Effects of animal type, handling, and transportation conditions on beef cattle temperament pre-slaughter

ABSTRACT This study aimed to investigate the effects of animal type, on-farm handling, and transport conditions on temperament of beef cattle pre-slaughter. The study evaluated 4,061 cattle batches, averaging 49 animals, with a total of 199,026 cattle. Temperament was evaluated through a behavioral assessment carried out based on the temperament shown by cattle when approached by humans. One of three different temperament scores were assigned to each batch: calm, anxious, or excitable. Excitable temperament was reported in animals subjected to long transport time and distance and high loading density. The degree of carcass fatness was higher in calm animals, followed by anxious animals, with the excited temperament animals having the lowest carcass fatness degree. Older animals showed more excitable temperament, while younger animals showed calmer temperament. The excitable temperament was more evident in horned animals in relation to polled animals. Animal temperament was more reactive in batches with older cattle, low carcass fatness degree, and with more than 20% horned animals. Good handling and loading facilities and procedures at the farm contributed to adequate cattle temperament expression prior to slaughter.


INTRODUCTION
Regarding livestock production, animal welfare is of great concern for consumers.In this sense, animal welfare should be considered, not only within the production system, but also during transportation and pre-slaughter animal handling within the slaughter industry.Pre-slaughter handling is the most stressful period for cattle (Stockman et al., 2012).Exposure to challenges including changes in social structure, water and feed deprivation, fatigue, and fear, leads to increased respiratory frequency and altered animal temperament, where animals become more reactive (Fergunson and Warner, 2008).
In beef cattle and other livestock species, temperament can be defined as the behavioral expression of animals in response to human interaction.In this sense, it is possible to classify cattle according to the level of responsiveness, from calm to excitable, through temperament evaluations (Burrow et al., 1988).Research has demonstrated the impact of temperament on growth, performance, feeding efficiency, carcass characteristics, and meat quality in beef cattle (Francisco et al., 2015;Willian et al., 2019).
More excitable temperament impacts not only meat quality (Moura et al., 2021) but it also has a direct effect on beef production efficiency (Francisco et al., 2015;Willian et al., 2019).Therefore, understanding the factors that negatively impact animal temperament is of great interest to minimize losses associated with undesired animal behavior, which can lead to carcass bruising and injury.
Despite the considerable knowledge gained in recent years on the association of beef cattle temperament to animal performance and meat quality, how factors such as: animal type, onfarm handling and transportation conditions affect cattle temperament pre-slaughter is still not fully elucidated.This study aimed to investigate the effects of animal type, on-farm handling, and transportation conditions on temperament of beef cattle prior slaughter.

