Review: castration – animal welfare considerations

ABSTRACT The castration of male cattle is an integral part of routine farm management. The nature and duration of an animal’s response to castration are dependent on a number of factors, including the method employed, the age of animals, the post-castration management, and whether or not pain relief is provided with the procedure. Scientific assessments of the impact of castration on cattle welfare, including pain and injury, stress, inflammation, immune, and production, are the subject of this review. The objectives of this review are to describe (1) the different methods of castration, (2) the pain responses associated with each of those methods, and (3) how age and pain mitigation strategies affect those responses. Research studies are presented that have addressed the challenges imposed by castration procedures on the welfare of cattle based on two main biological events: (1) the changes in biological functions required to cope with the procedure, and (2) the biological consequences to the animals. Indices of animal well-being are described that have objectively demonstrated: (1) the degree of noxiousness that an animal experiences following castration and the success of the coping mechanisms, and (2) the benefit of using pain management in modulating these responses.


Why castrate animals
Bulls are castrated to prevent sexual behaviour, reduce aggression, and increase animal handling safety (Warriss 1984;Tennessen et al. 1985).In addition, steers (which are castrated bovine animals from 1 d of age or more) have a reduced risk of dark-cutting meat at slaughter (Tarrant 1981;Warriss 1984) and can be comingled with heifers without the risk of unwanted matings in the herd.Almost 8 million tonnes of bovine meat are produced in the European Union annually (Hocquette et al. 2018).A high production of beef from steers is observed in the UK (50.9%) (Rutherford et al. 2021) and Ireland (84%) (McGee et al. 2022).The scientific community and producers worldwide (Spooner et al. 2012;Moggy et al., 2017) agree that castration is a painful procedure and the use of an anaesthetic in combination with an analgesic as a measure to alleviate pain during castration is recommended (Coetzee 2013b;Ede et al. 2022).

Pain
Pain in animals can be defined as 'an aversive sensory and emotional experience; it changes the animal's physiology and behaviour to reduce or avoid damage, to reduce the likelihood of recurrence and to promote recovery' (Molony and Kent 1997).Thus, pain is an important stimulus to alert an animal that tissue damage has occurred, is occurring or is likely to occur, which allows immediate escape, withdrawal or other avoidance behaviour (Martin et al. 2022).Painful experiences also help animals to learn to avoid any similar pain-causing circumstances.Perceived pain varies according to the characteristics of the noxious stimulus (e.g.location, duration, intensity) and according to other factors (e.g.experience, emotional state, individual variation) that modify the way in which the central nervous system processes the perception of pain (Mellor et al., 2000).The evaluation of pain in animals is only possible using indicators that can be detected by external observers.Most of these indicators are based on physiological or behavioural responses (Reviewed by Prunier et al. 2013;Coetzee 2013b;Tschoner 2021).
Physiological changes associated with pain occur mainly through two related pathways.Firstly, pain is a powerful stressor that directly stimulates the release of hormones from the hypothalamic-pituitary-adrenal axis (HPA) and sympathetic-adrenalmedullary axis (SAM).Secondly, tissue damage activates the immune system and the release of numerous inflammatory mediators (e.g.cytokines) which may also activate the adrenal axis (Prunier et al. 2013).Hence, physiological indicators of pain involve hormones from the adrenal and sympathetic axes, their metabolic and physiological consequences, plasma markers of an inflammatory state and mediators involved in the physiological mechanisms of pain.At present, a number of tools for the recognition of pain are being used in research studies, including use of algometry to measure mechanical nociceptive thresholds, accelerometers and pedometers to measure activity, the use of infrared thermography, and the assessment of heart rate and heart rate variability (Tschoner 2021).

Discrepancies in regulations between countries
While the use of anaesthetics is mandatory during castration of calves at any age in Belgium and Switzerland, calves can be castrated without use of anaesthetic up to a certain age in some countries (Table 1).Castration is a painful procedure, and pain relief is recommended when castrating calves at all ages (Stafford and Mellor 2005;Coetzee 2013a;2013b;Canozzi et al. 2017;Mijares et al. 2022).For cattle there are recommendations from the Council of Europe (CoE 1988) that allow husbandry management procedures such as castration, freeze branding, dehorning, disbudding, ear notching, nose ringing, vasectomies and chipping (Spoolder et al. 2016).The principle is that 'mutilations should be avoided'.While there are derogations to this principle, mitigation measures are always recommended (based on age of the animal, provision of pain relief, presence of a veterinarian, etc).Indeed, the legislation concerning the use of anaesthesia for castration in cattle varies considerably among different countries and depends on the method used and age of the animal (Table 1).

Castration methods
Castration procedures are generally divided into two categories: surgical and bloodless (Burdizzo and rubber rings/banding).

Surgical castration
Surgical castration mainly involves removal of the testes by splitting or removing the distal one third of the scrotum and removing the testes by severing the spermatic cord in a manner that minimizes bleeding, usually with an emasculator or Henderson castrating tool (Jennings 1984).One other method is the incision of the lateral scrotal walls with a scalpel or a Newberry knife to expose the testicles.Using the Newberry knife, both lateral walls and the median septum are simultaneously incised, thus facilitating drainage from the wound (Fubini and Ducharme 2016).In addition, the Newberry knife allows the scrotal skin to be split without danger of other tissues being cut.After the incision, the testes and spermatic vasculature are pulled and exposed (surgical pull) to allow complete removal of the testicles.

Bloodless castration
Bloodless castration is generally performed by using an emasculatome (i.e.Burdizzo) or elastic band.

Burdizzo castration (emasculatome)
When using a Burdizzo, the scrotum remains intact while the spermatic cord of each testicle (within the scrotum) is placed in the jaws of the tool and crushed.The resulting damage stops blood flow to the testes with eventual testicular atrophy within the scrotum.The Burdizzo is applied twice (the second crush is repeated distally with one cm space between the two) to each spermatic cord along the neck of the scrotum (Ting et al. 2003a).The jaws of the instrument are closed for approximately 10 s in a single (Robertson et al. 1994) or two crushes (Fisher et al. 1996(Fisher et al. , 1997;;Ting et al. 2003a) to ensure that both blood supply and nerves to the testes are irreversibly destroyed.

