Investigating the effects of lairage duration and transportation time on physiological stress indicators and meat quality of Boran and Nguni breeds reared on natural pasturesByAnelisa Guzi201206930A thesis submitted in partial fulfillment of the requirements for the degree ofMSc AGRICULTURE in ANIMAL PRODUCTION SCIENCEDepartment of Livestock and Pasture ScienceFaculty of Science and AgricultureUniversity of Fort HareP/Bag X1314AliceSouth AfricaSupervisor: Dr. Y. Z NjisaneCo- supervisor: Prof V.Muchenje 2018Table of Contents TOC o “1-3” h z u 1.Introduction PAGEREF _Toc523678371 h 61.1Problem Statement PAGEREF _Toc523678372 h 91.
2Justification PAGEREF _Toc523678373 h 101.3The specific objectives were to: PAGEREF _Toc523678374 h 111.4Hypothesis PAGEREF _Toc523678375 h 121.5Reference PAGEREF _Toc523678376 h 122Chapter 2: Literature Review PAGEREF _Toc523678377 h 172.
1Introduction PAGEREF _Toc523678378 h 172.1Stress Concept in Animal PAGEREF _Toc523678379 h 192.2Stress measurement PAGEREF _Toc523678380 h 192.
3Stress related factors PAGEREF _Toc523678381 h 222.3.1 Sound PAGEREF _Toc523678382 h 222.3.
2 Novel Environment PAGEREF _Toc523678383 h 232.3.4 Fighting PAGEREF _Toc523678384 h 232.
3.5 Overcrowding PAGEREF _Toc523678385 h 252.3.6 Restraint stress PAGEREF _Toc523678386 h 252.3.7 Temperature PAGEREF _Toc523678387 h 262.
4Factors affecting haematological factors of farm animals PAGEREF _Toc523678388 h 272.5Transportation of Farm Animal PAGEREF _Toc523678389 h 272.6How to measure well-being of animals in abattoir locations PAGEREF _Toc523678390 h 312.
7Effect of laraige on meat quality PAGEREF _Toc523678391 h 332.8Influence of stress on biochemical compounds PAGEREF _Toc523678393 h 36i.Animal stress related compounds (Cortisol and Creatine phophoskinase) PAGEREF _Toc523678394 h 372.9Effects of temperature on the secretion of stress hormone (cortisol) PAGEREF _Toc523678395 h 402.9.1 Thermal stressors PAGEREF _Toc523678396 h 402.9.
2 Heat stress PAGEREF _Toc523678397 h 402.10 Summary PAGEREF _Toc523678398 h 41Chapter 3 PAGEREF _Toc523678399 h 483.1Introduction PAGEREF _Toc523678400 h 483.
2 Materials and Methods PAGEREF _Toc523678401 h 503.2.1 Ethical clearance PAGEREF _Toc523678402 h 503.2.2 Animal description and management PAGEREF _Toc523678403 h 503.2.3 Experimental Site description PAGEREF _Toc523678405 h 513.
2.4 Transportation details PAGEREF _Toc523678406 h 513.2.5 Lairage duration and Slaughter procedure PAGEREF _Toc523678408 h 523.2.6Blood sampling PAGEREF _Toc523678411 h 533.2.
7Physiological Parameters PAGEREF _Toc523678412 h 5188.8.131.52Haematological Analysis PAGEREF _Toc523678413 h 543.2.7.
1Hormonal (Cortisol) Analysis PAGEREF _Toc523678414 h 5184.108.40.206Biochemical (Glucose and Creatine Kinase) Analysis PAGEREF _Toc523678415 h 543.2.
8Meat samples and quality tests PAGEREF _Toc523678416 h 5220.127.116.11 Determination of meat pH PAGEREF _Toc523678417 h 563.2.
8.2 Determination of meat color (L?, a?, and b?) PAGEREF _Toc523678418 h 563.2.
8.3 Thawing and cooking loss measurements PAGEREF _Toc523678419 h 518.104.22.168 Tenderness PAGEREF _Toc523678420 h 573.2.
9Statistical Analysis PAGEREF _Toc523678421 h 573.3Results PAGEREF _Toc523678422 h 593.3.1Plasma cortisol PAGEREF _Toc523678423 h 603.3.2Glucose and Ck PAGEREF _Toc523678424 h 613.3.
3Weight loss PAGEREF _Toc523678425 h 613.4Discussion PAGEREF _Toc523678426 h 623.5Conclusion PAGEREF _Toc523678427 h 653.6Reference PAGEREF _Toc523678428 h 66Chapter 4 PAGEREF _Toc523678429 h 684Introduction PAGEREF _Toc523678430 h 684.1Materials and Methods PAGEREF _Toc523678431 h 714.1.
1 Ethical clearance PAGEREF _Toc523678432 h 714.1.2 Animal description and management PAGEREF _Toc523678433 h 714.1.3 Experimental Site description PAGEREF _Toc523678435 h 714.1.
4 Transportation details PAGEREF _Toc523678436 h 724.1.5 Lairage duration and Slaughter procedure PAGEREF _Toc523678438 h 724.1.
6Blood sampling PAGEREF _Toc523678441 h 734.1.7Haematological Analysis PAGEREF _Toc523678442 h 744.2Results PAGEREF _Toc523678443 h 754.3Discussion PAGEREF _Toc523678444 h 764.4Conclusion PAGEREF _Toc523678445 h 774.5Reference PAGEREF _Toc523678446 h 785Conclusion PAGEREF _Toc523678447 h 83Chapter 1: Background of the studyIntroductionBeef cattle have an abundant importance in the economy and culture of rural populations in South Africa.
South Africa is among the leading countries of the world in terms of cattle populations and production potential. Meat obtained from cattle is about 14% in South Africa while it is about 13.8 % in Turkey and 1.6% in Europe (FAO, 2015). That notifies that beef is valuable and highly appreciated by consumers and is marketed as an extravagance product in several countries.
However, the majority of cattle are transported at least once in the lifetime either to the slaughterhouse, a commercial for markets, auction or to a new environment or other farms (Huertas et al., 2010). Therefore road transport of livestock animals is an unavoidable agricultural practice. It is often considered one of the main causes of stress (Adenkola and Ayo, 2010), as it presents an acute stage of animal production, frequently associated with different forms of stressors sustained by the animals which increase considerable interest both in animals welfare and economic situation (Grannetto et al., 2011). Also, Adentola and Ayo (2010) reported that management of interactions in the transporting vehicles and handling of individuals before and after transportation have different effects on animal welfare. In agreement, Warris (2012) shown that road transportation can result in live weight loss.
Transporting and slaughtering livestock are the last stressing stages of an animal production system aimed to produce high quality meat (Ekiz et al., 2012). However, before transporting or during pre-slaughter period, livestock animals are exposed to different stressors including human-interaction (handling), loading and unloading, adverse weather conditions, feed and water deprivation, lairage, length of travel, mixing with animals from other groups, crowding, restraint and fatigue (Knowles, 1998; Muchenje, 2008; Rey-Salgueiroa et al.
, 2018). Moreover, slaughter animals are exposed to different handling procedures at different production stages, and handling is defined as one of the stress factors. These stressors may lead to variation in blood constituents such as plasma concentration of creatine kinase (CK), lactate dehydrogenase (LDH), glucose, cortisol and packed cell volume (Petherick et al.
, 2009; Ekiz et al., 2012). Therefore, many authors use these parameters in order to evaluate stress regarding to pre-slaughter treatments in farm animals (Ferguson and Warner, 2008). However, Grandin (1980) and Bórnez et al. (2009) noted that the expediency of physiological indicators of animal welfare may differ depending on blood sampling time or the age of the animals, and lairage time.Multiple stress indicators have been identified.
Among one of these identified stress indicators, cortisol is one of the most reliable and used indicators of stress (Grandin et al., 2007). In cattle, cortisol has been measured from blood, feces, urine, and saliva.
Tadich et al. (2004) identified all situations that include handling of the animal as psychological stressors. The release of stress hormones and enzymes adversely affect meat quality (Muchenje et al., 2009). Tadich et al.