MATERIALS AND METHODS
The study was approved by the Ethics Committee on Animal Care and Use of the Federal University of Pelotas, Brazil, case number 3110.008794 /2013-31 (number 8794 CEEA).The study was carried out at a cattle slaughterhouse in south Brazil (53°77'97" W and 29°78'18" S) with Federal Inspection Service, (S.I.F.1733), from August 2019 to October 2021.
The study evaluated a total of 4.061 cattle batches, being 2.124 batches composed by male and 1.937 batches composed by female cattle, averaging 49 animals per batch (ranging from 6 to 457 animals), with a total of 199.026 animals.For statistical analyses each batch was considered as an experimental unit.Animals were from different regions of Rio Grande do Sul state, being 125.707 (63.16%) male cattle and 73.319 female cattle (36.84%), with slaughter weight and cold carcass weight averaging 495 and 250.0 to 225.0 kg, respectively.
Information regarding on-farm animal handling and loading procedures and facilities conditions, as well as information on transportation conditions were collected prior to slaughter.The data were collected using forms developed by researchers in agreement with the people responsible for animal purchase by the slaughterhouse.Regarding animal transportation, information including animal loading and trip conditions was taken by previously trained drivers.This information was monitored and added to the dataset daily.Inconsistent data or data missing independent variables measurements were excluded from the dataset.
Variables including animal gender, handling and loading facilities conditions and animal handling and loading procedures at the farm, length of transportation time (transport time) and the distance from the farm to the slaughterhouse (transport distance) were analyzed as possible factors impacting cattle temperament preslaughter.Loading density, unloading time, animal type, animal age and carcass fatness degree were also evaluated.
Temperament evaluation was done one hour after animals were placed in the lairage pens.This one-hour period was allowed to ensure animal acclimation to the facilities and water consumption.
Animal temperament was evaluated through a behavioral assessment carried out based on the temperament shown by the animals, by testing their reaction when approached by humans, this evaluation was carried out by two trained personnel.Three different temperament scores were assigned to each batch: 1) "calm": animal static, quiet with no resistance to approach; 2) "anxious": with some resistance and constant movement; and 3) "excitable": frightened movements, escape attempts, very agitated, wild movements.
Temperature-humidity index (THI) was calculated from noon to 2 pm on the day of animal loading.The THI was calculated according to official data from the meteorological database of the Federal University of Santa Maria, which is located near the slaughterhouse.
Unloading time at the slaughterhouse was the time, expressed in minutes, between arriving at the slaughterhouse and animal unload and accommodation in lairage pens.Slaughter handling was carried out according to Regulation of Industrial and Sanitary Inspection of Products of Animal Origin -RIISPOA (Brasil, 2008).
Animal age and carcass fatness degree were evaluated at slaughter by trained personnel right after removal of ride/skin and head by trained workers, following federal legislation from MAPA.Animal age was determined by examination of the teeth.Carcass fatness degree was determined by visual assessments scored on a scale of five fatness levels (1 -5 scale), where 1 indicates no fat cover and 5 indicates excess fat cover (Brasil, 2008).
Transport distances were evaluated in km, including the distance traveled on paved and unpaved roads.Length of transport time (transport time) was evaluated in minutes, including the time from animal loading at the farm up to the arrival at the slaughterhouse.Loading density was calculated diving total load weight (kg) by total occupied area (m 2 ) and expressed as kg/m 2 .All vehicles used for animal transportation belonged to the same transport company.Truck maintenance, trailer dimensions and layout, truck driver training, and engine potential were all standardized.
Three scores were used to classify handling and loading facilities conditions and handling and loading procedures at the farms: good, moderate, and poor (Mendonça et al., 2016).Handling and loading facilities conditions were evaluated through observation of their conservation conditions.Facility conditions were classified according to the presence of facility defects that could potentially contribute to animal stress during handling, following the procedures described by Mendonça et al. (2016).Animal handling and loading procedures were evaluated through the level of aggressive handling applied by handlers.Animal handling was classified according to the use of dogs, sticks, electric prods, canes, whips, or any other object during animal handling that could potentially cause animal stress and injuries (Mendonça et al., 2016).
Batches according to animal type were classified according to horn presence or absence into three groups: "horned", "mixed" and "polled".Batches with more than 20% of horned animals were classified as "horned" whereas, batches with less than 20% were classified as "mixed", and batches with no horned animals were classified as "polled".
Animal temperament data were submitted to ANOVA and means were compared by Student t test using lsmean package.Statistical significance was set at p ≤ 0.05.Initially, differences between male and female were tested, being the analyses realized combined for both genders.The variables animal gender and batch number of animals were included in the statistical model as covariables, according to the following model: where: Y ijklmnopqr = dependent variable, temperament; MI i = effect of ith handling and facilities conditions at loading (i=1 good; i=2 moderate; 3= poor); I j = effect of jth animal maturity (j=1 deciduos teeth; j=2 two teeth; j=3 fourr teeth; j=4 six teeth; j=5 eight teeth); G K = effect of kth carcass fatness degree (k=1 absent; k=2 scare, k=3 moderate, k=4 uniform, k=5 excess); T l = effect of lth transport time; D m = effect of mth transport distance; DE n = effect of nth loading density (n = 1, ..., 4 classes); TD o = effect of oth unloading time at the slaughterhouse; TG p = effect of pth cattle type Arq.Bras.Med.Vet.Zootec., v.75, n.4, p.735-743, 2023 (1= polled; 2= mixed; 3= horned); N q = effect of covariable batch number of animals; S r = covariable animal gender; and e ijklmnopqr = error term.

