Impacto da Idade de Desmame na Imunidade de Bezerros de Corte - Estudo Comparativo

Research in Veterinary Science 138 2021 5361 Available online 2 June 2021 00345288 2021 Elsevier Ltd All rights reserved Weaning at 30 75 and 180 days Comparison between immune responses of beef calves Odilene de Souza Teixeira a Marcela Kuczynski da Rocha a Amir Gil Sessim a Everton Dezordi Sartori a Yago Machado da Rosa a Maria Carolina Muniz de Oliveira a Julia Abud Lima a Maria Eugˆenia Andrighetto Canozzi b Jaime Urdapilleta Tarouco a Stella de Faria Valle c Concepta McManus d Júlio Otavio Jardim Barcellos a a Department of Science Animal Universidade Federal do Rio Grande do Sul Ave Bento Gonçalves 91540000 Porto Alegre Brazil b Instituto Nacional de Investigacion Agropecuaria Programa Produccion de Carne y Lana Estacion Experimental INIA La Estanzuela Ruta 50 km 11 39173 Colonia Uruguay c Department of Pathology and Veterinary Clinic Universidade Federal do Rio Grande do Sul Ave Bento Gonçalves 91540000 Porto Alegre RS Brazil d Institute of Biological Sciences Universidade de Brasília 70910900 Brasília DF Brazil A R T I C L E I N F O Keywords Age at weaning Beef cattle Cortisol Hematology Stress A B S T R A C T The interruption of the maternalfilial bond in young calves can alter the physiological responses due to increased stress at this moment Therefore we aimed to analyze the association between age at weaning and the immune responses of 64 beef calves weaned abruptly The animals were distributed in three weaning W treatments hyperearly W30 early W75 and conventional W180 The treatments agree with the weaning ages used in cowcalf systems in South America Blood samples were collected of all experimental animals on the day of weaning and days 1 2 and 7 postweaning day of the three treatments Blood samples were used to analyze hematological variables total plasma proteins TPP fibrinogen serum amyloid A protein SAA and cortisol The variables analyzed were influenced by the sampling day P 0001 and the calf gender P 005 and an interaction between treatment x sampling day was observed Over the study period high serum cortisol levels were observed for W30 calves The serum amyloid A protein was significantly influenced by sampling day When compared to W180 calves W30 and W75 animals showed prolonged concentrations in plasma fibrinogen In addition they presented neutrophilia and lymphopenia during the 180187 days period which resulted in a greater neutrophillymphocyte ratio NL ratio Calf gender resulted in changes in the number of blood leuko cytes P 005 This result provides an alert for weaning at an early age indicating that different management strategies must be tested to minimize physiological instabilities in critical periods of the life of animals 1 Introduction The intensification of production in the cowcalf system and the emergence of new nutritional technologies have made it possible to improve management practices such as weaning calves at early ages These techniques that aim to anticipate the weaning age are adopted to optimize the reproductive indices in the cowcalf operation mainly the indicator of the pregnancy rate of the cow Oliveira et al 2018 Once the maternalfilial bond is broken the ovarian cycle of the cow can be reestablished in a shorter time Orihuela and Galina 2019 This system also provides the calves with a differentiated diet to prepare them for rearing and finishing Given the above weaning at 30 days of age called hyperearly weaning presents itself as one of the most precocious techniques in beef cattle and has been used to improve efficiency within the livestock industry Nevertheless weaning is characterized by multiple stressors for the calf Lynch et al 2010 Lynch et al 2019 When recognized by the central nervous system as threatening homeostasis these stressors result in a combination of biological defenses presenting alterations in behavior and in autonomic nervous neuroendocrine and immune sys tems Moberg 2000 Thus when the stress modifies neuroendocrine system consequently the liberation of glucocorticoids occurs In sequence there can be changes in the hematologic variables and serum acutephase proteins OLoughlin et al 2014 These proteins are Corresponding author Email address odilenezootecnistagmailcom O de Souza Teixeira Contents lists available at ScienceDirect Research in Veterinary Science journal homepage wwwelseviercomlocatervsc httpsdoiorg101016jrvsc202106002 Received 11 January 2021 Received in revised form 10 May 2021 Accepted 1 June 2021 Research in Veterinary Science 138 2021 5361 54 recognized as physiological indicators of stress which are also sensitive to the neuroendocrineimmune network Murata 2007 In these circumstances it is assumed that weaning younger calves may result in even more marked changes in the immune system once these animals experience severe stress conditions The weaning at 30 days is characterized by increased vocalizations crosssucking walking activity and respiratory rate and a lower percentage of time in the feeder when compared to traditional weaning that occurs at 180 days of the age of calves Teixeira et al 2021 Allied to this it is also note worthy that at the age of hyperearly weaning there are important changes in the development