(2004) and Njisane et al. (2016) showed that there is a link between animal welfare and meat quality because stressors produced by pre-slaughter handling often elicit behavioral and physiological responses, and if they are extreme they also contribute to a reduction in the carcass and lean meat quality.When arriving at the abattoir most of the transported animals are put to lairage for certain reasons. Lairage period could allow animals to rest and recover from prior stresses experienced during the transport (Zhen et al., 2013). Recovery rate of animals during lairage period may be affected from several factors such as lairage time (Ferguson et al., 2007; Ekiz et al.
, 2012), environmental conditions (Lowe et al., 2017), social mixing (Beery and Kaufer, 2015) and provision of food and water ( HYPERLINK “https://www.sciencedirect.
com/science/article/pii/S0309174011003986″ l “bb0110” Goddard, 2008). On the other hand, stressful lairage conditions or dehydration resulted from food and water deprivation during the lairage can lead to an additional stress in farm animals (Devlin et al., 2017; Perez et al., 2017). Lairages perform several roles (Weeks, 2008), it permits recovery from physical and emotional stress initiated by previous transportation (Liotta et al.
, 2007) and makes evisceration easier, and also allow wet animals to dry so that the risk of bacterial contamination to carcass could be reduced (Tekke et al., 2014). Hence holding the cattle in lairage is suggested as it also eliminates the fatigue arising from transportation. Lyvers (2013) tentatively concluded that reducing livestock stress during handling and lairage will provide advantages of increasing productivity and improving meat quality. Meat quality is a multi-dimensional concept that follows to a great deal the general food quality model (Bekhit et al., 2014); meat is valued based on culture, religion and many other factors such as hygienic, ethical, organoleptic and nutritional. According to Liotta et al.
(2007) and Zhen et al. (2013), short lairage time is more appropriate for both animal welfare and in obtaining improved meat quality as it resulted in lower plasma cortisol, a decreased drip loss and higher values of tenderness. In agreement, Diaz et al. (2014) reported that as lairage time increases weight losses increases and Longissimus muscles decreases. The effects of different lairage times on both animal welfare and meat quality have been reported by many research groups, but the results are not well defined. Hence, the aim of this study is to assess the response of physiological parameters and meat quality after lairage at the slaughter of Nguni and Boran steers.Problem Statement Handling, transport, lairage, and slaughter are important components of beef production that can affect both animal welfare and meat quality (Dikeman, 2017).
The study of animal welfare continues to struggle with the problem about how to measure levels of stress in animals, most especially at the abattoir premises. When animals are transported to an abattoir, on arrival they are unloaded and regularly kept in lairage before slaughter. Duration for lairages varies depending on the schedules at the slaughter plant. They normally range from 1 or less when they are slaughtered immediately after unloading, to 24 hours or more.
Lairage involves several potential stressful situations, such as novelty of environment, starvation, and handling. As a result, prolonged exposure of cattle to different stressors will disturb the mobilization of energy and therefore affect the normal body functions of the animal and physiological reactions that are involved in stress response (Chulayo, 2015). In addition, Ekiz et al. (2012) and Chulayo (2015) noted that stressful events before slaughter including lairaging may results in changes in some blood constituents example, the plasma concentration of creatine kinase (CK), lactate dehydrogenase (LDH), glucose, cortisol, and catecholamineAnimals are kept in lairages for the purpose of being held in areas while waiting for slaughter, but it also allows animals to rest and recover after transportation (Diaz et al.
, 2014). In the present-days, there are strict rules and regulations set on slaughter conditions regarding animal welfare but do not include recommendations concerning the ideal duration for lairage. However, there are inconsistencies among researchers about the effect of different lairage duration and transport times on the physiology and meat quality of cattle.
Current literature does not offer proper guidelines and more knowledge about the exact hours during lairage that are suitable for the animal’s behavior and health. Other interrelated problems also evolve around; which haematological parameters that can be used to evaluate stress levels after transportation most especially in cattle. JustificationNow, since animal welfare studies are still an important factor of the meat production cycle and it’s impossible to eliminate handling and transportation, there is a need to monitor stress levels in slaughter animals at lairage in relation to the product quality. Therefore, use of physiological parameters after transportation to evaluate stress levels in cattle may bring the research a step closer to success. It is, however, recommended that handling equipment and lairage guidelines should be designed to minimize the levels of stress in animals that are held in an abattoir.
On the other hand, measuring changes in biological parameters such as biochemical and haematological at slaughter can be used as an indicator of stress and may be able to discover unobservable responses. Haematological parameters can be used to assess pre-slaughter stress (Earley and O’Riordan, 2006; Bornez et al., 2009; ?obanovi? et al., 2017), however inaugurating a clear relationship with meat quality needs more work.Measuring the effect of lairage duration using plasma constituents such as haematological parameters and plasma cortisol can be reliable and time-consuming. Analysis of cortisol concentrations has been presented as one of the best methods for stress assessment (Grandin, 2007). According to Negrao et al.
(2004), analyzing cortisol concentrations in blood would provide valuable information regarding the stress status of the sampled animal. But cortisol is not the only hormonal matrix that can be used for stress detention, evaluation of haematic profiles can also be useful.Different time spent in lairage prior slaughter have been introduced and presented by many authors, which some agree on short lairage and some on long lairage. Nevertheless, Moss and Robb (1978), Liotta et al.
(2007), and Teke et al. (2014) suggested that longer lairage is recommended after transportation in order to prevent the adverse effect of transportation meat quality. While some literature reported short lairage duration as the best for both plasma stress indicators and meat quality (Ferguson, 2007). Hence, it is very important to determine the exact hours that are more prone for cattle at lairage using plasma stress indicators haematic, cortisol, glucose, and creatine kinase) and meat parameters. The broad objective of the current study was to investigate the response and reaction of plasma stress indicators and meat parameters to slaughter following different lairage duration.
The specific objectives were to: Investing the response of cortisol, glucose and creatine kinase on meat quality parameters of slaughtered Nguni and Boran breeds after a 16 and 24-hour lairage durationInvestigating the effects of a 16 and 24- hour lairage period on haematological parameters of Nguni and Boran breedsHypothesisThe null hypothesis states that: Lairage duration (16 or 24) has no effect on haematological, cortisol, creatine kinase and glucose, and meat quality parameters of Nguni and Boran breeds Lairage duration (16 or 24) has no effect on haematological, cortisol, creatine kinase and glucose, and meat quality parameters of Nguni and Boran breedsReferenceAdenkola, A.Y. and Ayo, J.O., 2010. Physiological and behavioural responses of livestock to road transportation stress: A review.
African Journal of Biotechnology, 9: 4845-4856.Alcalde, M.J., Suárez, M.D., Rodero, E., Álvarez, R.
, Sáez, M.I. and Martínez, T.F., 2017. Effects of farm management practices and transport duration on stress response and meat quality traits of suckling goat kids. animal, 11: 1626-1635.
Bórnez, R., Linares, M.B. and Vergara, H.
, 2009. Haematological, hormonal and biochemical blood parameters in lamb: effect of age and blood sampling time. Livestock Science, 121: 200-206.Brunel, H.D.
S.S., Dallago, B.S.L.
, de Almeida, A.M.B., de Assis, A.Z.
, de Bento Calzada, R.J., de Alvarenga, A.B.B., Menezes, A.M.
, Barbosa, J.P., Lopes, P.R.
, González, F.H.D. and McManus, C.
, 2018. Hemato-biochemical profile of meat cattle submitted to different types of pre-loading handling and transport times. International Journal of Veterinary Science and Medicine.Beery, A.
K. and Kaufer, D., 2015. Stress, social behavior, and resilience: insights from rodents.
Neurobiology of stress, 1: 116-127.Costa, L.N., Fiego, D.L., Dall’Olio, S.
, Davoli, R. and Russo, V., 2002.
Combined effects of pre-slaughter treatments and lairage time on carcass and meat quality in pigs of different halothane genotype. Meat Science, 61: 41-47.Chulayo, A.Y., 2015.
