The study was conducted on a commercial dairy farm located in Passos, southern Minas Gerais, Brazil. For this purpose, one hundred Girolando female calves kept in a “tropical calf house system” were used. In this System, calves are kept in a pasture area, without contact with each other, individually attached to a chain, which is attached to a line (working as a corridor). There is a shade cloth, a trough for feed supply, and a bottle for milk and water intake, all for individual use.
All calves were conceived through IVF, from the cross between the Holstein and Gir breeds, resulting in calves that are 3/4 Holstein-Gir and ½ Holstein-Gir. For the IVF procedure, semen from 9 Holstein bulls (bull “A” to “I”) were used, they presented a homogeneous distribution between groups HBW and control, respectively (A: 19 vs. 20; B: 7 vs. 6; C: 16 vs. 18; D:1 vs. 0; E:1 vs. 0: F: 1 vs. 2; G: 2 vs. 1; H: 3 vs.2 and I: 0 vs. 1). The oocyte donors and recipients were Gir cows from the same property, so all were subjected to the same climatic conditions. All recipients were cows presenting an average milk production of 20 liters. The in vitro production system was the same throughout the study and fresh embryos were transferred from May to September 2017. Research on animals was conducted according to the institutional committee on animal use (28396-2018).
For the study, the animals were distributed into two groups, according to the birth weight criterion, since this is an important parameter that can infer on the rate of dystocia, disease incidence, and lower performance of calves in dairy farms, although its may vary between farms (Holland and Odde, 1992; Jacobsen et al., 2000; Glover et al., 2019). Given this, at birth, all calves were weighed using a tape measure for medium-sized animals, obtaining an average weight of 35.45 ± 4.6 kg, and a median weight of 35.50 Kg. From birth weight, calves were included into two distinct groups: Control (n = 50) - including calves that were born weighing 35 kg or less; and HBW group (n = 50) - including calves with a birth weight higher than 35 kg. For the composition of these two groups, the median was used as a reference measure.
The management of calves were described (Weiller et al., 2021). Briefly, until two ours after birth, calves received about 10% of the weight in volume of colostrum, via bottle, and after that, received transition milk daily and milk replacer (Nurture Prime®, Nutron, Brazil). Corn silage was offered on 60 days after birth, ad libitum (NRC, 2001).
In all calves, navel disinfection was done using 5% iodine solution, once a day, during the first three days of life, and disbudded with hot iron at 21 days after birth.
Assessing passive transfer of immunity
Failure of passive transfer of immunity (FPTI) was determined in the calves (n=100) through na assessment of their serum levels of total plasma protein (TP), according to Weiller et al. (2021).
Occurence of diseases
All calves were daily monitored by the responsible Veterinarian of the study, from birth to 80 days of life, in order to diagnose diarrhea, respiratory or other diseases (bloat, tick fever and omphalophlebitis). For the determination of the presence of diarrhea, the feces were classified on a scale from 0 to 4, according to the methodology recommended (Teixeira et al., 2015), with score 0 characterized by normal feces and score 4, profuse diarrhea with watery and bloody feces. Whenever calves had feces with a score ≥ 2, it was characterized like presence of diarrhea. Diagnosis of respiratory diseases was performed according to Love et al. (2014). Remaining diseases that occurred during experimental period were all classified as "other diseases", and included cases of omphalophlebitis, tympanism, bovine parasitic sadness, etc.
The diseases were treated in accordance with a protocol determined by the property, which was similar for both groups.
Based on the records, it was possible to determine: morbidity (number of animals that become sick by the total number of animals in the experiment), mortality (number of animals that died divided by the total number of animals), lethality due to diarrhea (number of animals that died due to diarrhea divided by the number of animals that had diarrhea in the experiment), and recurrence (number of animals that had two or more diarrheas over the course of the study).
The zootechnical evaluations were carried out in 41 calves, randomly selected (Control: 22; HBW: 19). These were measured at different times, as follows.
Body weight was determined at birth (between 24-48 h), weekly until 30 days age, followed by evaluations on days 42, 60, and 80. Calf weights were estimated using a heart girth tape graduated in kilograms, placed vertically at the point of the elbow (Wood et al., 2015). The thoracic perimeter, withers height and width of the croup were measured using a tape measure. Assessments were performed weekly, up to 30 days of life (including birth), followed by the last assessment at 60 days. From the body weight assessment, the average daily gain (ADG) was determined.
Total plasm proteins, albumin, triglycerides, cholesterol, and urea concentrations, as well as the activity of enzymes gamma-glutamyl transfererase (GGT), paraoxonase 1 (PON1), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were evaluated in the same 41 calves. For this purpose, blood was collected using a Vacutainer system and tubes without anticoagulant (Vacuplast CRAL, São Paulo, Brazil), via the jugular vein, at the following times: at birth (between 24 and 48 hours) and on days 7, 14, 21, 28, and 60. All collections were performed in the morning, before food intake. After blood collection, all samples were sent to the laboratory, centrifuged at 2183 x g, and immediately frozen for further analysis. Except for PON1, all samples were analyzed in an automatic biochemical analyzer Labmax Plenno (Labtest Diagnostica, Minas Gerais, Brazil) using commercial colorimetric kits from the same company. The procedures were performed according to the manufacturer's recommendation.
To determine PON1 activity, a previously described protocol was used (Browne et al., 2007).
Bodyweight, thoracic perimeter, withers height, width of the croup, and biochemical results were analyzed using analysis of variance with repeated measures, followed by the Tukey-Kramer multiple comparison test, considering the group and time of collection, as fixed effects, and the animal as a variable effect, as well as their interactions. Data that did not have a normal distribution were transformed to log10 (i.e. PON1, GGT, and cholesterol).
Categorical variables such as FPIT, morbidity, mortality, lethality, and disease recurrence were assessed using the Chi-square test. The data obtained were analyzed using the NCSS 97 statistical program, being considered significant when P < 0.05; and a tendency when 0.05 ≥ P < 0.1. The relative risk was calculated (incidence in the HBW group/incidence in the control group) using a 95% confidence interval. Values were described as mean ± standard error.