of the immune system Between the third and fourth week of life immunity obtained from maternal antibodies passive immunity is in decrescent levels and the antibodies produced by the own organism of calves active immunity are initiating their responses Hulbert and Moisa 2016 Therefore this period is charac terized as susceptible to diseases e may be severed by weaning stress consequences In this sense there is currently a gap in scientific research as it is not known how these immunological changes occur in calves weaned at young ages Most research is based on later weaning from 89 to 250 days of age Blanco et al 2009 Lynch et al 2010 OLoughlin et al 2014 In addition more prolonged effects have not been described yet as these studies are limited to 35 days after weaning Therefore our study tests the hypothesis that calves weaned at an early age may present unsatis factory immunological conditions resulting from physiological re sponses to stress Over the long term we objected to analyze the immune responses in beef calves weaned at 30 75 and 180 days once these ages are adopted in the South American beef industry 2 Material and methods 21 Ethics statement The Ethics Committee on the Use of Animals from the Faculty of Agronomy and Department of Animal Science Federal University of Rio Grande do Sul Porto Alegre Brazil approved all management proced ures performed on calves during this research license number 33439 22 Animals This study was carried out at the experimental farm of the Federal University of Rio Grande do Sul located in the city of Eldorado do Sul Rio Grande do Sul Brazil latitude 30 06 207 S and longitude 51 41 219 W We distributed 64 Brangus calves male and female born in the spring in three groups according to the age of weaning i hyper early weaning W30 n 22 10 males and 12 females the average weight of 5345 110 kg mean SE and age of 32 089 days ii early weaning W75 n 20 12 males and 8 females the average weight of 8463 205 kg and age of 77 095 days and iii con ventional weaning W180 n 22 13 males and 9 females the average weight of 15762 287 kg and age of 183 082 days Calves were born of cows aged 43 023 years old and under normal conditions of parturition Before weaning cows and calves were kept under the same dietary conditions on native pasture with 2200 kg DMha and culti vated millet pasture Pennisetum americanum with 1830 kg DMha At weaning cows and calves were walked to the management center After separation the cows were taken to a picket 26 km away from the calves to prevent them from hearing offspring vocalizations The calves were weaned abruptly with total physical separation between the dam and offspring Weaning management was the same for all treatments with W30 and W75 performed in the summer December and February respectively and W180 in the fall May For the weaning groups calves born from dams of the same mating and calving periods were used in each treatment as in Blanco et al 2009 23 Experimental design After weaning the calves from the same treatment were collectively allocated in pen Fig 1 This system was chosen as it allows calves to develop their natural behaviors as playing Also it was verified that maintain animals in groups results in increased body weight by 15 when compared to calves allocated individually Valníˇckova et al 2015 improving animal welfare Buˇckova et al 2019 In addition it comprehends the management used in farms that adopt cowcalf sys tems The pen was equipped with two feeders an automatic water fountain and an artificial shade made of polypropylene 80 shading The W30 and W75 groups were allocated in two pens with identical format until the animals were sent to pasture After this one of the pens was used for weaning the W180 calves Birth weight weight at 30 days of age and calf temperament score methodology adapted from Hickey et al 2003 were similar between groups at the beginning of the experiment Two management protocols were applied for blood collection The first one refers to the period before the cowcalf separation The day before the collections the cow calf pairs were led by two herders on horseback to a paddock near the management center The next day collection day the pairs were guided by the herders without using the horse to the management center Animals were allowed to rest for 30 min after which collection began The second protocol refers to animals that had already been weaned The pen where these animals were kept was within the man agement center so the animals were led to the collection chute No truck transport was employed during the experiment Blood collections started at 0800 am and were made through the puncture of the jugular vein Blood samples were collected of all experimental animals regardless of treatment on days 30 31 32 37 75 76 77 82 180 181 182 and 187 of age These collection moments correspond to the weaning day and days 1 2 and 7 postweaning day of each treatment W30 W75 and W180 respectively a protocol adapted from Blanco et al 2009 and OLoughlin et al 2014 According to Blanco et al 2009 the number of host defense cells returns to normal up to seven days after weaning therefore it was evaluated until seven days after weaning 24 Nutritional and sanitary management of calves Calves in the W30 