The effects of distance, lairage duration and animal-related factors on pre-slaughter stress indicators, carcass characteristics, nanostructure and technological properties of beef (Doctoral dissertation, University of Fort Hare).Cozar, A., Rodriguez, A.I., Garijo, P., Calvo, L.
and Vergara, H., 2016. Effect of space allowance during transport and fasting or non-fasting during lairage on welfare indicators in Merino lambs. Spanish Journal of Agricultural Research, 14: 0501.Dikeman, M.E.
, 2017. Understanding the effects of handling, transportation, lairage and slaughter on cattle welfare and beef quality Michael S. Cockram, University of Prince Edward Island, Canada.
In Ensuring safety and quality in the production of beef. 2: 157-202.Díaz, M.T., Vieira, C.
, Pérez, C., Lauzurica, S., de Chávarri, E.G.
, Sánchez, M. and De la Fuente, J., 2014.
Effect of lairage time (0 h, 3 h, 6 h or 12 h) on glycogen content and meat quality parameters in suckling lambs. Meat science, 96: 653-660.Ekiz, B., Ekiz, E.E., Kocak, O., Yalcintan, H.
and Yilmaz, A., 2012. Effect of pre-slaughter management regarding transportation and time in lairage on certain stress parameters, carcass and meat quality characteristics in Kivircik lambs. Meat Science, 90: 967-976.
Ferguson, D.M. and Warner, R.
D., 2008. Have we underestimated the impact of pre-slaughter stress on meat quality in ruminants?. Meat science, 80: 12-19.
Ferguson, D.M., Shaw, F.D. and Stark, J.L.
, 2007. Effect of reduced lairage duration on beef quality. Australian Journal of Experimental Agriculture, 47: 770-773.
Giannetto, C., Fazio, F., Casella, S.
, Marafioti, S., Giudice, E. and Piccione, G., 2011. Acute phase protein response during road transportation and lairage at a slaughterhouse in feedlot beef cattle. Journal of Veterinary Medical Science, 73: 1531-1534.Grandin, T.
, 1980. The effect of stress on livestock and meat quality prior to and during slaughter.Grandin, T. 2007. Livestock Handling and Transport (Third Edition), CAB International Publishing Colorado State University, United States of AmericaHuertas, S.
M., Gil, A.D., Piaggio, J.M. and Van Eerdenburg, F.
Transportation of beef cattle to slaughterhouses and how this relates to animal welfare and carcase bruising in an extensive production system. Animal Welfare, 19: 281-285. Knowles, T.A.
, Southern, L.L., Bidner, T.D., Kerr, B.J. and Friesen, K.
G., 1998. Effect of dietary fiber or fat in low-crude protein, crystalline amino acid-supplemented diets for finishing pigs. Journal of animal science, 76: 2818-2832.Warriss, P.
D., Brown, S.N., Edwards, J.E. and Knowles, T.
G., 1998. Effect of lairage time on levels of stress and meat quality in pigs. Animal science, 66: 255-261.Liste, G., Villarroel, M., Chacón, G.
, Sañudo, C., Olleta, J.L., García-Belenguer, S., Alierta, S. and María, G.A.
, 2009. Effect of lairage duration on rabbit welfare and meat quality. Meat science, 82: 71-76.
Liotta, L., Costa, L.N., Chiofalo, B.
, Ravarotto, L. and Chiofalo, V., 2007. Effect of lairage duration on some blood constituents and beef quality in bulls after long journey. Italian Journal of Animal Science, 6: 375-384.Lowe, J.
, McCann, R. and Greiner, L., 2017. Factors that influence mechanical transmission of porcine reproductive and respiratory syndrome virus at the time of unloading animals into slaughter plant lairage.
Journal of Swine Health and Production, 25: 19-23.Muchenje, V., Dzama, K., Chimonyo, M., Raats, J.
G. and Strydom, P.E., 2008. Meat quality of Nguni, Bonsmara and Aberdeen Angus steers raised on natural pasture in the Eastern Cape, South Africa.
Meat Science, 79: 20-28.Moss, B.W. and Robb, J.
D., 1978. The effect of preslaughter lairage on serum thyroxine and cortisol levels at slaughter, and meat quality of boars, hogs and gilts. Journal of the Science of Food and Agriculture, 29: 689-696.
Njisane, Y., 2016. Farm to abattoir conditions and their subsequent effects on behavioural and physiological changes and the quality of beef from extensively-reared Nguni and non-descript steers (Doctoral dissertation, University of Fort Hare).Pérez, M.P., Palacio, J.
, Santolaria, M.P., del Carmen Aceña, M., Chacón, G., Verde, M.T., Calvo, J.
H., Zaragoza, M.P.
, Gascón, M. and García-Belenguer, S., 2002. Influence of lairage time on some welfare and meat quality parameters in pigs.
Veterinary Research, 33: 239-250. Petherick, J.C., Doogan, V.
J., Venus, B.K., Holroyd, R.
G. and Olsson, P., 2009. Quality of handling and holding yard environment, and beef cattle temperament: 2.
Consequences for stress and productivity. Applied Animal Behaviour Science, 120: 28-38.Polycarp, T.N.
, Obukowho, E.B. and Yusoff, S.M., 2016.
Changes in haematological parameters and oxidative stress response of goats subjected to road transport stress in a hot humid tropical environment. Comparative Clinical Pathology, 25: 285-293.Reséndiz?Cruz, V., Ramírez?Bribiesca, J.
E., Mota?Rojas, D., Guerrero?Legarreta, I., Cruz?Monterrosa, R.
G. and Hernandez?Sanchez, D., 2018. The effect of stress on haematologic response and physicochemical parameters of muscle meat in rabbits. Journal of animal physiology and animal nutrition, 102: 403-412Rey-Salgueiro, L.
, Martinez-Carballo, E., Fajardo, P., Chapela, M.J., Espiñeira, M. and Simal-Gandara, J.
, 2018. Meat quality in relation to swine well-being after transport and during lairage at the slaughterhouse. Meat science, 142: 38-43.Zhen, S., Liu, Y., Li, X.
, Ge, K., Chen, H., Li, C. and Ren, F., 2013. Effects of lairage time on welfare indicators, energy metabolism and meat quality of pigs in Beijing.
Meat science, 93: 287-291.Chapter 2: Literature ReviewIntroductionOn-Farm handling and pre-slaughter stress factors including transportation, lairage, and slaughter are the significant components of beef production (King et al.,2006) that can affect both animal welfare and meat quality (Michael, 2017). However, differences in stages of pre-slaughter management and between cattle and how humans interact with the animals have significant results on welfare and meat quality (Voisinet et al.
, 1997). Grandin and Shivley (2015) and Grandin (2017) continues to report that an animals previous experience with handling at the farm can affect its reaction to being handled in the future. Crowding, loading and unloading, adverse weather conditions, feed and water deprivation, lairage, length of travel, mixing with animals from other groups, restraint and fatigue are known cattle stressors (Muchenje, 2013; Rey-Salgueiroa et al., 2018). Stress in cattle can be monitored through blood, saliva, urine, and on meat parameters. The most commonly used hormonal variables are those representing changes in the activity of the hypothalamic-pituitary-adrenal (HPA) axis, and cortisol concentration (Muchenje et al., 2009).
However, according to animal welfare, stress is biologically produced by an animal resulting from exposure to a situation or environment (Von Borell, 2001; Mormede et al., 2007; Chulayo et al., 2013).
More than millions of farm animals in South Africa and about 75% are transported by trucks to the abattoir (FAO, 2016). According to Ljungberg et al. (2007), an increase of animal transport is caused by the continuous structural alterations in slaughterhouses in relation to demands by markets. Road transport of farm animals involves the gathering and loading of animals from their original place, captivity on a vehicle, unloading, and lairage at their final destination (Muchunje, 2009; Schwartzkopf-Genswein et al.