RESULTS
Animal temperament was classified as "calm" (score 1) in 34% (1.381 batches) of the batches, whereas in 48.19% (1.957 batches) of the batches, animal temperament was "anxious" (score 2).Excitable temperament (score 3) occurred in 17.8% (723 batches) of the batches analyzed (Tab.1).Temperature-humidity index (THI) and unloading time had no effect on animal temperament (P >0.05;Table 1).  b,c Means of the same variable followed by different letters in the same row differ by T test at P < 0.05.
2 Significance was declared at P ≤ 0.05 3 Determined by visual assessments scored on a scale of five fatness levels (1 -5 scale), where 1 indicates no fat cover and 5 indicates excess fat cover (Brasil, 2008).
Animals showing excitable temperament were older than other animals (P=0.0001).Whereas the youngest age was reported in animals presenting "calm" temperament (P=0.0001).
Regarding the effects of handling and loading facilities and procedures at the farm on cattle temperament, batches of animals from farms scored as "good" presented a calmer temperament compared to batches of animals from farms assigned as "moderate" and/or "poor" scores for facilities conditions and animal handling procedures (Table 2; P=0.0013). 1 scores = 1) "calm": animal static, quiet with no resistance to approach; 2) "anxious": with some resistance and constant movement; and 3) "excitable": frightened movements, escape attempts, very agitated, wild movements.
2 On-farm handling and loading facilities and procedures, classes determined according to Mendonça et al., (2016).
3 Batches with no horned animals. 4Batches with less than 20% horned animals. 5Batches with more than 20% horned animals.
Animal temperament was more aggressive (excitable temperament) in horned animals compared to polled animals (P=0.05).Horned (more than 20% of horned animals) and mixed batches (less than 20% of horned animals) had more aggressive (excitable) and anxious temperament than polled batches, which showed calm temperament (Table 2).

DISCUSSION
Previous studies have reported a direct impact of weather conditions and temperature on animal behavior (Mader, 2003;Brown-Brandl, 2018) (Brandrão and Cooke, 2021).In addition to its effects on animal temperament, ageing time also impacts meat quality, as more mature and temperamental cattle produce reduced quality meat (Mendonça et al., 2017), and are more prone to carcass bruising (Mendonça et al., 2016;Bethancourt-Garcia et al., 2019;Vaz et al., 2023).The herein study compared animals from different farms, raised in different systems of beef production; therefore, it is likely that young animals at slaughter included in this study are from intensive systems.Beef cattle raised on intensive livestock operations, such as: intensive or semi-intensive beef finishing systems have regular contact with humans (Petherick et al., 2009;Paranhos da Costa et al., 2021;Brandão and Cooke, 2021).However, in this study, it is not possible to determine the effect of feeding systems on animal temperament, as this variable was not studied herein.Therefore, further studies evaluating how animal temperament differs among beef cattle operation systems (feeding systems) are needed to better understand factors within the production systems driving animal behavior.
Carcass degree of fatness is highly affected by feeding systems, mainly regarding nutritional level offered to the animals, which can lead to improved animal performance (Silveira et al., 2012) and contribute to adequate cattle temperament, in both, feedlot systems (Mader, 2003) and/or grazing systems (Francisco et al., 2020) Pedroza et al., 2021;Vaz et al., 2023).
Long distance and length of transport prior to slaughter causes detrimental effects on cattle welfare by causing stress (Burdick et al., 2010;Bethancourt-Garcia et al., 2019).These effects were observed in the herein study, as greater transport distance and length of time resulted in more temperamental animals, showing excitable temperament.During transportation animals suffer stressful conditions which are mainly associated with improper transport environment or conditions, restrictions due to confinement and poor road conditions.Animals under stressful situations spend part of the dietary energy to adapt to these adverse situations.In this case, the amount of energy lost in this processes is impacted by the length of time in which animals are exposed to stress (Mackay et al., 2013).In this study, cattle subjected to long transport time and distance and high loading density had excitable temperament, which agrees with previous studies that have reported that these factors are associated with stress, affecting animal welfare potentially leading to injuries (Bethancourt-Garcia et al., 2019;Vaz et al., 2023).Alterations to animal temperament, due to transport, are likely a result of physiologic changes faced by the animals.Burdick et al. (2010) (Mackay et al., 2013).
Inappropriate loading density (high or low) during transportation is also known to impact cattle temperament.Abnormal behaviors are seen at loading densities that fall outside the optimal bounds.Different space allowances in vehicles expose the animals to unknown situations, which can cause fear changing their temperament (Schuetze et al., 2017).In this sense, high loading density impacts air quality inside of the trailer and increases the risk of bruising when falls occur due to the animals' difficulty in standing up when space is limited (Vimiso and Muchenje 2013).These factors closely explain the excitable temperament reported herein in cattle subject to high loading density.Similarly, cattle subjected to low loading density are likely to fall and slip, which also leads to high risk of injuries altering animal temperament (Mendonça et al., 2016).
The quality of handling is directly associated with cattle temperament (Olmos and Turner 2008;Paranhos da Costa et al., 2021).While infrequent and/or poor handling practices (stressful interactions with humans) result in negative behavioral changes in cattle (Petherick et al., 2009), frequent and calm handling (regular pleasant contact with humans) reduce animal fear and reactivity (Paranhos da Costa et al., 2021;Brandão and Cooke, 2021).These factors reported in the literature regarding the association of on-farm handling procedures and facilities condition to temperament explain the results observed herein, where cattle subjected to good handling facilities and procedures had adequate temperament (calm).Grandin and Shiyley (2015) observed that cattle are responsive to handling and can recall challenging situations (stressful situations) for a long period of time.In this sense, pleasant interactions between cattle and humans from the beginning of their lives and especially during the daily handling bring significant, as well as lasting benefits to the production systems.Young cattle can change their behavior when they experience management situations considered more adequate (Brandão and Cooke, 2021).Temperament is impacted by the environment in which animals are raised.When subjected to places without protection from climate change, especially cold and windy rains, animals can become more reactive (Del Campo et al., 2021).Animals kept during prolonged time in unknown places in which they are not used to stay, as well as prolonged water and feed deprivation, are likely to change their behavior becoming more temperamental (Moura et al., 2021).
Excitable temperament of batches with high number of horned animals (more than 20% of horned animals) observed in this study, is probably due to greater defensive behavior in horned animals, as they are dominant in the hierarchy of the group, expressing aggressive behavior more easily than polled animals (Reiche et al., 2020).The presence of horned animals increases the risk of accidents during on-farm handling, which can injure other animals, as well as handlers.Chiquitelli Neto et al. (2015) reported that the presence of the horn often prevents animals from being restrained during handling, making medication application difficult, increasing the introduction of needles into the animal and the possibility of bleeding.These factors are stressors and affect cattle reactivity directly.In recent years, efforts have been made to reduce the presence of horned animals in cattle herds with the major goal of facilitating animal handling, on-farm, during transport and at slaughter.Except for some breeders of a few breeds who still value the presence of horns, this stressor factor should be minimized with the advancement of animal breeding techniques.Anim. Sci., v.96, p.2607-2621, 2018.MACKAY, J.R.D.; TURNER, S.P.; HYSLOP, J. et al.Short-term temperament tests in beef cattle relate to long-term measures of behavior recorded in the home pen.J. Anim. Sci., v.91, p.4917-4924, 2013.MADER, T.L. Environmental stress in confined beef cattle.J. Anim. Sci., v.81, p.e110-e119, 2003 Agrop. Bras., v.56, p.e02349, 2021.OLMOS, G.; TURNER, S.P.The relationships between temperament during routine handling tasks, weight gain and facial hair whorl position

Table 1 .
Means ± standard error of variables with potential effects on beef cattle temperament preslaughter

Table 2 .
Mean ± standard error for cattle temperament scores pre-slaughter according to handling and loading facilities and procedures at the farm and presence or absence.
a,b,c means of the same variable followed by different letters in the column indicate difference at P <0.05 by T test.
Environmental conditions including temperature and humidity, during loading have no effects on cattle temperament pre-slaughter.Temperament is more excitable in batches with high age cattle, low carcass fatness degree and high number of horned animals.Cattle transported at high loading density, long transport time and distance are more reactive showing aggressive temperament.Good handling and loading facilities and procedures at the farm contribute to adequate cattle temperament expression prior slaughter.This study design was reviewed and approved by the Ethics Committee on Animal Care and Use of the Federal University of Pelotas, Brazil, case number 3110.008794 /2013-31 (number 8794 CEEA).GRANDIN, T.; SHIVLEY, C. How farm animals react and perceive stressful situations such as handling, restraint, and transport.Animals., v.5, p.1233-1251, 2015.HULBERT, L.E.; CARROLL, J.A.; BURDICK, N.C.et al.Innate immune responses of temperamental and calm cattle after transportation.Vet.Immunol.Immunopathol., v.143, p.66-74, 2011.LITTLEJOHN, B.P.; RILEY, D.G.; WELSH, J.R. et al.Use of random regression to estimate genetic parameters of temperament across an age continuum in a crossbred cattle population.J.