Rubber ring and banding
Banding involves using an elastrator to place a heavy elastic band around the neck of the scrotum with both testes inside.The band disrupts blood flow to the testes and scrotum, which atrophy over a long period of time and slough off.Rubber ring and elastic band castration are considered bloodless castration since there is no incision of the scrotum (Becker et al. 2012).Small rubber rings (rubber ring castration) are used for calves less than one month (mo-) of age.For older calves heavy wall latex bands are used along with a grommet to securely fasten the mechanically tightened tubing at the appropriate tension (Pang et al. 2009a;2009b).Animals generally receive tetanus prophylaxis to minimize the risk of tetanus (O'Connor et al. 1993).The effect of banding and Burdizzo castration of continental × beef bulls (12.0 ± 0.2 mo-old; (mean ± SE); weight 341 ± 3.0 kg (mean ± SE) was investigated by Pang et al. (2009a).The authors found that banding compared to Burdizzo castration caused more inflammatory associated gene expression changes in the epididymis and scrotum.In a further study, Pang et al. (2009b) investigated measures of neutrophil function in response to banding or Burdizzo castration of bulls.Neutrophil functioning in terms of phagocytosis and respiratory burst and serum IL-8 concentration were not affected by banding, Burdizzo, and cortisol infusion.These findings indicate that non-surgical castration is unlikely to induce a severe acute systemic inflammatory response in terms of neutrophil function.

Immunocastration (ImC)
Immunocastration (ImC) uses immunological methods to destroy the hormone balance of the hypothalamic-pituitarygonadal (HPG) axis (also known as the reproductive axis).Using this approach, the hypothalamic gonadotropin-releasing hormone (GnRH) is reduced, which cause a decrease in luteinizing hormone (LH) and follicle-stimulating hormone (FSH), thereby reducing the level of gonadal hormone and eventually leading to gonadal atrophy and functional inhibition (Adams et al. 1993;Mazon et al. 2019).Studies conducted in Mexico (Pérez-Linares et al. 2017), Canada (Marti et al. 2015), andBrazil (Amatayakul-Chantler et al. 2013;Miguel et al. 2014) found that vaccinating cattle against gonadotropin-releasing hormone (GnRH) produces effects similar to traditional castration methods.However, Mazon et al. (2019) reported that ImC decreases the tenderness of fresh meat and negatively affects the overall liking and tenderness sensory ratings.A disavantage of the ImC method is that the effect of immunocastration is of short duration (4 months) and requires repeated immunization to maintain effective suppression of testosterone concentrations.A commercial vaccine is available for use in cattle in some countries, but not in Europe.

Chemical castration
Chemical castration involves the injection of lactic acid, directly into the testes, causing oedema and sloughing (Capucille et al. 2002).This method is reported to cause less pain and complications compared to physical methods of castration.However, it is recommended only for calves less than 70 kg (Skarda 1986).In addition, this method does not always provide a successful castration (Hill et al. 2010) and therefore chemical castration is not considered a useful technique for castrating calves (Coventry et al. 1989).Furthermore, Fordyce et al. (1989) found that scrotal necrosis occurred in 25% of chemically-castrated calves which may have been associated with leakage of the chemical from the testes under the high pressure of injection.The authors reported that healing time for chemical castrates was twice that for surgical castrates and also found that 18% of chemically-castrated calves retained one testis.

Castration induced pain
The age of an animal is a relevant factor which contributes to individual pain differences (Tucker, 2018).In most research studies, castration procedures are considered least stressful on calves when performed at the earliest age possible.Studies investigating castration-induced pain have been performed in calves at different ages (see reviews by Bretschneider 2005;Coetzee et al. 2012;Coetzee 2013a;2013b;Canozzi et al. 2017).These studies suggest that castration at a younger age is less painful, however, it is difficult to draw a general conclusion since there are many differences between studies, such as breed of calves, environment (presence of the dam or not), and plane of nutrition.Age could be an important factor influencing the changes in stress response between studies.Indeed, Stafford and Mellor (2005) in their review on castration stress stated that it is complex and needs to be studied rigorously to determine effect of breed and age of the animals, the methods used and how animals are reared before castration.
The pain experience of individuals differs according to their sensitivity to noxious stimuli, susceptibility to developing neuropathic pain after injury, and their analgesic response to pharmacological therapy (Mogil 2012).A better understanding of the basis of these differences in cattle may assist with our ability to recognize and manage pain effectively through all its stages.

Cortisol concentrations and inflammatory responses
Castration is considered both a stressor and a painful experience for animals (Fisher et al. 2001;Bretschneider et al., 2005;Stafford and Mellor, 2011).Animal welfare concerns thus warrant that castration procedures are performed in a way that causes the least stress and pain.One of the defining features of the stress response to castration in male cattle is the secretion of excess glucocorticoids (cortisol), and a biological consequence associated with this reaction is the temporal suppression of the adaptive immunity.A high peak of cortisol after a known stressor may be a sign of a well-functioning HPA axis with an established glucocorticoid cycle (Moberg and Mench, 2000;Blecha, 2000;Mitra et al., 2009;Hulbert and Moisá 2016).Castration by Burdizzo and surgical methods has been shown to result in a rapid and large increase in plasma cortisol concentrations (Fisher et al. 1996).It would appear that a significant part of the castration-induced cortisol rise is caused by pain as the provision of local anaesthesia reduced the cortisol surge for both burdizzo and surgical methods, while local anaesthesia alone had no effect on plasma cortisol (Fisher et al. 1996).Pain is difficult to quantify in animals; however, plasma concentration of the stress hormone cortisol is often used as an indicator of castration induced stress related pain.King et al. (1991) investigated the plasma cortisol concentration changes in beef bull calves castrated at 2.5 or 5.5 mo-old by either surgery or Burdizzo.In their study, uncastrated calves were restrained in the same manner as castrated calves for 2 min, which was the average length of time taken for each castration.The authors concluded that the cortisol response to surgical or Burdizzo castration was reduced by castrating calves at 2.5 mo-old compared with 5.5 mo-old, but no direct comparison was made between age groups due to the study design.Interestingly, uncastrated 2.5 mo-old calves had a greater increase in cortisol concentration after sham handling and restraint compared to the 2.5 and 5.5 mo-old Burdizzo castrated calves, indicating that restraint is also a stressor.In their study, all calves were separated from their dams for sham handling, castration and blood collection.It is likely that the abrupt separation of the calves followed by restraint resulted in an increased cortisol response in all calves regardless of treatment (King et al. 1991).A similar reduction in cortisol response in young dairy calves was found in other studies.Robertson et al. (1994) evaluated plasma cortisol responses of dairy calves castrated by Burdizzo, surgery or rubber ring castration at 6, 21 and 42 days old.At each age, the peak of cortisol concentration of Burdizzo and surgical groups increased sharply after castration (Robertson et al. 1994).Similarly, another study using Holstein bull calves castrated by Burdizzo at 1.5, 2.5, 3.5, 4.5 and 5.5 mo-old found that the plasma cortisol area under the curve (AUC) from 0 to 3 h after castration was significantly reduced in 1.5 and 4.5 mo-old calves by 47% and 34%, respectively, with intermediate reductions in 2.5 and 3.5 mo-old calves (27 and 23%, respectively), compared to 5.5 mo-old castrates.However, no significant changes were observed on area under the curve (AUC) from 3 to 12 h after castration between age groups (Ting et al. 2005).In a study by Dockweiler et al. (2013), plasma cortisol concentration of Holstein bull calves was increased in 6 mo-old calves relative to their 2-mo-old counterparts, regardless of treatment applied which included sham handling of uncastrated calves, and surgical (cut and clamp; cut and pull) and non-surgical (band) castration procedures.The authors suggested that younger calves may be less acutely stressed by handling and castration procedures than older calves.
However, further studies found no difference in the cortisol response of calves castrated at different ages.Petherick et al. (2015) investigated the effect of band and surgical castration on plasma cortisol concentration of Belmont Red calves at 3 and 6 mo-old.In their study, plasma cortisol concentrations increased immediately after castration but was not different between age treatments 30 min post-castration; only 6-moold calves castrated by band showed increased plasma cortisol concentrations above baseline 2 h post-castration.Similarly, Meléndez et al. (2017b) evaluated effect of band and knife castration on chronic indicators of pain in 1 week old, 2 and 4 moold Angus crossbred calves.They found no differences in salivary cortisol concentration at 60, 120 min, or 7 d after castration in 1 week-old calves.Additionally, the baseline salivary cortisol concentration for 1 week-old calves was greater than for 2 and 4 mo-old calves supporting the suggestion that calf-dam separation and handling of 1 week-old calves are cumulative stressors and that a high intensity of circulating glucocorticoids was present before castration (Meléndez et al. 2017a).However, the comparison between ages was not possible as the studies were conducted at different time of the year using different restraint conditions due to calf size.Furthermore, Sutherland et al. (2013) investigated behavioural and physiological changes following surgical castration, dehorning, or both of 3 mo-old Holstein calves (101.5 ± 1.45 kg; mean, SE) and found that the responses were cumulative when the two procedure were carried out simultaneously.Therefore, combining castration and dehorning procedures will exacerbate responses to the procedures and should be avoided in view of the negative welfare consequences.