, 2012; Chulayo, 2016). In general, it is accepted that the duration of the journey is an aspect of transport that can a?ect the welfare and meat quality of cattle. Therefore, Perez et al. (2002) in a retrospective study, found that behavior, physiological changes related to the stress response and meat quality can yield very useful informationAfter entry in the slaughterhouse, two vital elements factors that may influence the level of stress in cattle, and thus meat quality, are lairage time and handling technique immediately prior to slaughter. According to Liotta et al. (2007), lairage before slaughtering has three imperative functions: it allows animal’s sanitary control ante mortem, it permits recuperation from physical and nervous stress caused by previous transportation, and it ensures a constant supply to the slaughtering chain.
Dokmanovik et al. (2014) reported that lairage time was an important source of variation determining meat quality. At pre-slaughter operations including slaughter stations, animals may be challenged by many stressors. At slaughter stations there are stressors and challenge that trouble homeostasis. First, physiological responses associated with emotional reactivity (heart rate and respiratory frequency) are triggered, followed by behavioral changes.
Then, the activation of the hypothalamic-pituitary-adrenal axis (HPA) and the release of hormones activate the sympatho-adrenal component of the autonomic response increasing cortisol and glucose levels ( HYPERLINK “https://www.sciencedirect.com/science/article/pii/S0309174017312032” l “bb0220” Ferguson and Warner, 2008; HYPERLINK “https://www.
sciencedirect.com/science/article/pii/S0309174017312032″ l “bb0680” Romero et al., 2017).In consequence, if the stress response is strong enough, production and meat quality will be affected ( HYPERLINK “https://www.
sciencedirect.com/science/article/pii/S0309174017312032″ l “bb0290” Grandin ; Shivley, 2015), causing major economic losses to the industry. Overall, the poorer the welfare of the animals is, the greater the economic losses will be ( HYPERLINK “https://www.sciencedirect.com/science/article/pii/S0309174017312032” l “bb0370” Webster, 2001).Stress Concept in Animal Past an expansive decent variety of ideas, welfare alludes chiefly to the subjective psychological state of the animal, as identified with its internal and external condition.
In the early nineteenth century Claude Bernard defined stress as an agitation of the constant state of the internal environment (millieu interieur). The term was differently difined by Canon (1929) using a modified term homeostasis. On the other hand, Selye (1973) expanded its definition, redifining it as nonspecific response of the body to any demand made upn it. Animal welfare Stress has been extensively examined, using a range of strategies and from a diversity of perspectives including the biomedical and animal sciences (Ralph and Tilbrook, 2016). Stress significantly affects animal health and efficiency and open impression of animal stress can impact industry practices.
Understanding stress reactions in livestock may help diminish stressful management procedures and encourage the determination of stress-tolerant animals.Stress measurement2.2.
1 Biochemical and haematological measures Measuring welfare in a commercial slaughter environment can ascertain challenging, because some of the most sensitive physiological criteria for measuring welfare such as heart and respiratory rate (Wigham et al., 2018) are not readily feasible to be assessed in the sensitive environment of the slaughter area. Grandin (1980) reported that the easiest method of measuring severe stress is comparing baseline levels for heart and breathing rate and body temperature with those obtained under stress conditions, but this only worked for physical activities. It was discovered that blood catecholamines and glucocorticoids can also be used for determinations of stress levels, since this hormones are involved in the body’s reaction and adaptation to stress (Kilgour, 1978 and Stermer, 1978). Recently, biochemical parameters have been mostly used to assess stress in animals. One of these parameters is cortisol, and although there are different parameters, cortisol is the most used in animals. Cortisol concentrations have been widely used to measure stress, and hence the a?ective state, in di?erent species including cattle (Stafford and Mellor, 2005; Bristow and Holmes, 2007).
Besides being an indicator of an animal’s response to stress situations, the hormone cortisol modulates the activities of various systems in response to environmental conditions (Cornin et al., 2013).There are some individual factors such as epinephrine, glucose, creatine kinase and lactate that are also related to pre-slaughter stressors and health in cattle. According to Grandin (1980) epinephrine levels are considered as the most complex animal’s response to chronic stress such as fear resulted due to handling methods used in the slaughterhouse or stunning pen. Yet according to Boissy and Neindre (1997), creatine kinase and lactate are muscle specific and hormones, respectively which indicate levels of stress and can indicate leakage from the muscle as a result of suffering in critical conditions such as pre-slaughter stressors; especially during handling (human-interaction) and physical exercise (Grandin. 2000; Grigor et al.,2004).
Moreover, lactate levels have been used as the indicator of physical stress and fatigue (Broom, 2003; Petherick et al., 2009). During these critical conditions creatine kinase regenerate Adenosine Triphosphate (ATP) to maintain energy homeostasis (Warris et al., 1998), because in the process hypothalamic-pituitary adrenal corticoid become influenced by the use of energy and protein imbalances (Bristow and Holmes, 2007). However, production of glucocorticoid as a result of stress can lead to rapid glycolysis, resulting in elevated blood lactate (Probst et al., 2012). Measuring changes in biological factors such as haematological parameters can be used as the proof of stress and may be able to notice unobservable reactions (Wigham et al.
, 2018). Haematology is the study of the numbers and structure of cellular elements of the blood namely; the red cells (erythrocytes), white cells (leucocytes), and the platelets (thrombocytes) and the use of these results for treatment of the blood and diagnosed diseases (Etim et al., 2014).
However, haematological parameters are good indicators of physiological status of animal (Docan et al., 2017). Furthermore, haematological components help in monitoring feed poisonousness especially with feed ingredients that affect the blood () and also the health status of farm animals (Chineke et al., 2006). According to NseAbasi et al.
(2014) heamatological values can work as baseline information for differences in conditions of physiological and health status of farm animals. In addition, the measurement of plasma constituents in a blood sample can provide information about the stress of the animal during the various management operations (Bornez et al., 2009 and Etim et al., 2014). As reported by Isaac et al. (2013) animals with good blood composition are likely to show good performance. Laboratory tests on the blood are vital tools that help detect any deviation from normal in the animal or human body (Webster, 2001).
Table 2.1: Haematological Values for Farm AnimalsCow Sheep Swine Rabbit PigPCV (%) 24 – 48 24 – 45 32 – 50 30 – 50 37 – 48Hgb (g/dl) 8 – 15 8 – 16 10 – 16 10 – 15 11 – 15MCV(fl) 40 – 60 23 – 48 50 – 68 78 – 95 67 – 77MCH(pg) 11 – 17 8 – 12 17 – 23 – -MCHC(g/dl) 30 – 36 31 – 38 30 – 36 27 – 37 30 – 34WBC (?100) 4 – 12 4 – 12 7 – 20 4.5 – 11 5 – 8.9Lymphocytes 40 – 70 40 – 70 40 – 60 40 – 80 39 – 72Monocyte 1 – 6 0 – 6 2 – 10 1 – 4 2 – 6Eosinophils 0 – 4 0 – 10 0 – 10 0 – 4 0 – 5Basophils 0 – 2 0 – 3 0 – 2 1 – 7 0 – 3Adopted from: Etim et al. (2014)2.2.2 Heart Rate Heart rate is one of the ways to measure cardiac activity.
In cattle heart rate can be used to measure stress from physical and emotional origins (Borell et al., 2007). The calves wear the heart rate monitors for an hour to facilitate to become accustom to the equipment (Borell et al, 2007) and get proper HR readings. Stress related factors2.3.1 SoundA study by Lyvers et al.
(2013), shows that cattle have more sensitive hearing than humans, and noises that are lower to humans are quite loud to cattle. Ames (1974) also reported that cattle are known to have low frequency hearing compared to any other mammal. Therefore, it was picked up that this increased sensitivity makes noises such as equipment rattling, metal on metal sounding, and noise created by humans, prime areas for potential hearing stressors (Lyvers et al., 2013). Moreover, to livestock unexpected loud or novel noises can be extremely stressful.
Sheep exposed to exploding firecrackers or noises in a slaughter plant were found to have an increased thyroid hormone level and elevated cortisol (Pearson et al., 1964).Talling et al.
