsin Scoring System. Treatments followed established farm protocols and varied according to the condition. For BRD, the standard protocol included intramuscular antibiotic therapy with enrofloxacin (Kinetomax®, 3 mL/40 kg for 3–5 days), flunixin meglumine, a non-steroidal anti-inflammatory (Niglumine®, 1 mL/22.5 kg for 3 days) and bromhexine hydrochloride mucolytic (Aliv V®, 8 mL for 3 days). For diarrhoea, treatment depended on the presence of fever (>39.4°C). Febrile calves received intramuscular antibiotic with trimethoprim and sulfadoxin (Borgal®, 10 mg/kg for 3 days), non-steroidal anti-inflammatory – meloxicam (Maxicam®, 0.5 mg/kg for 3 days), oral probiotic (Floravac®, 5–10 g for 3–5 days) and intravenous fluid therapy (lactated Ringer’s solution). Non-febrile calves were treated with Maxicam®, Floravac® and oral rehydration (Hidralac®) for 3 days. All treatments were recorded on individual calf sheets, and at weaning, data from each calf in the study groups were collected and transferred to an Excel spreadsheet.
Performance: weight gain
The estimation of body weight of the calves was conducted on the day of birth, at 28 days, and at weaning. For weight measurement, a birth hoofometer tape for bovines was used at birth (Calfscale, Iowa USA), and a thoracic weight tape was used for the other weight measurements of the calves (Heinrichs and Hargrove, Reference Heinrichs and Hargrove1987). Overall average daily gain (ADG) was calculated as the difference between weaning and birth weights divided by the total number of milk feeding days.
Statistical analysis
For the descriptive analysis, the data were tabulated in an Excel spreadsheet, and means were calculated for continuous variables and proportions for categorical variables. To address the objective of the study, generalised regression models were used to evaluate the association between the product intake and the variables of interest. For each model, confounding factors were identified and included as covariates (e.g. birth weight as a covariate in the model assessing the effect of treatment on the final weight gain of the calves). The animal was the experimental unit, so for models that included repeated measures on the animal, it was included as a random effect. Estimated means were extracted using the least squares method. All statistical analyses were performed in R software through the RStudio interface (RStudio Inc., 2022). P-values <0.05 were considered statistically significant. P-values between 0.05 and 0.10 were considered to indicate a trend.
Results
Performance: weight gain
No differences were observed between calves receiving the supplemented product and those in the CON group for weaning weight or ADG during any of the evaluated periods (birth to 28 days, 28 days to weaning and overall) (Table 2).
Weaning weight and average daily gain (ADG) at different periods in 126 dairy calves according to treatment group (TRT and CON)
Legend: CON: calves that did not receive the nutraceutical. TRT: calves that received 100 g of the nutraceutical per milk feeding. ADG, average daily gain; d, days; BRD, Bovine Respiratory Disease.
* The severity of disease was measured according to the maximum faecal score of each calf.
Health: number of treatments during pre-weaning
There was a difference in the total number of antibiotic treatments between the groups (P = 0.01). On average, calves in the CON were treated more during the pre-weaning period than calves in the TRT (TRT: 1.53 ± 0.11; CON: 1.90 ± 0.10; P = 0.01). This difference was influenced by the number of treatments for BRD, as the CON calves were treated more for BRD than the TRT calves (Table 2). The total number of treatments for diarrhoea showed no difference between the groups (Table 2).
Health: diarrhoea
Calves in the TRT group experienced the first case of diarrhoea earlier than those in the CON group (Table 2). No difference was observed in the number of diarrhoea events requiring antimicrobial use (OR: 0.89; 95% CI: 0.70–1.12; P = 0.31). Likewise, there was no difference in diarrhoea severity, as measured by the maximum faecal scores (Table 2).
Health: BRD
There was no difference between groups regarding the age at first BRD occurrence or the average age at first BRD treatment. Nevertheless, CON calves received more BRD treatments compared to TRT calves. No differences were observed in maximum Wisconsin scores or in pulmonary consolidation scores at 30 days of age. However, at 75 days of age, calves in the TRT group presented higher pulmonary consolidation scores compared to the CON group. All data are presented in Table 2. As a result, CON calves showed a greater reduction in pulmonary consolidation scores from 30 to 75 days of age, and the odds of recovery from pulmonary consolidation at this time point were significantly higher in the CON group (OR: 3.00; 95% CI: 1.31–76.16; P = 0.01).
Discussion
The inclusion of the nutraceutical in the calves’ liquid diet did not result in a significant reduction in the incidence of clinical diseases or improvements in weight gain compared to the CON group. Although a lower number of clinical treatments was observed in the supplemented group, given that no differences were identified in the other evaluated parameters, this outcome may not be directly attributed to the nutraceutical itself. Instead, it is possible that the reduced treatment frequency was influenced by management-related factors, such as staff perception of the product’s effectiveness.
Although our initial hypothesis was that the additive would enhance calf performance, no differences in ADG or weaning weight were observed between groups. Similar findings were reported by Frizzo et al. (Reference Frizzo, Soto, Zbrun, Signorini, Bertozzi, Sequeira, Armesto and Rosmini2011), who found no effect of probiotic supplementation on calf growth, emphasising that such outcomes are highly dependent on variables like environmental conditions, pathogen pressure and stress levels. One possible explanation for the lack of nutraceutical effect is the high nutritional quality of the milk replacer used, which likely supported elevated growth rates even in the CON group. Notably, this milk replacer already met or exceeded NRC (2021) recommendations for key nutrients – providing, for example, 85 mg/kg of zinc (vs. the recommended 24 mg/kg) and 5,000 IU/kg of vitamin D (vs. 3,200 IU/kg). Once the animals’ requirements are met, excess vitamins and minerals are typically excreted – even when supplemented with the nutraceutical – potentially minimising the scope for additional benefits. This highlights how the efficacy of such additives may be constrained by the baseline nutritional adequacy of the diet.