group remained confined from 30 to 110 days of age Fig 2 From 30 to 90 days they were fed ad libitum with initial pelleted feed 1 R1 composed of 200 gkg of crude protein CP 60 g kg of crude fiber CF and 740 gkg of total digestible nutrients TDN and alfalfa hay 167 gkg of CP and 456 gkg of NDF average total consumption of 10 of live weight based on the dry matter from 30 to 40 days of age and total average consumption of 23 from 41 to 90 days of age From days 91 to 110 calves received the pelleted com mercial growth feed 2 R2 180 gkg of CP 120 gkg CF and 720 gkg of TDN and alfalfa hay with an average total consumption of 31 of live weight Food consumption on the day of weaning was lower for the W30 calves as seven calves did not search for food on that day as well as five W75 calves and one W180 calf However on the day after weaning there was an increase in feeding activity for all treatments and on the third day all calves were eating properly In the three treatments food consumption was measured collectively considering the difference be tween the quantity offered and the quantity of leftovers on the following day The food uneaten was removed and weighed daily to adjust the amount to be supplied providing a 5 surplus of natural matter Regardless of the treatment no animals received supplementation before weaning Calves of W75 were weaned and remained until 110 days of age in feedlot From 75 to 90 days of age they received R1 and alfalfa hay with an average consumption of 10 of live weight Between 91 and 110 days the calves received R2 and alfalfa hay with consumption of 220 of live weight The calves of W30 and W75 were fed three times a day O de Souza Teixeira et al Research in Veterinary Science 138 2021 5361 55 0800 am 0200 pm and 0600 pm After 110 days of age calves of both treatments were conducted to Urochloa pasture U decumbens U brizantha and U ruziziensis The bromatological composition of the pasture was 58 gkg of CP and 665 gkg of NDF and forage mass of 2270 kg of DMha with an average feed consumption of 15 of the live weight per day The calves remained on this pasture until 190 days of age and were supplemented in the morning once a day 0800 am The W180 calves were weaned at 180 days and received the commercial pelleted R2 and alfalfa hay with an average total consumption of 14 of live weight per day The calves received a preventive vaccination for clostridiosis at 40 days of age with a booster dose at 90 days Also they were medicated monthly for endo and ectoparasites This sanitary protocol was adopted because the regions climate is subtropical Hence the combination of high temperature and high humidity contribute to parasitic prolifera tion putting the health of calves at risk After weaning the incidence of nasal and ocular discharge and diarrhea Larson et al 1977 were monitored visually during feeding daily Rectal temperature was ob tained after blood collection or when there was an abnormal clinical sign The measurements were taken on all calves weaned or not For the animals that had not been already weaned the supervision was carried out twice a week as the animals were maintained at pasture with their dams in an extensive production system 25 Blood collection Blood samples for cortisol and serum amyloid A SAA analyses were collected immediately after the animal immobilization using a 10 mL vacutainer tube BD Franklin Lakes NJ USA with a clot activator These samples were centrifuged 2000 g for 20 min conditioned in microtubes and frozen in a cylinder containing liquid nitrogen 196 C For blood count and fibrinogen analyses blood was collected in 4 mL vacutainer tubes BD Franklin Lakes NJ USA with dipotassic ethylenediaminetetraacetic acid EDTA K2 anticoagulant 26 Assays of physiological parameters and blood count Total plasma proteins TPP and plasmatic fibrinogen concentrations Fig 1 Diagram of the confinement of beef calves A Plant B Front plant C Crosssection The stalls were equipped with feeders water fountains and an arti ficial shade O de Souza Teixeira et al Research in Veterinary Science 138 2021 5361 56 in EDTAblood samples were estimated by refractometry and heat pre cipitation Willard and Tvedten 2004 Cortisol was determined using the chemiluminescence method and a commercial kit Beckman Coulter and read by Access II Beckman Coulter For serum amyloid A SAA protein it was used the Elisa method specific commercial kit for cattle Bioassay Technology Laboratory and read in a Thermo Fisher Scientific machine Blood samples were processed using an automatic analyzer Procyte DX Idexx Laboratories White blood cell differential count was performed manually in a stained smear DiffQuick Stain using optical microscopy 100 x under immersion oil The neutrophil lymphocyte ratio NL ratio was obtained for all animals from this analysis 27 Statistical analysis In a first moment data were subjected to a principal component analysis PCA PAST 30 program Hammer et al 2001 to indicate how the immunological variables of the calves are ordered in treatments weaning groups and sampling days Then a PERMANOVA analysis was used to test differences in orders of immunological responses considering a 5 level of significance PAST 30 program Hammer et al 2001 In this way it was possible to create