(1996) reported that high-pitched sounds had a greater effect on an animal’s heart rate than low-pitched sounds. Lainer (2000) also found that the stimuli that were most effective for causing a startle response in cattle were sporadic, high-pitched sounds and sudden movements. Domestic animals like pigs are most sensitive to noise at 500 Hz (Lyvers et al.
, 2013; Talling, 1996).This current study suggest that novel sound is an affecting stimulus that primarily triggers the animals’ defense mechanisms. The authors acknowledged that reduction of all forms of noise during handling is needed to help decrease the level of fear in beef cattle.2.
3.2 Novel EnvironmentThe two main central stress-responsive neuroendocrine systems that play a critical role in the activation and regulation of energy changes are the hypothalamic–pituitary–adrenocortical (HPA) and the autonomic nervous system (Ferguson and Warner, 2008; Muchenje, 2008). An autonomic reaction is usually initiated in response to acute stressor such as human handling, and adaptation to new environment (Micera et al., 2007). The HPA axis is revealed by the release of glucocorticoids (cortisol) from the adrenal cortex (Ferguson and Warner, 2008).
It is also worth noting that HPA axis impacts feeding behavior, pancreatic hormone secretion (Muchenje, 2008). Moreover, rapid secretion of glucocorticoids increases the mobilization of energy induced by the catechlomines (Moberg, 2001; Miranda et al., 2012).
When animals are moved to another place they can be exposed to a variety of challenging stressors. These challenges disturb the animal’s homeostasis and as a result of these pre-slaughter stimuli, an animal may experience fear, unusual physical behavior and injuries. Furthermore, the lack of ability to sufficiently resolve some changes may aggravate psychological distress.2.3.4 FightingWhen animals are handled they are subjected to inevitable husbandry practices. Grandin (1990) reported that the strength of an animal’s response to some stressors depends on many factors, including degree of tameness and adaptation to the climate.
The basic hormonal mechanisms underlying stress include the secretion of epinephrine, which activate the animal for the classic “fight or flight” response, and the secretion of glucocorticoids (Grandin, 1990; Moberg, 2001). According to Carreras et al. (2017) glucocorticoids are released later and help to maintain energy supplies and to resist the stress. Furthermore, exposure to humans is one of the potentially most frightening events that many farm animals experience in their life.
As shown by glucocorticoids and corticosteroids it has been reported that human interaction with animals does not only affect animal productivity but also their physiology (Carreras et al., 2017). At all times, before slaughter, cattle may experience stress from a range of handling practices and some preslaughter stresses, such as fighting, fasting, loading and transport, mixing, and human interactions (Faucitano, 2018). In cattle fighting has been reported as the key cause of dark cutters (Deesinga, 1997) and the major cause of death losses (Grandin, 1970).
When different animals are mixed together they fight in order to determine the new group leader. Having different breeds in the same group, most especial between steers is a major animal welfare issue. When mixing strange cattle, aggression occurs as they create a new social hierarchy. The cattle which had dark cuter were often either the lightest or the heaviest animals in the pen (Grandin, 1970).However, this indicates that social order is related to stress since the heavier animals are usually dominant. The higher occurrence of dark cutters in lighter weight cattle is possibly due to aggressive small animals which continued to fight with larger animals.
Therefore the body size of an animal, age, size and shape of the pens may also be considered as a factor (Grandin, 1980). Von Borell (2001) reported that, animals were in a vast pen and were watched running at each other and butting at high rates of speed. Furthermore, in many occasions where the dark cutters were the heavier animals, the movement level in the pen was high and dominant animals exhausted a great deal of vitality (Grant and Albright, 2001).However,in the gathering where the dark cutters were the lighter weight dairy cattle, perceptions showed that the docile lighter steers experienced issues staying away from the dominant animals in the little swarmed pen (Grandin, 1980; Broom, 2003). More research is expected to decide the ideal space necessity.2.3.
5 OvercrowdingOvercrowding is an overpopulation of animals in one area of an environment. The high density of animals on the farm results to stress due to the fights, and bullying that takes place in the crowd, however, overcrowding reduce an individual’s ability to avoid aggression from others (Beery and Kaufer, 2015). Furthermore, crowding reduces access to valued resources such as food or resting spaces, increasing competition between animals (Beery and Kaufer, 2015; Kathryn and Gregory, 2015). Therefore, for cattle, increasing animal density per unit feeding space increases agonistic behavior during feeding (Proudfoot et al., 2009).2.3.
6 Restraint stressRestraint is a method used when handling cattle and many other species. There are many situations where farm animals have to be handled, restrained and or transported (Grandin and Shivley, 2015). Chen (2016) reported restraint as a stressor that causes behavioral changes. Restraint stress has negative effects on productivity of cattle and on the secretion of cortisol by adrenal axis (Chen, 2016). There are apparently contradictory studies based on the effects of restraining stress on cortisol levels.
For example Szenci et al. (2011) demonstrated that in cattle, restraint increases Adrenocorticotropic hormone (ACTH) response, elevates serum cortisol concentrations and also alert the immune response. Similar findings were demonstrated by Grandin and Shivley (2015) that when some animals are restrained, they react with greater nervous behaviour and higher cortisol concentrations. Although, Chen et al. (2015) showed that placing a mask over the face of an animal during restraint significantly reduces cortisol concentrations. Restraint for Veterinary procedures may be very stressful for one animal and for another animal it may be relatively low stress (Chen et al., 2015; Grandin and Shivley, 2015).
Source: FAO2.3.7 TemperatureDuring periods of critical weather, optimum conditions for animal comfort and performance are compromised (Mader and Griffin, 2015).
The combinations of humidity, precipitation and heat radiation are the effective ambient temperatures that lead to increased levels of stress. Hot weather can strongly affect animal bioenergetics (Hahn, 1999), with adverse effects on the performance and well-being of beef cattle (Piao et al., 2015). All of that increases the levels of stress in animals resulting in reduced feed intake and growth production. Although Curtis et al. (2017) reported that environmental stressors may not have an immediate effect on production variables, like feed intake, but environmental stressors lead to stress and when animals are stressed there is a rapid release of chemicals ( cortisol, creatine phosphokinase and lactate).
When animals release more chemical compounds they lose more energy. Hence, use of alternative supplementation programs need to be considered for livestock challenges by environmental conditions and in the case of heat provision of shades on the farm is needed.Factors affecting haematological factors of farm animalsThere are genetic and non-genetic factors that have been observed which affect haematological parameters of farm animals (McGlorie, 1999 and Chineke et al., 2006). These factors includes physiological, environmental conditions, starvation, and drug administration (Adenkola and Ayo, 2010; Gupta et al., 2014; Etim et al.
, 2015). On the other hand, Onasanya et al. (2015) showed that factors such as breed, sex, age, health of the animal, and degree of activity of an animal also affect the blood constituents of animals. Earley and O’Riordan (2006) agreed that the most factors that impact the haematological factors are sex, age, management, diseases and stress factors. Micera et al. (2007) reported that besides the physiological and the environmental factors that might affect the blood constituent transport, exercise, methods of breeding and housing has also been recognized.Transportation of Farm AnimalThe meat production network is an imperative aspect in the farming and meat industry that incorporates different critical stage such as transportation, loading and slaughter of the animals (?obanovi? et al.
, 2017). Transportation of animals may differ depending on the source of the animals and in South Africa, steers that are sold at closeout markets can either be transported straight to the slaughterhouse for coordinate slaughter or can be taken to ranches or feedlots where they are held before discharged for slaughter (Vimiso and Muchenje, 2013). And transportation is also known to have a huge impact on animal welfare (Frimpong et al., 2014).As it is widely known and scientifically proven that pre-slaughter stress commences during occasional handling of the animal as well as meat products. Nevertheless, Wigham et al. (2018) reported that reduction of muscle glycogen due to pre-slaughter has found to have a strong effect on several key meat quality attributes such as ultimate pH, tenderness, color, water-holding capacity and sensory indicators ( HYPERLINK “https://www.
sciencedirect.com/science/article/pii/S0309174017312032″ l “bb0530” Mounier et al., 2006). Chulayo et al. (2016) showed that during transportation period, animals develop means to cope with the environmental changes that end-up affecting even their physiology.