By contrast, studies that reported growth improvements with supplementation often involved more restrictive rearing conditions, including early weaning (e.g. at 56 days), limited milk allowance and lower initial performance levels – as observed in Zanetti et al. (Reference Zanetti, Schalch and Rossini1999), where calves averaged only ∼ 380 g/day in ADG. These comparisons suggest that nutraceuticals may be more effective in settings where nutritional or environmental challenges are present.
It is also important to consider the method used to estimate weight. In our study, calf body weight was assessed using a heart girth tape – a practical but indirect approach. As noted by Heinrichs and Hargrove (Reference Heinrichs and Hargrove1987), this method may reduce measurement precision, potentially limiting our ability to detect subtle differences in weight gain between treatments.
We identified a high treatment rate for the disease, considering that the farm protocol includes antibiotic therapy. This management may have a significant impact on the intestinal microbiota, eliminating commensal bacteria and spreading resistant bacteria, promoting dysbiosis (Modi et al., Reference Modi, Lee, Spina and Collins2013), as well as compromising the availability of probiotic bacteria, increasing the severity and clinical recovery of diarrhoea cases. Additionally, in this study, we believe this affected the product's efficacy. The probiotic dose may also have been another factor why we did not obtain good results at the enteric level. A study conducted with probiotics had positive results on performance and reduction of diarrhoea cases (Alawneh et al., Reference Alawneh, Barreto, Moore, Soust, Al-harbi, James, Krishnan and Olchowy2020), but the doses used were higher than those applied in this study.
Calves that received the commercial nutraceutical developed diarrhoea earlier than those in the CON group. This outcome may be associated with the product’s high lactose content – around 40% of its formulation – which, when added to the milk replacer, likely increased the overall intestinal osmolality. Milk replacers already tend to be slightly to highly hypertonic (ranging from just over 300 mOsm/kg to more than 450 mOsm/kg), whereas whole bovine milk is isosmotic, with osmolality near 300 mOsm/kg (McGuirk, Reference McGuirk2003). Elevated osmolality has been shown to increase gastrointestinal permeability, potentially compromising intestinal barrier integrity and predisposing calves to enteric disturbances (Wilms et al., Reference Wilms, Berends and Martín-Tereso2019).
The use of nutraceuticals or probiotics remains controversial, as their efficacy under field conditions is influenced by numerous factors. Moreover, many products have not been evaluated in well-controlled experimental or field trials. As a result, the effectiveness of many probiotics – whether administered as single bacterial or yeast strains or as multi-strain cocktails – remains unsubstantiated by rigorous scientific evidence (Parreño et al., Reference Parreño, Saif, Strain and Chase2022). Probiotics can exert varying effects, and their therapeutic use must be tailored to the specific disease or clinical context. Furthermore, probiotic cocktails – often composed of varied combinations – may counteract or neutralise the beneficial effects of certain individual strains within the mixture (Kandasamy et al., Reference Kandasamy, Vlasova, Fischer, Chattha, Shao, Kumar, Langel, Rauf, Huang, Rajashekara and Saif2017). Different products have distinct modes of action and efficacy profiles, which help explain the variability observed when translating probiotic research findings into real-world farm settings (El Jeni et al., Reference El Jeni, Villot, Koyun, Osorio-Doblado, Baloyi, Lourenco, Steele and Callaway2024).
The CON group calves were treated more for BRD than the TRT group calves. We believe this influenced the results related to the severity of the cases and the likelihood of recovery, as the CON group had a lower pulmonary consolidation score than the TRT group at 75 days of age. This difference may suggest that the fact that the CON group was treated more resulted in reduced pulmonary consolidations. Another hypothesis could be that the calves consuming the product had a later onset of pulmonary disease or presented a milder or even subclinical condition and, consequently, were not identified and treated by farm staff, resulting in the observed outcomes.
Another hypothesis is that the staff responsible for diagnosing and treating the calves daily may have influenced the number of treatments and consequently the recovery of disease cases. The TRT group calves were marked, which may have led the staff to assume that these animals already had increased immune support and, therefore, could resolve their cases without medication. However, the farm staff was not aware of the hypothesis of this study. These considerations are important for interpreting the study results and understanding how different factors may influence the perceived effectiveness of the treatment.
It is important to highlight that this study was conducted on a commercial dairy farm, where animals were exposed to real-life conditions rather than a fully controlled research environment. The farm used in this study already utilised a high-quality milk replacer containing additives such as probiotics, which could have masked any additional effects of the treatment. Still, all calves faced typical management and health challenges commonly found in large-scale commercial operations, including varying infection pressures and environmental stressors. These factors may have limited the ability to detect subtle differences between treatments. Despite these, we consider this study highly relevant, as it evaluates the performance of commercial products under practical, field-like conditions. Testing interventions in real-world scenarios is essential to understanding their actual applicability and benefits in commercial production systems.
Conclusion
The commercial product did not impact calves’ weight gain or enteric health and showed no effect on the overall incidence of diseases. Although a lower number of treatments for BRD was observed in the supplemented group, this reduction may not be directly attributed to the nutraceutical itself, especially in the absence of differences in other health or performance parameters. Management-related factors, such as staff perception of the product’s effectiveness, may have influenced treatment decisions. It is also important to consider that not all nutraceuticals will necessarily produce direct effects on the evaluated variables, and the specific formulation and combination of components may play a critical role in their efficacy.
Funding statement
The funder of this study had no participation in the data collection, analysis and interpretation of our results. All authors have contributed significantly to be included in the authors list.
Competing interests
The authors declare no conflict of interest.