a general pattern for the immunological response The mathematical model for PERMANOVA was γijkl τj ρk τρjk ηι τηjl ρηkl τρηjkl εijkl where γijk dependent variables τj effect of the jth treatment ρk effect of the kth collection day τρjk interaction between the jth treatment and kth collection day ηι effect of the lth gender male or female τηjl interaction between the jth treatment and lth gender ρηkl interaction between the kth collection day and lth gender τρηjkl interaction between the jth treatment kth collection day and lth gender εijk random error error b Changes in blood variables were evaluated over time from 30 to 187 days of age for all the calves A general linear model was used to analyze variance with repeated measures considering the age of the dam as a covariate using SPSS version 200 software IBM Armonk New York 2011 The model included the effect of age at weaning W30 W75 and W180 days 0 1 2 and 7 postweaning and gender male or female as fixed factors as well as the interaction between them The calf was included as a random factor The GreenhouseGeisser correction was used which allows testing the repeated measures even if sphericity is not met Dunnets T3 test was used to compare within factors The sig nificance level was 5 but cortisol showed a tendency of P 006 on day 77 The mathematical model for repeated measures was γijkl μ τj φi τj ρk τρjk ηι τηjl ρηkl τρηjkl εijkl where γijk dependent variables μ mean of all observations τj effect of the jth treatment φi repetition within treatment error a ρk effect of the kth collection day τρjk interaction between the jth treatment and k th collection day ηι effect of the lth gender male or female τηjl interaction between the jth treatment and lth gender ρηkl interac tion between the kth collection day and lth gender τρηjkl Inter action between the jth treatment kth collection day and lth gender εijk random error error b 3 Results 31 Principal component analysis PCA The PCA shows the order of the evaluated factors of component 1 axis 1 and component 2 axis 2 Fig 3 In axis 1 neutrophils and the NL ratio were the main causes of variation while in axis 2 these causes were plasma fibrinogen and SAA From this order PERMANOVA showed a difference in the immunological variables per treatment P 0001 per day P 0001 and interaction between treatmentsam pling day P 0001 The first two components contributed 4266 of the total variation 32 Physiological variables Interaction between treatmentsampling day P 0001 was seen for cortisol analysis The W30 calves showed higher cortisol levels on days 31 32 180 and 181 compared to W75 and W180 Similarities were observed on day 77 between W30 and W75 P 005 and on day 187 between W30 and W180 P 005 On days 77 P 006 and 82 P 004 W75 calves showed higher cortisol levels than W180 ones Fig 4 The TPP differed only for sampling day P 0001 despite the lower level at day 75 of age 624 007 gdL and the higher level in the subsequent days with a mean of 700 013 gdL at day 187 Fig 2 Nutritional management before and after weaning calves W30 hyperearly weaning from 30 to 90 days animals received feed 1 R1 and from 91 to 110 days they received feed 2 R2 in confinement W75 early weaning from 75 to 90 days animals received R1 and from 91 to 110 days they received R2 in confinement The W30 and W75 calves were conducted to the pasture with supplementation after 110 days of age W180 conventional weaning from 180 to 190 days animals received R2 in confinement O de Souza Teixeira et al Research in Veterinary Science 138 2021 5361 57 The influence of treatmentsampling day interaction P 001 for plasma fibrinogen was observed for W30 and W75 treatments which presented higher levels at 32 180 and 182 days of age than W180 one Calves from W30 demonstrated high fibrinogen levels on day 37 compared to those from W75 and W180 and higher levels on day 75 day than W75 46363 3325 mgdL v 35000 2856 mgdL The SAA levels were altered only due to sampling day P 003 and the peaks were identified between days 82 and 187 ranging from 3201 370 to 3696 430 mgL 33 Hematological variables Interactions between treatmentsampling day P 0001 were observed for lymphocytes neutrophils NL ratio and monocytes Fig 5 On the other hand there was no effect on eosinophils P 005 Calves from W30 and W75 groups had lower percentages of lymphocytes on days 180 182 and 187 compared to W180 calves The percentage of neutrophils was lower for W30 calves on day 37 1654 195 compared to the W75 3415 248 and W180 4081 284 ones There was a higher percentage of neutrophils for W30 and W75 calves on days 75 77 180 182 and 187 than for W180 Because of this W30 and W75 calves obtained a higher NL ratio P 005 on days 75 77 182 and 187 On day 82 the percentage of monocytes was higher for calves from early weaning P 0001 1035 108 compared to hyperearly 669 049 and conventional 472 040 weaning On day 180 of age we observed the highest percentage in monocytes for calves from W30 814 137 in comparison to those from W75 255 048 and W180 359 044 treatments Male calves had higher percentage of lymphocytes than females P 0003 7037 076 vs 6576 088 respectively However fe males showed a higher concentration of neutrophils than males P 0002 2773 083 vs 2350 073 respectively and NL ratio