Cattle react to stressful factors with increased concentrations of lactate, creatine kinase and catecholamines activity (Terlouw, 2005; Chuyalo et al., 2016). In addition, pre-slaughter stress factors such as ambient temperature, transportation, poor occasional handling, distance and lairage duration cause animal to release hormones and enzymes (creatine kinase, cortisol and catecholamine) into the bloodstream leading to a series of secondary processes that involve energy metabolism (Beerda et al., 1997; Ferguson and Werner, 2008; Chulayo and Muchenje,2012). A study by Frimpong et al. (2014) showed that amount of stress hormone (cortisol) decreased with an increase in transport time while lactate and creatine kinase increases.
Furthermore, it was also reported that long transportation hours in poor condition transportation vehicles may be unfavourable to animal welfare (Njisane, 2016). Transportation affects the susceptibility of animals to infection. This is because exposure to stressors during transportation is related with a decrease in the function of the immune system of animals (Hulbert et al., 2011; Adenkola and Ayo, 2010) through increasing susceptibility to respiratory diseases in cattle (Grandin, 2017), which may result to release of stress hormones (Llonch et al., 2015).
Moreover, transportation at high stocking density was described to elevate plasma cortisol (Kadim et al., 2009). McKenna (2017) reported that the increase of stress hormones pick in animals may be due to handling and the novel environment (when arrived at the abattoir) during the sampling. Nevertheless, assuring great transportation does not just guarantee good animal welfare and meat quality; it is likewise of economic importance (Anderson et al., 1999; Bornez et al., 2009; Costa et al.
, 2015).Transportation is an intimidating event in the life of livestock animals; it involves a series of handling and limitation situations which can lead to distress, injury or even death of the animal (Tarrant and Grandin, 2000). During transportation, animals have to cope with several multifactorial stressors (Miranda et al.
, 2010). The transport and handling of animals are vital factors in the meat production chains. According to Ljunberg et al. (2007), transport and handling of slaughter animals are one out of many events that cause an increase of cortisol stimulation and unfavorable conditions on beef steers, increasing the chances of spread of disease and also reducing the meat quality (Chulayo et al., 2013). In many studies, it was discovered that during transportation animal uses more energy (Grandin, 2010; Miranda et al.
, 2014). Chulayo et al. (2016) reported that the use of energy during this critical production stage (transportation) is influenced by hypothalamic-pituitary-adrenal axis leading to a secretion of cortisol (Tarrant and Grandin, 2000; Chulayo et al., 2016). A common observation following transportation is a temporary increase in blood cortisol concentration, many factors may add to this stress response, including human-animal interactions immediately prior to and after transportation (Chen et al.
, 2015). The difficult combination of stressors associated with transportation proceedings have been related with a wide kind of physiological responses (Chen et al., 2015) including altered immune function, behavioral response (Njisane et al., 2016) and changes muscle physiology (Chulayo et al., 2016). Table 2. 1: Commonly used physiological indicators of stress during transportationStressor Physiological variableMeasured in blood or other body fluids Food depreciation ? FFA, ? ß-OHB, ?glucose, ? ureaDehydration ? Osmolality, ? total protein, ? albumin, ? PCVPhysical exertion Motion sickness ? CK, ? lactate? Vasopressin Fear or Arousal Other measuresFear/arousal and physical ? Cortisol, ? PCV? Heart rate, heart rate variability ?, ? respiration rateHypothermia/ hyperthermia Body temperature, skin temperatureModified from: Broom (2003)The transported animals are often exposed to a variety of physical and psychological stimuli, many of which are novel and some are aversive (Rajion et al.
, 2001). Physical and psychic exertions occurring during road transportation of food animals disrupt their homeostasis and metabolism and consequently, increase activity of enzymes and hormones (Ayo and Oladele, 1996; Mstl and Palme, 2002; Adenkola et al., 2009b). Also Bauer et al. (2001) and Saunders and Straub (2002) demonstrated that many stressors induced the activation of the autonomic nervous system as earlier reported by (Spencer et al.
, 1984) with the pituitary-adreno-cortical system also playing an important role in the modulation of stress response (Armario, 2006). Plyaschenko and Sidorov (1987) reported an increase in adrenal cortical activity in pigs transported by road by demonstrating an increase in blood level of 11hydroxycorticosteroids (11-oxy) 30 min after the onset of transport. In a recent study, Odore et al. (2004) observed the activation of the hypothalamic-pituitary-adrenal axis and the catecholaminergic system in long-term transporttation of calves by road.
They observed a rise in blood hormone levels, lymphocyte, glucocorticoid and adrenergic receptor, cortisol and catecholamine concentrations. The increased values, however, returned to normal at 24 h and one week after arrival. Adenkola et al. (2009b) demonstrated that road transportation induced leucocytosis, neutrophilia, lymphocytosis, eosinophilia in control pigs that were not supplemented with ascorbic acid while total protein, alkaline phosphatase, aspatate amino transferase and neutrophil lymphocyte ratio was found to decrease significantly in pigs administered with ascorbic acid as compared to control group after 4-h of road transportation during harmattan season in Northern Guinea Savanna zone of Nigeria in pigsKnowles et al. (1999) researched the impacts of road transportation on cattle for up to 31 h and found that plasma creatine kinase expanded maximally after 26 h. Elevated amounts of cortisol were found in those animals that rests amid the adventure.
Plasma protein and egg whites ascended with expanding venture, plasma glucose expanded yet diminished after 21 h of transport and plasma urea expanded with expanding venture time. Stuffed cell volume (PCV) expanded amid the initial 12 h of recuperation, yet diminished after 72 h. Galipalli et al. (2004) observed that Tasco seaweed (Ascophyllum nodosum) supplementation expanded cell reinforcement movement and invulnerable reactions following road transportation in goats. They demonstrated that plasma cortisol and glucose expanded because of transportation, however diminished essentially in the wake of holding and that plasma creatine kinase exercises and neutrophil check expanded. In spite of the fact that lymphocyle tally diminished fundamentally amid the transportation, eosinophil and monocyte checks were not impacted by transportation stress.How to measure well-being of animals in abattoir locationsIn livestock stress is now regarded as a response that take place ineluctably when animals are introduced to adverse environmental conditions, which could be the cause of many unfavorable consequences, starting from discomfort to death (Alcalde et al., 2017). At slaughter plant or house animals are handled and controlled, that can cause stress which affect both animal welfare and meat quality. However, there is substantial awareness and published information aboutloin meat. As a result in some countries (including United Kingdom) pressure has been applied on slaughterhouse to significantly restrict the use of electric rods on livestock during lairage (Wigham et al., 2018). Therefore, establishments of reliable and valid repeatable welfare assessment procedures could allow the e?ects of welfare improvement measures to be quanti?ed. Furthermore, measuring welfare in a commercial slaughterhouse can demonstrate challenging, because some of the most sensitive physiological criteria for quantifying welfare such as heart and respiratory rate (Grandin, 1980; Miranda, 2013) are not readily reasonable to be assessed in the environment of the slaughter area. There are significant animal based welfare measures that should be assessed at slaughter such as animal scoring systems, animal behavior, weighted procedures and or animal based standards, and biochemical and haematological measures (Grandin, 2010; Wigham et al., 2018 ). Based on four main principles which are good feeding, maintained housing, good health and appropriate behavior, procedures have been designed to assess welfare in di?erent livestock species in a range of environments including abattoirs (Miranda-de la Lama et al., 2012). Slaughter plants have been undergoing drastic alterations in current years due to the need to increase competence and add in new technologies for the improvement of the infrastructure, animal well-being, and the quality of the product. Grandin (2010) reported that the use of animal based scoring systems has resulted in great improvements in pre-slaughter stressors when it was used to audit animal welfare at slaughter plants. Five freedoms were established in 1965 by Animal Welfare Council (Brambell, 1965) and are considered as one of the first animal scoring system