Behaviour
Several postural and behavioural indicators of pain have been reported in animals, such as avoidance and defensive behaviours, vocalisations, behaviours directed towards the painful areas.The pain of castration occurs first as acute, short-term pain associated with the actual castration procedure.Chronic pain is the longer-lasting pain that occurs in the days following castration until the injury is healed, associated with inflammation and neural injury.Boesch et al. (2008) examined the short-term pain response of 30 calves less than 1-week of age to Burdizzo castration with and without local anaesthesia.Twenty minutes before castration, 10 ml lidocaine (n = 10), bupivacaine (n = 10) or saline solution (n = 10) was injected into each spermatic cord and distributed subcutaneously in the scrotal neck.The behaviour of the calves and response to local palpation was assessed on the day before and after castration during 8 h periods.Plasma cortisol levels were determined on the day of castration.The struggling behaviour exhibited by the calves during castration and their response to local palpation of the spermatic cords indicated that the procedure was painful.Nevertheless, postures and behavioural elements thought to be associated with pain were not readily apparent after castration in the majority of calves.The pain caused by injection of the anaesthetic was considered minimal.Local anaesthesia reduced the immediate pain response during castration as evidenced by less struggling during castration and lower cortisol levels.However, some calves struggled during the procedure and had distinct pain-indicating signs afterwards.There was no apparent difference between the analgesia provided by lidocaine and bupivacaine.Molony et al. (1995) concluded that burdizzo castration, when performed correctly, was probably less stressful to young calves than surgical castration, and that castration using a rubber ring induced abnormal behaviour for up to 42 d, possibly associated with chronic pain.Nogues et al. (2021) compared two castration methods; surgical (n = 10 calves) and rubber ring (n = 11).Pre-weaned dairy calves were castrated at 28 days of age using multimodal pain control.During the 8 week period post-castration, wound healing, local inflammation, body weight, milk and calf starter intake, lying time, and wound-directed behaviour were recorded.Surgical wounds were fully healed on average 4 weeks after the procedure, but only 1 calf in the rubber ring treatment fully healed within the 8-week study period.Inflammation was greater after rubber ring castration; skin temperature in the area around the lesion was higher (+1.7 ± 0.35 °C; mean ± sd) than for the surgical treatment.Compared with surgically castrated calves, those castrated by rubber ring gained less weight over the study period (on average 11.9 ± 5.1 kg less), a difference due in part to lower intake of calf starter (on average 1.8 ± 0.6 kg less).Calves in the rubber ring treatment spent less time lying down (on average 4.2 ± 1.2% fewer scans per day) and licked their lesions more frequently (on average 16.0 ± 3.3 more licks per day).The authors concluded that the rubber ring calves experienced more pain in the weeks following the procedure and thus recommend that surgical castration be favoured for pre-weaning dairy calves.Thuer et al. (2007) investigated the behavioural and cortisol responses of calves as indicators of pain to assess short-and long-term effects of bloodless castration methods with and without local anaesthesia.Seventy calves, aged 21-28 days old, were control handled (n = 20) or castrated using the Burdizzo (n = 25) or rubber ring technique (n = 25).Either 10 ml lidocaine or NaCl was distributed in both spermatic cords and the scrotal neck.The plasma cortisol response was recorded for 72 h, and behavioural and clinical traits monitored over a three month period.Local anaesthesia reduced the level of indicators of acute pain after both the Burdizzo and rubber ring techniques.It did not, however, result in a totally painless castration.As there was evidence of chronic pain lasting for several weeks after rubber ring castration, the Burdizzo method was considered to be preferable to the rubber ring technique based on the animal's behavioural and physiological responses.Indeed, rubber ring castrated calves responded up to 4 weeks longer than Burdizzo calves on local palpation and continued to show an increase in proportion of abnormal postures after the first week following castration.Lambertz et al. (2014) evaluated the effect of castration on the stress response assessed by behavioural observations as well as blood traits and performance of calves weaned at the same time of castration or 4 weeks afterwards.The treatments consisted of bulls that were (1) castrated and concurrently weaned in week 0 after housing; (2) castrated in week 0 and weaned in week 4; (3) bulls weaned in week 0; and (4) bulls weaned in week 4. Weaning was found to have a greater effect on the number of vocalizations, standing/walking and lying behaviour and ADG compared with Burdizzo castration.The authors concluded that, with undertaking the procedures separately, concurrent castration and weaning did not affect behaviour and haematological parameters or impaired animal performance.Petherick et al. (2015) investigated effect of band and surgical castration on behaviour of tropical breed calves (Belmont Red) castrated at 3 and 6 mo-old, by band or surgery.In their study, some behavioural indicators of restlessness/activity were influenced by calf age, being greater in 3-mo-old than 6-mo-old calves on the day of castration.These greater levels of restlessness in the younger compared with older calves may have been due to a greater motivation for calves to reestablish contact with their mothers, which is a response frequently reported in studies where the calf and dam are separated for some time.In the same study, during the 4 to 7 h period post castration, by which time calves had been separated from their mothers for at least 4 or 5 h, there were greater levels of vocalization by the control (not castrated) calves compared with the castrated calves (Petherick et al. 2015).Although vocalization behaviour can be used to assess pain, in this case, calves may be experiencing the least pain and showing more normal behaviours, by trying to establish contact with their mothers.In fact, these authors concluded that in experimental situations where unweaned calves are temporarily separated from their mothers for data collection, pain-related behavioural responses may be attenuated due to calves being motivated to re-establish contact with their dams, and this in turn may switch their attention from pain (Petherick et al. 2015).
Two studies evaluated the effects of band and knife castration on acute (Meléndez et al. 2017b) and chronic (Marti et al. 2017a(Marti et al. , 2017b) ) indicators of pain, such as behaviour, in 1 week old, 2 mo-old and 4-mo-old Angus crossbred suckler calves.Investigating acute signs of pain in 1 week old calves, knife castrated calves showed a greater number of pain-related indicators on the day of castration (stride length and tail flicking), whereas calves castrated by elastic bands exhibited pain-related behaviours on days 2 and 3 (lying duration and standing and lying bouts) The authors commented that the lack of differences between tail flicking in banded and control calves may be due to high individual variation for tail flicking compared to the rest of the behaviours.In 2 mo-old calves, knife castrated calves had greater standing percentage, and walking duration, as well as decreased eating and lying behaviours on the day of castration.In addition, greater standing and lower lying percentages were observed during the first 7 days after castration in knife castrated calves, suggesting that knife castration is more painful than band castration in 2 moold calves.In 4 mo-old calves, band castrated calves presented greater restless behaviour 2 to 3 days after castration.However, knife castrated calves had greater frequency of leg movement, and vocalizations at the time of castration, shorter stride length immediately after castration, a greater number of tail flicks 2-4 h after castration, and greater standing activity for the first 5 d after castration.This indicates that behaviour responses associated with pain lasted for a longer period after knife castration.
Using the same animals as the previous study, the same authors found no differences in standing or lying, or duration of lying bouts among treatments, and no chronic effect of castration method on stride length, eating time, or behaviours related to pain (tail flicks, foot stamping, head turning, or lesion licking) throughout the trial in 1-week and 2-mo-old calves (Marti et al. 2017a(Marti et al. , 2017b)).However, in 4 mo-old calves, lying time after castration differed between treatments; band castrated calves spent more time lying compared to knife and control calves (Marti et al. 2017a(Marti et al. , 2017b)).In those studies, cow-calf pairs were kept together in the same pen (Marti et al. 2017a(Marti et al. , 2017b;;Meléndez et al. 2017b).Throughout the day, pain-related behavioural responses can be reduced by the calf shifting it's attention elsewhere (mediated by conscious awareness of pain) (Gentle 2001).Consequently, the presence of the dam with the calf can shift their attention from the pain and change the expression pattern of pain-related behaviours.