P 0 02 053 004 vs 040 002 respectively Fig 6 There were interactions between treatmentsampling day P 0001 for hematocrit with higher values after weaning The W30 calves showed higher values on days 32 3781 075 and 37 4072 075 than W75 ones 3335 117 and 3175 124 Similarly the highest hematocrit percentage for W75 was observed on days 77 3520 100 and 82 3530 076 compared to W30 3045 075 3232 074 respectively and W180 3086 084 3010 081 respectively For W180 calves the higher values for hematocrit were obtained on days 182 3350 092 and 187 3368 077 compared to those from W30 2959 072 and 3100 076 respectively 4 Discussion The influence of early 30 or 75 days or traditional 180 days weaning on immune responses in calves was evaluated Our hypothesis was that calves weaned at an early age face unsatisfactory immuno logical conditions resulting from physiological responses to stress Our results showed that the younger the calf is the greater the immuno logical challenge suffered by this animal These reactions are mainly characterized by higher levels of cortisol fibrinogen neutrophilia and late lymphopenia resulting in a higher NL ratio The release of cortisol and immunological changes are due to the activation of the hypothalamicpituitaryadrenal axis HPA during stress Blecha 2000 Under these conditions the HPA axis acts by secreting corticotrophinreleasing hormone CRH from the hypothala mus which subsequently binds to receptors on the pituitary gland This hormone stimulates the release of the adrenocorticotropic hormone which conditions the secretion of cortisol and other hormones into the bloodstream Axelrod and Reisine 1984 Herman and Cullinan 1997 Algamal et al 2021 As a result these hormones activate the acute phase proteins Murata et al 2004 Murata 2007 and inhibit the cells of the immune system such as leukocytes Merlot 2004 Finally the levels of cortisol in the HPA coordinate the immune responses to protect the organism against stress Borghetti et al 2009 In hematological terms neutrophils are the main line of defense of the body Burton et al 2005 and lymphocytes act on innate and cellular immunities as well as on antibody production Tizard 2009 Thus changes in the concentrations of these cells presuppose an insta bility of the immune system For W30 calves the lowest percentage of neutrophils occurred 7 days after weaning which may be related to the incidence of diarrhea observed in these animals The occurrence of neutropenia resulting from the onset of diarrhea was reported by Novo et al 2017 during weaning in milk calves The change of feeding regime can generate imbalances in the gastrointestinal tract mainly in very young calves Although no differences were found between neutrophils and lym phocytes for immediate postweaning sampling we observed that calves separated earlier from their dams W30 and W75 showed a late immune alert status compared to conventional weaning ones W180 It was characterized by neutrophilia and lymphopenia representative of a stress leukogram which is associated with management practices in farm animals Kelley 1980 It can be related to the sensitivity of neu trophils under conditions of elevated glucocorticoids cortisol as observed in the present study showing an imbalance This evokes a possible increase in the production and release of neutrophils from the bone marrow Burton et al 2005 along with the retraction of neu trophils from other organs into the bloodstream Merlot 2004 The occurrence of it is due to neutrophils need the Lselectin protein expression to migrate from the bloodstream to the tissues However during stress glucocorticoids are believed to act by reducing Lselectin gene expression so neutrophils decrease their circulation remaining in the bloodstream Weber et al 2001 Thus once neutrophils reach the bloodstream they are prevented from returning to tissues diapedesis which justifies the increase in neutrophils after stressful events The opposite occurs with lymphocytes Under stress the action of the corticotrophinreleasing hormone suppresses the proliferation of lym phocytes Jain et al 1991 Also those lymphocytes recruited into the bloodstream are encouraged to move to the lymphoid tissue Merlot 2004 resulting in a decrease in circulating lymphocytes Therefore the lower levels of lymphocytes up to 28 days after weaning demonstrates Fig 3 Main component 1 PC1 versus 2 PC2 of the principal component analysis PCA of the immunological variables for the different ages at weaning Treatments W30 hyperearly weaning W75 early weaning and W180 con ventional weaning Days 30 31 32 37 75 7677 82 180 181 182 and 187 refer to the age of beef calves Dots individual animals ellipses treatments O de Souza Teixeira et al Research in Veterinary Science 138 2021 5361 58 the prolongation of the immune response to stress caused by weaning Lynch 2010 The comparable higher levels in neutrophils after abrupt weaning are considered a stress biomarker along with the NL ratio OLoughlin et al 2014 Blanco et al 2009 tested the influence of age at weaning on beef calves abruptly separated at 90 and 150 days of age Although there were no differences in terms of age the effects of weaning caused high levels of neutrophils and low levels of lymphocytes In another study evaluating the influence of weaning at 120 and 210 days of age re searchers found no differences for leukocyte subpopulations or NL ratio Smith et al 2003 For OLoughlin et al 2011 the NL ratio remained altered until 11 days after weaning the maximum limit of the study evaluation In our research the percentage of monocytes was above the maximum limit for W75 calves at 82 days This parameter can vary from 2 to 7 for cattle Jones and Allison 2007 In addition the calves also had higher rectal body temperature on the same day 399 C However this clinical sign was monitored and no disease was diagnosed There fore the temperature rise may have been caused by the weaning stress since it was measured 7 days after the separation of the cowcalf pair For Johnston et al 2015 this expression of monocyte levels after the management practice suggests higher sensitivity to weaning and con siders it a critical moment in the life of the calf In the present study a different biological response was found due to calf gender when considering neutrophils lymphocytes and NL ratio However there is no consensus about this subject in the literature For OLoughlin et al 2011 gender did not change the total number of neutrophils or NL ratio after weaning However Hickey et al 2003 found a higher proportion of neutrophils and NL ratio for male weaned calves than females an opposite result to the one found in our research Moreover this lack of clarity is also evidenced in investigations on an imal behavior in the postweaning period Under natural weaning con ditions the cow interrupts the maternalfilial bond about two months before in the case of a female offspring compared to a male one not allowing suckling behavior to occur Reinhardt and Reinhardt 1981 Castanheira et al 2013 In addition males receive greater protection from the dam Stˇehulova et al 2013 On the other hand Stˇehulova et al 2017 highlighted that females vocalize more than males during separation showing that this behavior is one of the most sensitive to characterize postweaning stress Thus our findings also indicate that females undergo greater stress represented by the higher percentage of neutrophils and NL ratio However the mechanisms of immune re sponses to stress vary according to age nutrition genetics and gender Kim et al 2011 and therefore different results are likely to be found High concentrations of TPP were observed from 75 to 180 days of age As age advances there is an increase in total proteins Bueno et al 2003 due to a higher concentration of immunoglobulins in adulthood Plasma fibrinogen an acutephase serum protein presented a rise in blood concentrations for calves in the W30 and W75 treatments How ever even though these concentrations are higher in the W30 and W75 groups than in the W180 one they are close to the reference values for beef calves Edmonds et al 1975 According to Qiu et al 2007 fluctuations in the concentration of acutephase proteins may be due to Fig 4 Means SEM of cortisol plasma proteins fibrinogen and serum amyloid A in beef calves according to the age at weaning and sampling day Treatments W30 hyperearly weaning W75 early weaning and W180 conventional weaning O de Souza Teixeira et al Research in Veterinary Science 138 2021 5361 59 handling such as weaning and animal transport For W30 calves the high values obtained for fibrinogen after weaning are related to the impulses of the neuroendocrineimmune network which induces the synthesis of this protein in the liver Murata 2007 In the literature the maximum period of assessment of fibrinogen in calves after weaning corresponded to 35 days showing increased responses and demon strating the prolongation of stress Lynch 2010 The results presented for the serum amyloid A protein were not ex pected because as an acutephase protein there should be a rise at the weaning moment which did not happen The inaccuracy of SAA was reported by OLoughlin et al 2014 which indicated that this protein seems not to be a good indicator for postweaning calf stress Other studies are needed biochemical molecular and cellular biology to elucidate its effect under stress conditions On the other hand the late high 187 days in SAA levels observed in this study should not be dis regarded as the peak of this protein usually occurs at 7 days of age and subsequently the levels decrease and stabilize at 24 days of age Orro et al 2008 As this did not occur it is assumed that the repeated blood collections may have caused a high level of this protein as SAA is sen sitive to handling stress calves Alsemgeest et al 1995 Given the late changes in fibrinogen cortisol and leukocyte cells in calves at younger ages we hypothesized that early exposure to stress may generate suffering memories for the management events to come Consequently it may trigger the complex neuroendocrine network with immune responses Studies of this nature are carried out in rats Their exposition to stressors affects several brain areas and may lead to post traumatic disorders due to distressing or aversive memories Camp and Johnson 2015 In other words after the rats have experienced a moment of stress contextual memories of fear are observed in subse quent exposures since the mechanisms that promote the formation and storage of memory are mediated