and most widely recognized welfare model (Grandin 1997; Phillips, 2008; Wigham et al., 2018). However, it has had an important influence on animal welfare (OIE, 2006). The five freedom states as follow (Webster, 2001; Grandin; Wigham et al., 2018)Freedom from thirst, hunger and malnutrition – by ready access to water and a diet to maintain health and vigourFreedom from discomfort – by providing an appropriate environment including shelter and a contented resting area Freedom from pain, injury and disease – by prevention or rapid diagnosis and treatmentFreedom to express normal behaviour – by providing sufficient space, proper facilities and company of the animal’s own kindFreedom from fear and distress Freedom from fear and distress – by ensuring conditions and treatment, which avoid mental su?eringEffect of laraige on meat qualityAnimals can be exposed to a series of stressors during preslaughter period. Many factors related to preslaughter handling affect meat quality such as transportation and lairage duration (Li et al., 2018 ). Despite the fact that lairageing is to empower animals to rest and to recover from transportation stress, it can be a notable source of meat quality issues. Animals may experience the ill effects of various level of bruising and injury because of attacking or overcrowding (Miranda et al., 2014). Lairage can also act as repositories of disease by pathogenic bacteria and there is prove that longer holding times increase the risk of carcass contamination (Adzitey, 2011). However, poor handling of animals in the lairage, for example, the utilization of electrical rods, abstracting the movement of animals through race, beating and firm grasp of the coat with the hand, contact of animals to microbial pollutions will antagonistically have impact on the meat quality (Jarvis et al., 1996). R reported that conditions in the lairage therefore need to be favourable in order to prevent diseaeses and so as to avoid further or more stress after transportation. However, Rey-Salgueiroa (2018) reported that animals transported for 15 min showed signi?cant lower pH in muscle and also a tendency to show most acid muscle compared with animals transported for 3 h. Furthermore, a study by Perez et al. (2002) showed that pigs subjected to short journeys have higher tendency to produce PSE (Pale soft exudative) meat than pigs transported longer. A study by Teke et al. (2013) showed that, cattle which had shorter lairage duration were more stressed at slaughter, and their carcass had higher pH than those of long lairage duration. These findings agree with Liste et al. (2011) who found that pre-slaughter stress including lairage duration had a significance effect on meat quality, because the meat of those who were short lairaged had higher pH values. In contrast to these findings Dokmanovik et al. (2014) and Liota et al. (2007) reported that animals undergoing long lairage duration had significantly higher pH values compared to short lairage duration. The meat of animals that rested for a longer time showed grater redness compared to those rested for a short time (Gallo et al., 2003), these results are in accordance with the observations of Rey-Salgueir et al. (2018) about the negative effect of lairage period on meat quality. However, a study by Ekiz et al. (2012) and Li et al. (2018) found that, both the rate of pH decline and the ultimate pH of post mortem muscle are vital for meat quality development, which influence meat tenderness, water holding capacity and colour (Chulayo, 2016)Table 2.2: effect of pre-slaughter handling on carcass and meat quality of beef cattleSpecies Pre-slaughter handling Carcass and meat quality effects ReferencesCattle OverloadingHeld overnight in noisy yardsTransportationTransportationMore than 24 hours of fastingCattle transported for 2h Prolonged stressPoor handlingOverloadingLonger journeyLonger lairage timeHigh stocking densityLong distance road Injury and damageBruiseSpread of pathogens on the hidesShedding of Salmonella sppCarcass yield was reduced by 1.7% 1.5 % loss in carcass yieldMeat appear darker and heat ring is formed4.1 % of dark cutting beef Animal injury and damage to carcassAssociated with a significantly larger live weight lossDecreased muscle luminosity and increased DFDIncreased bruising and reduce dressed carcass weightLost weight Tarrant (1990)Eldridge (1988)Avery et al. (2002)Barham et al.(2002)Price (1981)Smith et al. (1982)Buyck et al. (1985)Brown and Bevis (1988)Tarrant (1990)Gallo et al. (2003)Gallo et al. (2003)Tarrant and Grandin (2000)Marahrens et al. (2002)Influence of stress on biochemical compoundsThe way animals respond to pre-slaughter handling and distress are aspects that disturb glycogen reduction in cattle and quality of meat aspects that include the color, pH, cooking loss and juiciness of the meat (Muchenje et al., 2009).Hence it must be taken into considerations that pre-slaughter stress may result in biochemical and physical changes of an animal. In addition, these biological and physiological changes have a bad impact on animals leading to distress (Chulayo et al., 2016 ).On the other hand, before slaughter, and during slaughter procedures, animals may experience stress caused by many factors including handling, restraint, weather conditions, fatigue, hunger and thirst (Grandin, 1990; Muchenje et al., 2009). Moreover, the pre-slaughter stressors may result from new adaptations on the farm, due to feeding withdrawal when animals are being transported for many hours (Ferguson and Warner, 2008; Muchenje et al., 2009) overcrowding of animals, different breeds and gender of animals, long time standing on the transport (Chulayo et al., 2016), no accessibility to grass, and noise and off-loading (Grandin, 1990). During these critical situations the use of energy and protein imbalance is influenced by the hypothalamic- pituitary adrenal corticoid (HPA) axis (Bristow and Holmes, 2007; Chulayo et al., 2017; Romero et al., 2017). However, when an animal is stressed there is a rapid release of stress-related hormones such as cortisol, creatine phosphokinase and lactate. And during stressful situations, cortisol is secreted at higher concentrations.Animal stress related compounds (Cortisol and Creatine phophoskinase)Stress is a concept that has been discussed since the 1950s (Proudfoot et al., 2015) but is often difficult to interpret. Moreover, to many research, stress is described as a response ascending from real or apparent environmental stressors that can be assessed threatening depending on the adaptive coping resources to an individual (Kathryn and Gregory, 2015). According to Kathryn and Gregory (2015) stressors initiate two main biological pathways; the sympathoadrenal medullary pathway and the hypothalamic-pituitary-adrenal pathway. Between these two biological pathways, one of the most commonly measured immediate physiological responses to stress is activation of the hypothalamic-pituitary-adrenal axis (Jama et al., 2014; Beery and Kaufer, 2015; Chulayo et al., 2016).Hormonal variables have been widely used to assess the levels of distress experienced by cattle exposed to a range of stressors (Moya et al., 2013), such as climate and common management procedures (handling) such as transport, castration, dehorning or branding (Tarrant et al., 1992; Petrei et al., 1996; Gonzalez et al., 2009). However, unlike with other species, measures obtained from restrained or handled beef cattle are confounded by the effects of animal handling, which itself can induce stress (Moya et al., 2013; Moberg and Mench, 2000). The most commonly used hormonal variables are those representing changes in the activity of the hypothalamic-pituitary-adrenal (HPA) axis, and cortisol concentration (Schwartzkopf-Genswein et al., 2012).Cortisol is a glucocorticoid hormone produced by the adrenal cortex that is released routinely and has a number of constructive functions (Warriss et al., 1998; Perez et al. 2004; Bristow and Holmes, 2007; Choir et al., 2012), it also a time-dependent measure. The hormone cortisol is not only an indicator of an animal’s response to stressful situations but can also control the activities of several systems in response to environmental changes (Muchenje et al., 2009; Cornin et al., 2013). However, when an animal is stressed there is a rapid release of stress-related hormones such as cortisol, creatine phosphokinase and lactate. Several authors reported that creatine kinase and lactate are muscle specific and hormones, respectively which indicate levels of stress and can indicate leakage from the muscle as a result of suffering in critical conditions such as suffering; especially during physical exercise (Boissy and Neindre, 1997; Grandin, 2000; Grigor et al., 2004; Jama et al., 2014). Stafford and Mellor (2005) agreed that the release of cortisol is often increased in response to stress. However, the perception of stress directly stimulates the hypothalamic-pituitary-adrenal axis (Lay et al., 1996) which results in the release of high cortisol (Brown and Vosloo, 2017). Conversely, there are several factors that cause changes in cortisol concentration at the farm including flies, castration, and adaptation.Availability of flies most especially Stomoxy calcitrans causes