Wound healing and sensitivity
Petherick et al. ( 2015) investigated the impact of band and surgical castration on wound healing of tropical breed (Belmont Red) calves castrated at 3 or 6 mo-old, by band or surgery.The scrotal wounds of calves castrated at 3-mo-old took longer to heal compared to calves castrated at 6-mo-old.In contrast, Norring et al. (2017) found no difference in wound healing after surgical castration of beef calves at 3 d or 2.4 mo-old calves.In their study, younger calves reacted to lighter pressure of Von Frey monofilaments (48% more sensitive) compared to castrates at 2.4 mo-old especially in the first stages of the healing process, and there were other signs indicative of inflammation processes in this region at this time (Norring et al. 2017).The incisions of younger calves healed more quickly than older ones (fully healed, median (95% confidence interval); 39 (32-61) v. 61 (61-77) days), however, they had relatively increased swelling in the days after castration.

Scrotal temperature and circumference
The presence of heat and oedema following tissue trauma, such as castration, are used as signs of inflammation.Ting et al. (2005) investigated the changes in scrotal temperature and circumference following Burdizzo castration of calves at different ages.In their study, the increase in scrotal circumferences of calves castrated at ages between 2.5 and 4.5 mo was not different from the increase in scrotal circumference in the 5.5 mo-old calves.However, the 1.5 mo-old castrates had little change in scrotal circumference on the first day following castration compared with all other castrates, and less change on days 7, 21 and 28 post-castration compared with either 4.5 or 5.5 mo-old castrates.Since the younger calves had the least genital tissue development, castration at younger ages could be done to minimize tissue inflammation, as suggested by Ting et al. (2005).There is evidence to show that surgically castrated older calves take longer to heal their wounds and to resolve swelling compared with younger calves.Healing after surgical castration can take between 10 days (Molony et al. 1995) and 4-6 weeks (Fisher et al. 2001;Stafford et al. 2002;Mintline et al. 2014;Marti et al. 2017aMarti et al. , 2017b)).

Thermal nociception threshold
The physiological development of the calf and responses to stressful stimuli at a young age was also evaluated.Ting et al. (2010) assessed the thermal nociception threshold of calves castrated at different ages (1.5, 2.5, 3.5, 4.5 and 5.5 mo-old) to a heat spot laser which was directed to the lower leg of the calves after Burdizzo castration.The thermal nociception was not significantly increased following Burdizzo castration at any age.However, the study showed other age-related differences between calves.The skin temperature of the hind legs of calves 1.5 mo-old were lower before and after castration compared to calves 2.5, 3.5, 4.5 and 5.5 mo-old.The reaction time to the heat spot laser increased in all calves after castration.However, calves 1.5 mo-old, which also had a lower skin temperature, tolerated the heat spot laser for a longer duration than the older calves.The lower skin temperature and more tolerance to the heat spot laser suggests that younger calves are less capable of showing behavioural signs of pain (Ting et al. 2010).
Variations in skin temperature are likely due to changes in tissue perfusion, metabolism, and blood flow in the superficial veins, and sympathetic nerve activity.However, the question remains to be addressed concerning the physiological developmental stage of calves at 1.5 mo of age versus older calves (2-6 mo-old) and their responses to castration procedures, since the laser-based thermal nociceptive assay used in the study by Ting et al. (2010) was influenced by the initial skin temperature and the age of calves.This finding suggests that the assessment of pain should be multimodal and based on physiological and behavioural indicators.