by glucocorticoids with different re sponses for males and females Kulp et al 2020 However this hy pothesis needs to be tested by assessing brain areas affected by weaning stress in very young calves The rise in the hematocrit immediately after weaning is a relevant observation in all three weaning ages According to Wright et al 2000 this increment is indicative of dehydration Thus even though we did not measure water intake by the calves ingestive behavior was evalu ated Among the activities analyzed visits to the drinking fountain represented the shortest occupational time of the animals regardless of age at weaning Teixeira et al 2021 Also despite the treatment it is evident that on the day after weaning there was a decrease of 53 in the frequency of visits to the drinking fountain when compared to the weaning day Dehydration can have serious consequences especially in calves which demonstrates the importance of monitoring these animals and treating any expressing clinical signs that may occur which was not diagnosed in this study In the following days the animals reestablished water intake However the consumption decrease on the second day Fig 5 Percentage SEM of hematologic variables neutrophils lymphocytes NL ratio monocytes and eosinophils in beef calves according to age at weaning and sampling day Treatments W30 hyperearly weaning W75 early weaning and W180 conventional weaning O de Souza Teixeira et al Research in Veterinary Science 138 2021 5361 60 was sufficient to interfere with the hematocrit result In summary weaning at young ages generated greater potential for damage to the immune system which at times could have been inter preted as a possible diagnosis of disease due to the multiple variations presented More research is necessary to associate the effects of age at weaning to the longterm immunological effects on beef calves as they are littleknown or understood It is explained by the limitation of studies on the analysis of these parameters at weaning Studies should prioritize the comparison of animals at all ages regardless of the weaning strategy as performed in the present study since fluctuations in immunological parameters are also conditioned to animal growth Thus blood samples at different ages may not accurately represent what is being tested In addition contradictory and inconsistent results compose the nature of this theme which is partly understood by the different perceptions of animals to stressors as they are dependent on life experiences breed age and psychological state Moberg 2000 5 Conclusion Calf weaning at 30 or 75 days of age results in different immuno logical responses compared to calves weaned at 180 days Weaning at younger ages triggers changes in the defense cells resulting from physiological responses to stress These changes are associated with a higher level of cortisol in the bloodstream neutrophilia and late lym phopenia in animals weaned at 30 or 75 days Also there is a differen tiation between genres in the primary defense cells contradictory to previous studies This evidence provides opportunities for weaning management changes to avoid physiological instabilities in critical pe riods in the life of the animal Therefore the influences of weaning on the immune system in young calves is an emerging and necessary theme in the search for practices with a lower impact on calf welfare Declaration of Competing Interest The authors declare that there is no conflict of interest Acknowledgments We thank the Research Support Foundation of the State of Rio Grande do Sul Brazil FAPERGS protocol 17255100012771 the National Council for Scientific and Technological Development Brazil CNPq protocol 43127720184 and 30789120194 the Coordina tion for the Improvement of Higher Education Personnel Brazil CAPES protocol 001 PROEX PPGZOOTECNIAUFRGS program and the company Animal nutrition company Puro Trato Brazil which supported this research We are grateful to all participants of the Center for Research on Systems of Beef Cattle Production and Supply Chain NESPro Brazil and the experimental farm team at the Federal Uni versity of Rio Grande do Sul for the development of this study References Algamal M Pearson AJ HahnTownsend C Burca I Mullan M Crawford F Ojo JO 2021 Repeated unpredictable stress and social 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Corah LR Stokka G Blecha F 2000 Effects of preweaning vitamin E selenium and copper supplementation on the performance acute phase protein concentration and immune function of stressed beef calves PAS 16 111120 O de Souza Teixeira et al Resenha do artigo Weaning at 30 75 and 180 days comparison between immune responses of beef calves O artigo intitulado Weaning at 30 75 and 180 days comparison between immune responses of beef calves de autoria de Odilene de Souza Teixeira et al está elaborado ao decorrer de 9 páginas das quais apresentamse divididas em títulos como Introdução Material e métodos Resultados Discussão Conclusão com seus respectivos subtítulos seguido dos agradecimentos e referências bibliográficas O artigo tem como objetivo a comparação do desmame aos 30 75 e 180 dias e as respostas imunológicas desencadeadas em cada tempo de desmame Em Introdução os autores abordam a respeito do desmame