irritation, muscle flicking, muscle twitching tail switching and led stamping (Lehane et al., 1997) with the intentions of chasing flies away. Additionally, it has been scientifically proven that Stomoxy calcitrans increase levels of cortisol and discomfort in cattle (Vitela-Mandoza et al., 2016) because, in a stressful situation, corticotrophin releases stress hormone. The other significant factor that causes changes in the concentration of cortisol is castration. Canozzi et al. (2017) stated that castration is one of the common management procedure used by farmers that also increases cortisol levels and changes animal behavior. Specific methods of castration and pain relief can be used to prevent and minimize the adversarial effects on beef cattle. Recently, it has been presented to be highly significant to be acquainted with the causes of cortisol changes during handling, in order to find approaches to improve stress-related factors in animals and resolve these issues. Moreover, poor operational techniques, unsociable settings and or sampling method used in pre-slaughter often results in high secretion of creatine phosphokinase (Beerda et al., 1997; Jama et al., 2014). In agreement, Poleti et al. (2015) reported that Creatine kinase is an index of physical stresses which response rather rapidly. The effects of creatine kinase are to regenerate Adenosine Triphosphate (ATP) to maintain energy homeostasis (Warris et al., 1998). According to Jama et al. (2014), Adenosine Triphosphate is the primary energy source in living organisms for energy-demanding processes in cardiac and skeletal muscles, brain, retina and primitive-type spermatozoa (Grandin,2013 Chulayo, and Muchenje, 2013; Jama et al., 2014). More use of ATP gives rise to a specifical commotion of the enzyme called creatine kinase (Chulayo and Muchenje, 2013). The production of glucocorticoid and catecholamine as a result of stress can lead to rapid glycolysis and increased lactate production, resulting in elevated blood lactate (Probst et al .,2012). According to Probst et al. (2012), lactate is frequently used in addition to cortisol to measure stress-related reactions as these indicators rapidly respond to stressors. Lactate level has been used as an indicator of physical stress and fatigue occurring in livestock (Petherick et al., 2009; Broom, 2003). Hoffman and Laubscher (2011) reported that the high secretion of cortisol, creatine phosphokinase and or stress related indicators result in decreasing meat quality. Similarly, Carreras et al. (2017) reported that human handling does not only influence animal welfare, as it increases corticosteroid rates (Aghwan et al., 2016) but also productivity and meat quality. Moreover, Brown and Volsoo (2017) found adverse effects affecting meat quality including environmental stressors. It has been severally proven that perception of stress from either an internal or external stimulus by livestock results in severe changes in its physiology (Vena et al., 2010; Burdick et al 2011; Aghwan et al., 2016; Baird et al., 2016; Jama et al., 2016;Carreras et al., 2017; Brown and Vosloo, 2017). Effects of temperature on the secretion of stress hormone (cortisol)2.9.1 Thermal stressors In a hot climate, high ambient temperatures are found to be the environmental stressing factors that may cause strain on livestock animals including beef cattle. The survival that is well-being and performance of beef steers are strongly affected by climatic conditions (Mader, 2013). Livestock surviving in a natural environment experience different temperature all day long. Animals experience thermal stressors, including heat and cold stress. Although heat stress has been mostly studied in dairy cows has been found that it also affects the secretion of stress-related indicators or stress hormones in beef cattle (Grandin, 1980). Management system and skills are needed that include information and strategies on how cattle respond to climatic conditions (Mader, 2003). 2.9.2 Heat stressIn most environments, cattle are raised on pasture. According to Moons et al. (2014) pasture-based farms are preferable for the welfare of animals. Even though animals reared on pasture in farms are more exposed to climatic conditions including heat and cold climate. In addition, cattle feeding on pasture experience more heat stress than cattle that have access to graze on shades. However, providing shade on farms will protect cattle against heat stress. Moons et al. (2014) and Grandin (2016) reported that cattle living and grazing under shade and or shelter release lower concentrations of corticosteroids due to lower stress compared to those exposed to hot climate.The effect of heat stress on those animals that handling has been more accustomed is preserved compared to those that are less handled (Young, 1993). However, there is been little information and discussion on literature regarding the relationship between heat and secretion of stress-related hormones mostly in beef cattle raised on natural pastures. According to Silanikove (2000), welfare is a characteristic of an animal, which differs from poor to good and can be distinct by separate parameters such as changes in hormone levels and body temperature.Heat stress has resulted in major losses in animal production, including dairy cattle, beef cattle, swine and poultry (Nejad et al., 2016). In many studies, it was found that behavior, diseases incidence, body temperature and metabolic processes are adversely affected by heat stress (Romero and Butler, 2007; Jama et al., 2016; Nejad et al., 2016).The effect of heat stress in beef cattle has not been typically measured but heat stress has been reported by (Mader, 2013) to cause changes in concentrations of hormones and enzymes including cortisol, creatine phosphokinase and lactate. Similar findings were reported by Jama et al. (2015) on pigs that high secretion of stress-related indicators which include cortisol and creatine kinase is increased at high temperatures, and that can impede heat. Gonzalez et al. (2007) agreed that heat stress is a consequence and a stress factor that induce secretion of cortisol more especially during transportation with high stocking densities and poor ventilation.2.10 SummaryPre-slaughter handling and lairage may bring about increasing plasma concentration of the stress hormones (adrenalin, noradrenalin, and cortisol) and certain biochemical (glucose, creatine kinase, lactate dehydrogenase) and hematological factors (Knowles et al., 2014) which give data about stress levels of livestock. On the other hand, lairage of livestock is a commercial practice after transportation and the length of lairage period changes relying upon the schedules at the abattoir (Schwartzkopf et al., 2012). However, past investigations in regards to the connection between lairage length and stress reactions have exhibited variable outcomes (Liu et al., 2012). Lairage period can be an added stress factor because of a novel environment meanwhile by giving sufficient resting period to livestock in lairage unit before slaughter can be a guide for the rehydration and recuperation from stress reactions. Moreover, stress related with pre-slaughter handling influences muscle glycogen digestion and consequently increases the danger of diminishing meat quality (Dokmanovic et al., 2014). Extreme meat pH has been utilized broadly as an indicator of fresh meat quality as it might influence the meat colour, water holding capacity and tenderness (Sommavilla et al., 2017). In addition, the impact of lairage period on meat sensory characteristics was less talked about. Concentrates that joined physiological stress reactions with item quality can expand the achievement of setting up handling practices to enhance animal welfare and meat quality Lessening livestock stress during handling will create advantages of increasing productivity and improving meat quality. As seen from current studies that there are plenty of reasons and factors that interact and affect meat quality, and the consumer opinion of meat eating quality. The factors range from the way the animals are raised, transportation to the abattoir, post-slaughter handling and the keeping of meat in butcheries, shops and home. Different factors at every stage should be measured to improve quality of meat.ReferenceAnderson, B.H., Watson, D.L. and Colditz, I.G., 1999. The effect of dexamethasone on some immunological parameters in cattle. Veterinary research communications, 23: 399-413.Adenkola, A.Y. and Ayo, J.O., 2010. Physiological and behavioural responses of livestock to road transportation stress: A review. African Journal of Biotechnology, 9: 4845-4856. Broom, D.M., 2003. Transport stress in cattle and sheep with details of physiological, ethological and other indicators. Deutsche Tierarztliche Wochenschrift, 110(3), pp.83-88.Blum, J.W., Kunz, P., Leuenberger, H., Gautschi, K. and Keller, M., 1983. Thyroid hormones, blood plasma metabolites and haematological parameters in relationship to milk yield in dairy cows. Animal Science, 36: 93-104.Bórnez, R., Linares, M.B. and Vergara, H., 2009. Haematological, hormonal and biochemical blood parameters in lamb: effect of age and blood sampling time. Livestock Science, 121: 200-206.Chineke, C.A., