Prophylactic use of an ultra-diluted complex on polymorphonuclear leukocyte function and respiratory scores of weaned Holstein calves immediately after grouping

The aim of the study was to evaluate the effect of an ultra-diluted complex supplementation as a prophylactic strategy on immunity, performance, and respiratory scores of weaned Holstein calves immediately after grouping. Thirty-six weaned Holstein female calves (80.4±1.3 days old; 105.6±10.4 kg) were allocated to 6 pens (n=6 per pen) in a completely randomized design experiment in a double-blind, placebo-controlled trial. During a 28 days period, animals received a total mixed ration and were enrolled into two different groups (n=18 per group): 1) Control (basal diet + calcium carbonate, top-dressed at 30 g/animal/d – ultra-diluted placebo vehicle), or 2) ultra-diluted complex (basal diet + TopVitaTM-Real H, top-dressed at 30 g/animal/d – Sulphur:10 + Viola tricolor:10 + Caladium seguinum:10 + Zincum oxydatum:10 + Phosphorus:10 + Cardus marianus:10 + Colibacillinum:10 + Podophyllum:10 + Vehicle: calcium carbonate; q.s. 1kg). Blood samples were collected from each heifer at enrollment and 28 days later to assess polymorphonuclear leukocyte (PMNL) function and blood cell count. Body weight was assessed at enrollment and 28 days later at the end of the study. Regarding respiratory-screening process, a calf scoring system modified for calves in group pens was used. There was no effect of prophylactic ultra-diluted treatment on PMNL, nor it affected lymphocytes count and its ratio. Besides, the ultra-diluted product did not affect body weight and ADG. Further, no effect was observed in respiratory scores throughout the study period. In conclusion, the ultra-diluted complex did not improve blood cells count and PMNL function, nor it had impact on the performance of weaned Holstein calves after grouping.


Methodology
The methodologic approach herein used may be classified as a quantitative method. A quantitative or numerical data is collected due to the use of measurements of quantities, which is obtained through metrology (numbers with their respective units; Yin, 2015). All procedures performed in this study were approved by the Animal Care and Use Committee (IACUC, protocol #20018-02). Based on previous blood samples analysis from the studied herd, baseline neutrophils count in weaned calves housed in superhutches was anticipated to average 4 x 10 6 cells/mL and standard deviation = 1 x 10 6 cells/mL, with an assumption that ultra-diluted treatment would reduce neutrophil count by at least 25% (1 x 10 6 cells/mL unit). To detect this reduction, 17 calves per treatment for each group was required to ensure a power (1-β error probability) of 0.80. To ensure this number of animals per treatment, herein we enrolled 18 animals each group.
Between February 2020 and March 2020, 36 clinically healthy weaned Holstein calves were randomly selected at a commercial dairy located in west Texas, USA. The dairy milked 3700 Holstein cows three times daily in a 70-stall rotary milking parlor. After being weaned about 60 ± 1 d of age, the calves remained at hutches until 80.4 ± 1 d of age, when the calves were moved to the group pen. The calves were housed in outdoor, plastic superhutches with an attached pen. Superhutches, is a large calf hutch (6 × 3 m) that provides transitional housing for a small group of 6 weaned calves per group. A total of 6 superhutches (6 animals each) were enrolled for this study. The superhutches were bedded with cottonseed hulls and equipped with a feed bunk that provided space for 6 calves and 1 water bowl. When moved into groups, the calves continued to receive the same starter fed in the milk-feeding period. The animals had ad libitum access to water and starter. The experimental period lasted 28 days after enrollment date.
Blood samples were collected from each heifer at enrollment and 28 days later, from the jugular vein using a Vacutainer tube with lithium heparin, a Vacutainer tube with potassium EDTA, and a 20-gauge x 2.54-cm Vacutainer needle (Becton, Dickinson and Company, Franklin Lakes, NJ). After collection, the heparinized blood was stored in an ice chest without any ice to preserve their phagocytic and oxidative burst capacity (Sellers et al., 2013). The EDTA and coagulated blood samples were transported to the laboratory on ice. The heparinized and EDTA blood samples were processed in the same day of collection for measures of hematology and ex vivo PMNL responses.
Leukocyte count and differentials were performed using a hematology analyzer (IDEXX Procyte DX, Westbrook, ME), and the variables of interest were neutrophil count and neutrophils to lymphocytes ratio. Flow cytometry was used to determine the phagocytosis and oxidative burst capacity of peripheral PMNL and the quantification of the adhesion molecule L-selectin Research, Society andDevelopment, v. 10, n. 11, e281101119582, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i11.19582 (CD62L), with minor modifications (L. E. Hulbert et al., 2011). To measure the phagocytic and oxidative burst capacity, 100 μL of heparinized whole blood were incubated for 15 min in an ice bath. After this initial incubation, 20 μL of a 100 μM solution of dihydrorhodamine (Invitrogen, Carlsbad, CA), and 20 μL of a 10 9 cfu/mL propidium iodine labeled E. coli suspension were added to the blood, and then incubated in a 38.5°C water bath for 10 min (negative controls were incubated in an ice bath for 10 min). Then, samples were immediately placed on an ice bath for 5 min, and erythrocytes were hypotonically lysed, and washed with PBS. Dual-color flow cytometry was performed using an Attune flow cytometer (Life Technologies, Thermo Fisher Scientific Inc., Waltham, MA). The PMN population was gated using forward and side scatter plots. The mean fluorescence intensity (MFI) and proportion of PMNs that performed phagocytosis and oxidative burst were acquired using the optical filters After a 1 h incubation in an ice bath, erythrocytes were hypotonically lysed. After centrifugation, the leukocyte pellet was resuspended in a 50 μL solution of fluorescein-labeled secondary antibody at a 1:400 dilution (F(ab′)2 anti-mouse IgG:FITC; AbD Serotec Raleigh, NC) and incubated for 1 h in an ice bath. After a PBS wash, samples were analyzed using single-color flow cytometry. The PMN population was gated as previously described, and the MFI for L-selectin was gathered using BL-1.
Data were analyzed using Attune Cytometric software (Life Technologies, Thermo Fisher Scientific Inc.).
Body weight was assessed at enrollment and 28 days later at the end of study. Through the practical and reliable technique, body weight was estimated using calibrated weight tapes measuring heart girth circumference (Heinrichs et al., 1992).
These measurements were used to calculate the body weight average daily gain (ADG) during study period (final body weight -initial body weight / 28 days).
To respiratory-screening process, a calf scoring system modified for calves in group pens was used according to McGuirk and Peek (2014). This group pen respiratory scorer is available at https://www.vetmed.wisc.edu/fapm/svm-dairyapps/group-pen-respiratory-scorer/. The categorization was made scoring nasal discharge, ocular discharge, ear tilt, and coughing. This modified scoring system identify diseases of the group instead of individual animals. If the group/pen is classified into having more than 25% of the animals with abnormal in 2 or more categories scored, this group would be classified as a high risk to develop respiratory disease. Abnormal scores are stated as score 2 or 3 from 0 to 3-point scale. This tool is used in farm´s calf health-screening program to obtain early diagnostic of respiratory diseases in calves (McGuirk & Peek, 2014).
The effect of ultra-diluted treatment on PMNL function, cells count, body weight, and growth (ADG) was performed using the MIXED procedure of SAS (v.9.4, SAS Institute Inc., Cary, NC). The model included the fixed effects of treatment and random effect of pen. The random effect of pen was used to recognize the experimental unit for treatment group. In addition, repeated observations within a pen were modeled using a compound symmetry residual variance-covariance structure.
Respiratory scores were modeled using a categorical multinomial distribution fitted with a cumulative logit link function in GLIMMIX procedure of SAS (v.9.4, SAS Institute Inc., Cary, NC). Least square means and proportions are reported for all parameters evaluated. For all the analyses, differences detected at P ≤ 0.05 were considered significant.