Electroencephalogram responses
Dockweiler et al. ( 2013) investigated the age-related differences in pain response of Holstein bull calves subjected to surgical and band castration at 8 weeks and 6-mo of age.Desynchronized electroencephalogram (EEG) and electrodermal activity readings (both indicative of pain response) were greater in 6-mo-old compared to 8-week-old calves after castration.However, the absence of desynchronization across castration methods in young calves does not imply that they do not require analgesia at times of castration.The cortical function in these young calves are not developed enough to show the same EEG responses observed in older calves.However, this does not necessarily indicate they are not experiencing pain.This study illustrates the importance of measuring different variables, whenever it is possible.Lehmann et al. (2017) investigated the mitigating effects of administration of local anaesthetic or systemic meloxicam on the EEG responses during surgical castration of bull calves.The EEG changes indicated nociceptive responses in all three groups during surgical castration, being greater in calves receiving local anaesthetic compared to calves that received no preoperative analgesia and those that received preoperative meloxicam.Lidocaine and meloxicam administered prior to castration attenuated these responses in bull calves.Bergamasco et al. (2021) investigated effect of unmitigated surgical castration on the EEG responses of male Holstein calves in three age categories [<6 weeks, 3, 6 mo of age; 10 calves per age group].Calves were subjected to a simulated castration session (sham) followed 24 h later by surgical castration without analgesia.The EEG total power decreased, and median frequency increased relative to sham in 6 week and 3 mo-old calves only following treatment.The authors reported variation in EEG responses following unmitigated surgical castration in calves and suggested that the responses could be age specific.

Substance P (SP)
There is some evidence to suggest that SP might be a suitable biomarker for nociception in cattle, but results of research are heterogenous.Bergamasco et al. (2021) also investigated effect of unmitigated surgical castration on the plasma substance P concentrations in calves of different ages under the same experimental conditions, as described above.Substance P concentrations decreased in the castrated treatment compared to the sham treatment at the later times; 6 week old calves showed lower substance P concentration at castration relative to sham.The authors reported variation in SP concentrations following unmitigated surgical castration in calves which may be age specific.In a recent systematic review, Tschoner and Feist (2022) concluded that SP concentrations of calves and adult cattle differ throughout studies and that further research is warranted to investigate factors others than nociception which might influence the SP concentrations in the circulation.The authors stated that although studies showed that SP concentrations differed significantly by age, with 6-months-old calves showing higher concentrations than 8-week-old calves (Dockweiler et al. 2013), there was no consistency among age groups of animals included in studies.Even within the same gender and age group, high between-and within-calf variations were found (Coetzee et al. 2008).

Acute phase proteinshaptoglobin and fibrinogen
Acute phase proteins (APPs) are induced primarily in the liver and often described as positive (up-regulated) or negative (down-regulated) acute phase proteins in response to the challenge (Murata et al. 2004).The former have important roles in the inflammatory response, whereas the latter are important carrier proteins such as albumin, corticosteroid binding protein, and transferrin (metal-binding protein).Examples of positive APPs include haptoglobin, fibrinogen, serum amyloid A, C-reactive protein, and α−1-glycoprotein.
While some studies have found an increase in plasma haptoglobin concentration after a stressor (Murata and Miyamoto, 1993;Morrow-Tesch and Whitehead, 1998), others have failed to demonstrate any effect of stress on the haptoglobin response, even in the presence of increased levels of other acute phase proteins such as serum amyloid A (SAA) and fibrinogen (Alsemgeest et al. 1995;1996;Hickey et al. 2003).It therefore appears that the haptoglobin response to stress is complex.A reduction in cortisol and APP production following castration with analgesia would indicate that the 'noxiousness' associated with the stimulus has been effectively alleviated, and hence impact on the welfare of castrated animals would be minimized (Earley and Crowe 2002).

Effect of castration on animal performance
There is a belief that delaying castration could extend the production advantages of keeping animals as bulls until weaning or beyond puberty.However, a number of studies have shown that there is no advantage in delaying castration of bulls from 5 to 7 mo of age up to 17 mo in terms of liveweight, growth rate, or carcass weight at slaughter (Keane 1999;Knight et al. 1999aKnight et al. , 1999b)).Keane (1999) reported that Burdizzo castration of spring-born calves in their first autumn (complete castration) at 5-6 mo-old did not significantly affect the overall 347 d liveweight gain compared with: (1) delayed unilateral castrationthe right testicle removed in autumn and left testicle the following spring with ∼178 d apart; or (2) split castration in spring with about one month interval between removal of each testicle.Furthermore, in that study, no interaction between castration treatment and breed type (Friesian versus Charolais × Friesian) was found.Knight et al. (1999aKnight et al. ( , 1999b) ) reported that the age at surgical castration (7-15 versus 17 mo-old) of post-pubertal bulls had no effect on either liveweight or carcass weight when the animals were slaughtered at 22 mo-old.Pang et al. (2008) assessed the effect of banding or Burdizzo castration, performed on farms, on plasma testosterone, acutephase proteins, scrotal circumferences, growth, and well-being of bulls.The 243 Continental bulls (12 mo-old; 399.2 ± 5.72 kg; mean ± SE) used in the study were on three different commercial farms.The bulls were allocated at random, after stratification on weight within breed type, to one of three treatment groups: banding castration, Burdizzo castration and controls (intact).The authors reported that banding and Burdizzo castration significantly reduced plasma testosterone concentration; reduced average daily weight gain mainly during the first 2 weeks, which was not compensated during the subsequent 16 weeks; increased withdrawal of stored energy and increased plasma protein concentration.Burdizzo showed an advantage over banding in growth during days 15-28 following castration.Theurer et al. (2019) investigated the distal splitting of the scrotum at time of banding of bulls, ranging in weight from 306 to 552 kg (average 436 kg), and reported improved average daily gain, healing time, and increased ribeye area compared to leaving the scrotum intact.
Castration has been shown to elicit a reduction in growth to varying degrees.Fisher et al. (1996) reported that surgical castration of 5.5 mo-old calves resulted in lower 35 d growth rates compared with Burdizzo castration, and the depressive effect occurred mainly during the first week after castration.However, the animals used in their study were maintained in individual tie stalls which may influence their growth compared with animals raised in the feedlot or pasture environment.King et al. (1991) found no effect of either surgical or Burdizzo castration on the liveweight or daily gain of 2.5 mo-old calves compared with bulls over a three-month period after castration.In contrast, Fenton et al. (1958) found no difference in either surgical, Burdizzo, or elastrator castration procedures in 7-weekold calves in terms of liveweight gain five weeks after castration, but the control calves had higher gains than castrates.However, the authors reported a significant retardation of growth for the elastrator group on the 28th day after castration due to chronic pain and sepsis (based on visual assessment and palpation of the scrotum) proximal to the ring.This is supported by the findings of Molony et al. (1995) who showed trends for lower 36-d growth rates in rubber ring, and combined rubber ring plus Burdizzo castrated 1-week-old calves compared with intact, surgical or Burdizzo castrated calves.Bretschneider (2005) reviewed the effects of age at castration on performance of beef cattle and found that castration at birth or close to birth drastically reduced weight loss associated with castration.Later, studies designed to compare the impact of age at castration on performance agreed with his findings.Norring et al. (2017) found that beef calves surgically castrated at 2.4 mo had a reduction of 57% on ADG (average daily gain) from 1 to 77 days after castration compared to calves castrated at 3-d-old (Table 2).In agreement with the previous study, the study by Petherick et al. (2015) found that surgery or rubber ring castrated calves at 3 mo had superior weight gains compared with calves castrated 6 mo-old calves.Meléndez et al. (2017a) found no difference on ADG from d −1-7 of calves castrated at 1-week or 2-mo-old, by either band or surgically, compared to intact calves within respective age group.However, in the same study, calves surgically castrated at 4-mo-old had a significant lower ADG, from d −1-7, compared to intact calves at same age.
Conversely, other studies found no difference in performance of calves castrated at different ages.Ting et al. (2005) found no difference in growth trends up to 42 days after Burdizzo castration of Holstein-Friesian calves castrated at 1.5, 2.5, 3.5, 4.5, and 5.5 mo-old.In another study, Micol et al. (2009) evaluated the effect of age (2 or 10 mo) at castration by rubber band on performance of Charolais steers and their muscle characteristics and meat quality traits.They found no difference in liveweights and average daily gains according to castration age.In addition, meat quality traits of tenderness,  juiciness and flavour were equivalent for the two age groups of steers (Micol et al. 2009).These studies suggest that the effect of the age at castration on performance of the calves also depends on the castration method with surgical castration having a greater impact on performance mainly in older calves.