precoce de bezerros sendo esta uma técnica utilizada para melhorar o índice de prenhez da vaca fazendo com que seu ciclo reprodutivo seja mais rapidamente restaurado aumentando então a sua vida reprodutiva O desmame hiperprecoce do bezerro aos 30 dias ocorre a partir de dietas específicas para esta finalidade Porém o processo de desmame envolve mecanismos do sistema nervoso central o que faz com que sejam desencadeadas respostas imunológicas e neuroendócrinas o que faz com que sejam liberados glicocorticoides capaz de alterar as proteínas séricas de fase aguda por exemplo esta uma indicadora fisiológica de estresse Sendo assim o desmame aos 30 dias pode ser observado como sendo altamente estressante ao bezerro e caracterizado por alterações imunológicas importantes o que pode prejudicar o correto desenvolvimento do sistema imune propiciando dificuldades em combater doenças características dessa fase de vida do bezerro Nesse contexto o artigo em questão busca a pesquisa experimental de diferentes tempos de desmame a fim de analisar as respostas imunológicas quando realizado aos 30 dias de vida 75 e 180 dias desmame tradicional Como material e métodos os autores se atentam à autorização do Comitê de Ética no Uso de Animais da Universidade Federal do Rio Grande do Sul para dar início à pesquisa Autorizada a equipe utiliza de 64 bezerros da raça Brangus sendo eles divididos em 3 grupos desmame aos 30 75 e 180 dias tendo os pesos corporais os mais homogêneos possíveis entre animais do mesmo grupo As vacas foram afastadas dos bezerros a uma distancia de 26 km para que não houvesse interferência Após os períodos de desmame bezerros pertencentes ao mesmo grupo foram mantidos separado aos depois grupos a fim de que cada grupamento pudesse desenvolver seus comportamentos naturais sem a interferência de bezerros desmamados em um período diferente ao seu Assim os animais foram alocados em baias contendo 2 comedouros bebedouros e com área de sombreamento Ainda os animais de todos os grupos tiveram sangue coletado em mesmas quantidades e mens dias independente do tratamento 30 75 ou 100 dado ao animal Quanto à alimentação foram oferecidas rações inicial peletizada e feno de alfafa para os bezerros S3 feno e alfafa para os bezerros S75 e a partir de 110 dias alimentados com pastagens de Urochloa Já os bezerros S180 receberam ração R2 peletizada e feno de alfafa O consumo médio dos animais e grupos foram mensurados através da diferença entre alimento fornecido e sobra Ressaltase que todos os animais foram vacinados aos 40 e 90 dias como forma de prevenção a clostridioses além de medicação de endo e ectoparasitas Ao que se segue os pesquisadores abordam os materiais utilizados para a coleta de sangue bem como o método para determinação de cortisol e amiloide sérico contagem de glóbulos brancos e relação entre neutrófilo linfócito Os dados coletados foram submetidos a analises estatísticas em modelo matemático PERMANOVA a fim de se obter um padrão matemático eficiente a todos os grupos Em Resultados foram obtidas as análises de componentes principais sendo este a relação entre neutrófilo linfócito no qual foi possível observar diferenças estatísticas nas variáveis imunológicas entre os tratamentos Quanto as variáveis fisiológicas pode ser observado maiores níveis de cortisol nos animais S30 quando comparado aos animais S75 e S180 Houve ainda comparações entre porcentagem de monócitos dos quais foram variáveis entre os grupos Ao que concerne à discussão do experimento os autores apresentam a hipótese sendo esta a de que animais desmamados precocemente possuem respostas imunológicas menos eficazes em consequência da resposta fisiológica ao estresse onde há sobretudo liberação de cortisol fibrinogênio e altos níveis de neutrofilia e linfopenia tardia Observouse que em relação a separação materna os bezerros separados mais cedo S30 e S75 apresentaram o alerta imunológico mais tardio em comparação aos animais S180 Os autores seguem a discussão apresentando revisões de literatura sobre os parâmetros e respostas observadas a fim de dar embasamento aos resultados encontrados e identificar se destoam daqueles encontrados na literatura Como conclusão temse que bezerros desmamados aos 30 e 75 dias apresentam respostas imunológicas diferentes quando comparados aos bezerros desmamados aos 180 dias Observase que desmame precoce tem como consequência a alteração das células do sistema imune em razão da liberação de cortisol devido ao estresse fisiológico Sendo assim sugere a modificação no manejo do desmame precoce para que o mesmo não desencadeie processos fisiológicos dos quais tenham como consequência o prejuízo imunológico do animal O artigo a que se refere apresenta dados altamente relevantes para o entendimento do processo de desmame que embora seja favorável a vida reprodutiva da vaca fazendo com que a mesma tenha o seu ciclo reprodutivo recuperado mais rapidamente faz com que os bezerros sofram alterações imunológicas significativas e importantes para o correto desenvolvimento do seu sistema de defesa Assim traz a informação embasada em pesquisa da necessidade de reaver os processos de desmame precoce dos quais necessitam de melhoria do manejo do bezerro para minimizar suas alterações a níveis imunológicos