Results and Discussion
Several management changes are imposed to the animals over the heifer-raising period. The most cited changes reported are introduction to a new feeding and housing system, regrouping, social isolation, and diet modifications. These changes require extensive behavioral resiliency of the animals to acclimate to them (De Paula Vieira et al., 2012). A study about a profile of calves' management practices in the U.S. related that about 80% of farms move calves into group pens around 8 weeks of age.
Further, large dairies have moved their calves to group pens later than small operations in order to decrease the spread of pathogenic microorganisms in milk-feeding period (NAHMS, 2007). In an excellent review, Bøe and Faerevik (2003) reported that grouping of unfamiliar animals increases aggression, social stress, and locomotor behavior in calves. Additionally, this practice may negatively impact feed intake, body weight ADG, and immune functionality of dairy calves after weaning (Lindsey E. Hulbert & Ballou, 2012).
We did not detect any effect of ultra-diluted supplementation on immune function and blood cells count of weaned dairy calves after grouping (Table 1). Additionally, even though any effect was detected on BW ADG, satisfactory gains was detected between the groups (1.0 and 0.99 kg/d for controlled and ultra-diluted treated group, respectively). Differently of Hulbert and Ballou (2012), our findings may be explained through the absence of stress effects on neutrophil functionality at this lifetime of the calves, similarly as mentioned by Leblanc (2020), who reviewed the immune function of transition dairy cows under social stress. This author reported that the association between stress (acute or chronic) and impaired immune function has not been confirmed in controlled trials, concluding that competitive displacement at the feed bunk in overcrowded groups may have no effect alone on immune function (phagocytosis and oxidative burst) of transition dairy cows. To date, there is no well-designed studies verifying the effects of ultra-diluted products on immune function of weaned dairy calves to perform relevant discussion among ultra-diluted strategies.   Lundborg et al. (2005) and Svensson and Liberg (2006) studies indicate that size of the group after commingling matters, whereas calves housed in large group pens had higher risk for respiratory disease. In this study, prophylactic supplementation of an ultra-diluted complex did not alter the respiratory scores in weaned dairy calves after grouping ( Figure 1). As mentioned earlier, to date, there is no randomized, double-blind, placebocontrolled trial reporting the effect of an ultra-diluted medicine on heifer health. Additionally, low quality studies have been reported for livestock in some literature reviews both in animals and humans (Doehring & Sundrum, 2016;Mathie & Clausen, 2015

Conclusion
In conclusion, prophylactic use of an ultra-diluted complex did not alter PMNL function, growth, nor it had effect on respiratory score of weaned growing calves during 28 d immediately after grouping.