Effect of castration on animal health
To our knowledge, detailed epidemiological studies on the effects of castration per se on health are unavailable, however, earlier work by Addis and colleagues in the mid 70s indicated that castration of calves causes adverse effects on animal health, particularly when it was performed at any time near weaning or shipment (cited in Zweiacher et al. 1979).This supports the theoretical framework developed by Moberg (2000) who postulated that exposure to multiple stressors leads to an adverse 'cumulative' effect on the health and welfare of an animal.Zweiacher et al. (1979), in two separate studies, found that calves (mean bodyweight; 180 kg) purchased as steers had less health problems than those purchased as bulls and subsequently castrated by surgical methods on arrival at a feedlot (mean percentage of animals requiring treatment for sickness = 17.5% for steers [n = 97] and 38.4% for surgical castrates [n = 104]).These health effects were not related to the time (castration on arrival versus after 1 week or after 2 weeks) or methods of castration (surgery with either use of elastrator band for ligation, or crimping of testicular cords with an emasculator to prevent the blood loss).However, the death losses were greatest in bulls castrated on the day of arrival at the feedlot compared with bulls castrated one week, or two weeks after arrival (mean percentage death loss was 8.5% for bulls castrated on arrival [n = 35], 0.0% for bulls castrated after one week [n = 35], and 3% for bulls castrated after two weeks [n = 34]).These animals were also branded, dehorned, wormed and vaccinated on arrival at the feedlot.In their second study, surgical castration occurred on the day of arrival and prophylactic treatment with oxytetracycline for the first three days appeared to reduce, but not prevent the higher occurrence of sickness in the castrates (mean percentage of animals requiring treatment for sickness was 2.6% for steers [n = 38] and 13.0% for surgical castrates [n = 85]; Zweiacher et al. [1979]).These findings are supported by Berry et al. (2001) who found that calves (mean bodyweight, 166 kg) purchased as bulls and subsequently castrated by surgical method the day after arrival at the feedlot had greater incidences of sickness (requiring treatment for respiratory disease) than calves purchased as steers.
However, in another study, these authors did not find any differences in terms of health response between the steers, banded calves or surgically castrated calves on arrival.The main problems associated with the studies on the effects of castration on animal health reported so far are that there were numerous other confounding factors involved (e.g.transport, branding and dehorning).Thus more detailed and specific epidemiological studies may be warranted in order to draw a more definitive conclusion.Furthermore, Sutherland et al. (2013) investigated behavioural and physiological changes induced by surgical castration, dehorning, or both and found that the responses were cumulative when carried out together.

Attributed to age at castration
A misconception that castration is less stressful for younger animals likely arose as older animals have a greater peak of plasma cortisol after castration, and calves did not gain as much weight after castration as older animals (Bretschneider 2005;Stafford and Mellor, 2011).In fact, Murray and Leslie (2013) suggest that pain may be even greater among neonatal animals compared with mature animals since their nervous system and HPA axis are not developed.Kampen et al. (2006) reported that immune parameters in young calves differ from what is found in older calves and adult animals, therefore these age-related factors should be considered when assessing immunological responses in young calves to castration.Robertson et al. (1994) examined the effect of rubber ring, Burdizzo and surgical castration in Ayrshire bull calves at different ages (6, 21 and 42 days old) on behavioural responses.In their study, the younger calves showed less tail wagging and foot stamping and more head turning and spent more time lying normally than the older calves (21 and 42 days old).The total time spent in abnormal postures was increased following castration by all methods, and the 21 and 42 d old calves spent less time eating and suckling than non-castrated controls, indicating that the pain induced by castration temporarily dominated their behaviours.The time spent in abnormal standing postures increased in Burdizzo and surgical castrates and lasted for 24 min in 6 d old calves and up to 120 min in 21 and 42 d old calves, indicating that older calves were experiencing discomfort for a longer time.
Effect of age at castration on physiological, immunological stress indices, and behavioural responses using 60 Holstein-Friesian bull calves was examined by Ting et al. 2005Ting et al. , 2010. .Calves were sourced such that they were in one of five age groups for Burdizzo castration on day 0 (n = 10 per treatment), 1.5, 2.5, 3.5, 4.5, and 5.5 mo of age, or were sham castrated at 5.5 months of age (Ting et al. 2005(Ting et al. , 2010)).Castration was shown to be stressful across all ages between 1.5 and 5.5 mo-old, as indicated by the increased (∼180 to >300% mean increase) integrated plasma cortisol response for the first 3 h after treatment relative to the intact controls.However by reducing the age at castration, the integrated cortisol response was markedly reduced (by nearly half) in the 1.5 mo-old and by onethird in 4.5 mo-old castrates, but the ∼20% reduction observed in the 2.5 and 3.5 mo-old castrates were not significant.The peak cortisol responses to castration were reduced by castrating calves at younger ages.However, there was no evidence to suggest that the welfare (including performance and immune responses) of calves was adversely affected by Burdizzo castration from 1.5 to 5.5 months of age (without use of local anaeshetic) (Ting et al. 2005).This is an important consideration; the cortisol response was short-lived, there was no adverse effect on immune variables and performance at the age range studied.Ting et al. (2005) also reported that plasma concentration of the acute phase proteins (APPs), haptoglobin and fibrinogen, were reduced by castrating calves at younger ages.Plasma haptoglobin concentration on the third day after castration was markedly reduced by castrating calves at 1.5 and 2.5 moold (reduction of 75 and 45%, respectively); but no significant changes were found in haptoglobin concentration of calves castrated at 3.5 and 4.5 mo-old, compared to calves castrated at 5.5 mo-old (Ting et al. 2005).In the same study, plasma fibrinogen concentration was reduced on the third day after castration at 1.5, 2.5 and 3.5 mo-old (reduction of 29, 19 and 17%, respectively) compared to 5.5 mo-old castrates (Ting et al. 2005).This indicated that the inflammation caused by castration was reduced by castrating calves at a younger age.Marquette et al. (2021b) examined effect of age at castration on stress indicators and performance of 40 crossbred suckler beef calves.Calves were assigned to two age groups and two castration treatments; calves 2.5 or 5.0 mo-old (mean body weight (SD) = 120.8(29.3), 218.1 (30.8) kg, respectively) were sham (control) or Burdizzo castrated in a 2 × 2 factorial design.Following castration, peak plasma cortisol concentrations were greater in 2.5 and 5.0 mo-old calves compared with corresponding controls, while peak cortisol concentrations in control animals were greater in 5 mo-old compared with 2.5 mo-old calves.The integrated cortisol responses for the first 4 h after castration were not different between 2.5 and 5.0 mo-old calves.However, the integrated cortisol response was greater in 5.0 mo-old calves from 4 to 9 h post treatment.The increase in scrotal circumference after castration was greater in 5.0 mo-old calves, and abnormal postures were observed more often in 5 mo-old castrates.There was no effect of castration on haematology profiles, haptoglobin, metabolites, body temperature and growth performance.
Following castration, calves spent more time standing during the first hour, regardless of the treatment.Duration spent lying was only affected by time which was decreased in the first hour compared with the second and third hour block after treatment.There was no difference in time spent standing in abnormal position between 2.5 or 5.0 mo-old control calves for any sampling time.However, calves castrated at 5.0 mo-old spent more time standing in abnormal position (928 s) compared with calves castrated at 2.5 mo-old (358 s), in the first hour post-castration.Castrated calves exhibited more foot stamping compared with control calves, regardless of the age at castration.Before treatment (−24 h), the mean latitudinal scrotal circumferences was greater for 5.0 mo-old calves compared with 2.5 mo-old calves.Both latitudinal and longitudinal scrotal circumferences were increased in castrated calves at 24 and 48 h after treatment compared with their respective baseline.Calves castrated at 5.0 mo-old had a greater increase in latitudinal scrotal circumference at 24 and 48 h compared with 2.5 mo-old castrates.No difference was observed in longitudinal scrotal circumference between 2.5 and 5.0 mo-old castrated calves.
The increased plasma cortisol concentration following castration observed in this study suggests that Burdizzo castration is stressful for both 2.5 and 5.0 mo-old suckler beef calves.However, an increase in abnormal postures, scrotal swelling and a prolonged plasma cortisol response observed in older calves suggest that these calves suffered more stress induced by handling and castration.Therefore, castration of younger suckler calves is preferable from an animal welfare point of view.The age-related differences in stress response found in this study need to be considered when recommending the use of anaesthesia and analgesia during castration, especially for older calves.The timing of pain relief (analgesia) administration should be planned to effectively reduce the stress response according to the age of the calf.The practical implications of the study are that if calves are to be castrated without analgesia or anaesthesia, then it would be preferable to do this at 2.5 mo-old rather than later, in order to minimize the stress associated with Burdizzo castration.
In cattle, most of the immune system maturity is seen by 5-8 months of age.For example, T cells (CD4+, CD8 + and TCRγδ+ cells) do not reach peak levels until the animal is 8 months of age (Cortese, 2009).Research has demonstrated that from birth, there is a decrease in immune responses until day 3 in calves, when they are at their lowest levels (Cortese, 2009).By day 5, these responses are back to the level of immune responses seen on the day of birth.Procedures like disbudding, castration and movement need to be considered as stressors that have the potential to decrease immune system function temporarily in younger calves.

Attributed to pain mitigation strategy
The use of analgesia after castration of calves differs with age and breed.A Canadian survey reported that 6.9% of beef calves and 18.7% of dairy calves under 6 mo of age and 19.9% of beef calves and 33.2% of dairy calves over 6 mo of age, received some form of anaesthesia at the time of castration (Hewson et al. 2007), but very few received post-operative analgesia.In a 2010 survey of veterinarians in the United States, Coetzee et al. (2010) reported that approximately 20% of respondents used some form of pain relief for surgical castration in cattle.Similarly, Fajt et al. (2011) reported that approximately 30% of respondents provided some analgesic drugs when castrating calves.A UK survey of veterinarians by Remnant et al. (2017) found that 67% of respondents used local anesthesia in calves undergoing castration.
Research studies have demonstrated that local anaesthesia alone or combined with systemic analgesic drug administration prior to castration mitigates physiological, and neuroendocrine changes usually associated with pain and distress (Fisher et al. 1996;Earley and Crowe 2002;Ting et al. 2003aTing et al. , 2003b)).Calves administered ketoprofen, a non-steroidal anti-inflammatory drug (NSAID), prior to castration exhibited increased feeding and rumination activities and fewer pain-associated behavioural responses than those castrated without ketoprofen (Ting et al. 2003a).Local anaesthesia prolonged the increase in acute-phase proteins suggesting that LA administration may further exacerbate inflammatory reactions associated with castration, including pain responsiveness.Ketoprofen was more effective than lignocaine or epidural in decreasing cortisol and partially reversed the reduction in average daily gain following castration.The use of ketoprofen or epidural was more effective than local anaesthesia in decreasing painassociated behavioural responses observed during the first 6 h after treatment.Systemic analgesia with ketoprofen, was more effective in reducing inflammatory responses associated with castration than local or epidural anaesthesia.However, the authors did not investigate the pain caused by the epidural injection in uncastrated bull calves.
The effect of carprofen administration before banding or Burdizzo castration of bulls on plasma cortisol, in vitro interferon-gamma production, acute-phase proteins, feed intake, and growth was investigated by Pang et al. (2006).Overall, the integrated cortisol response was greater in the castrates than in control, whereas carprofen treatments tended to reduce this response compared with banding and Burdizzo castration without carprofen treatment, respectively.The authors concluded that carprofen (1.4 mg/kg of BW) tended to reduce the integrated cortisol response, and it reduced cortisol secretion in banded animals between 6 and 12 h post-castration.There was an increased acute-phase protein production following castration; this response was effectively reduced by the administration of carprofen before castration.Pang et al. (2011) investigated the effect of Banding or Burdizzo castration of bulls on the gene expression profile of a range of peripheral leukocyte inflammatory cytokines (IL-1, IL-6, IL-8, IL-10, interferon-γ and tumor necrosis factor-α) and determined the response to administration of carprofen before castration.Thirty Holstein-Friesian bulls (5.5 months old; 191 ± 3.7 kg, mean ± SE) were blocked by weight and randomly assigned to one of five treatments: (1) Untreated control (Con); (2) Banding castration at 0 min; (3) Banding following an i.v.injection of 1.4 mg/kg BW of carprofen (C) at −20 min; (4) Burdizzo castration at 0 min; or (5) Burdizzo following 1.4 mg/kg BW of carprofen at −20 min.Generally, there were no differences among treatment groups in haematological variables following castration.Cortisol concentrations were unchanged throughout the experimental period in Con bulls.Burdizzo animals had greater cortisol concentrations than banded and controls at 6 h post treatment.Of particular interest is IL-6, an inflammatory cytokine, which plays a critical role in the development of pathological pain.At 24 h post-castration, Pang et al. (2011) found the relative quantity of IL-6 mRNA was greater in Burdizzo calves than banded calves at 24 h post-castration.This finding is in agreement with their previous study (Pang et al. 2006) which found that banding was associated with a greater cortisol secretion than Burdizzo at 12 h post-castration (Pang et al. 2006) and that of Molony et al. (1995) that banding caused more chronic inflammation than Burdizzo.
Others NSAID's have been studied, for example, nalbuphine hydrochloride (Coetzee et al. 2012), flunixin meglumine (Nordi et al. 2019), and meloxicam (Roberts et al. 2015;Vindevoghel et al. 2018;Melendez et al., 2017aMelendez et al., , 2018aMelendez et al., , 2018bMelendez et al., , 2019)).Stilwell et al. (2008) showed that epidural anaesthesia with lidocaine will reduce temporarily the pain caused by Burdizzo castration.Inflammation, pain-related behaviours and high levels of cortisol were still present at greater than 48 h after clamp castration and were only abolished after the use of a long-acting NSAID (Pang et al. 2006;Stilwell et al. 2008).Several studies have assessed the efficacy of different analgesic protocols.Thuer et al. (2007) showed that a local anaesthetic, injected into the spermatic cord and subcutaneously at the neck of the scrotum, reduced acute pain during and immediately after calves were castrated by use of a castration clamp.Therefore, providing calves with pain relief, in the form of local anesthetic and an NSAID, can markedly reduce both the behavioural and physiological response to castration.As evidenced, pain management for castration is achieved by pharmacological interventions.However, factors affecting the development of pain and inflammation, such as the age of animals, the techniques for castration and associated severity of injury, and the post-operative animal management could be be optimized in future studies to reduce the adverse effects of castration.

Conclusion
Depending on the techniques used (Burdizzo, surgical, banding (typically using either a rubber ring or band for constriction)), and the age of animals, castration of male cattle causes varying degrees of acute distress by inference from increased plasma cortisol concentrations, acute-phase protein production, leukocytosis with lymphopenia, temporal suppression of immune function, increased scrotal swelling, depressed temperature gradient between the core body and scrotal skin, and increased expression of pain-associated behaviours.In this review, studies also suggest that effect of calf age at castration on cortisol response depends on the calf breed types (beef versus dairy).Reduced cortisol responses were found in studies with dairy breed calves while studies with beef calves found no difference among age groups.The findings of these studies could be attributed to the different husbandry management of dairy and beef calves.Beef calves are typically separated from the dams for castration, which could increase the stress response, regardless of the age of the calf.Early separation of the dairy calves from their dams occurs within a few hours of birth which makes it easier to measure the effect of castration alone on those calves.
The pain, stress and inflammatory responses to tissue injury elicited by castration procedures constitute an acute challenge in cattle of all ages.Therefore, it is desirable to devise measures to alleviate the adverse effects of castration through optimizing pain management.While there is clear evidence from research studies that multimodal analgesic protocols are associated with a significant decrease in plasma cortisol concentration after castration, the practical implications of these interventions at farm level require further investigation.In future studies it would be of interest to develop a comprehensive view of the dynamic inter-and intracellular cytokine fluxes and associated changes in cell signaling pathways following castration, in order to elucidate basic mechanisms involved in the modulation of pain including 'inflammatory-anti-inflammatory' cascades.The subjective emotional experience of castration pain is probably one of the most significant determinants of acute distress in cattle.However, suitable models for measurement of the emotional distress associated with the perception of castration pain in cattle have yet to be fully developed.Furthermore, the behaviour-based approach is likely to be the most practical, non-invasive method for the assessment of castration stress and pain in cattle.
Studies investigating the effect of age at castration on stress response are important to draw recommendations on what is the optimum age to castrate calves.In addition, understanding how calves castrated at different ages respond to pain is also helpful to recommend the optimum timing of pain relief.When considering how age at castration affects calf welfare, the consensus is that the younger the calf is at time of castration, the less impact castration may have on its welfare.Research has indicated that the pain associated with castration of younger calves is at a lower level and has less of a negative impact on the calf in terms of welfare, health and performance.
In practice, there is no advantage of banding over burdizzo castration in adult animals.The administration of anti-tetanus vaccination and antibiotics at castration add to the financial cost.There are also some contradictory reports on growth suppression associated with castration stress, which may be due to the ages / weights of the animals studied.It appears that banding or burdizzo castration had less impact on growth in 5.5 months old than in 14 months old castrates.A combination of banding and burdizzo techniques has been evaluated in young lambs and indicates that there is a benefit in terms of reduced stress responses.However, only one study in the literature (Molony et al., 1995) has attempted to address this as an alternative technique for castration of calves and this was done at a very young age (5-7 days).Further evaluation of combined burdizzo / banding approaches to castration is warranted in cattle.
Regarding the impact of castration on immune function and inflammation, transcriptomics and proteomics could be employed to demonstrate an integrated and thorough profile of leukocyte (and /or other cells) functioning (probably in terms of gene expression) during and post castration.
Castration has been shown to elicit physiological stress, inflammatory reactions, pain-associated behaviour, suppression of immune function, and a reduction in growth to varying degrees.The scientific assessment of animal health and welfare has been significantly advancing through multidisciplinary approaches including physiology, behaviour studies, immunology, qualitative and quantitative molecular biology, and functional genomics.This should allow the impact of castration on cattle welfare to be further assessed using biological indices of welfare.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Table 1 .
Legislation concerning the use of anaesthesia for castration in calves.
a Rubber ring/band are not allowed.

Table 2 .
Summary of the scientific literature examining the effect of age at castration (cx) on welfare outcomes.
Abbreviations: Trt treatment; AUC, area under the curve; WBC, white blood cells; ADG, average daily gain in body weight.a WBC percent changes were calculated between day 1 and day 2 of the same treatment group.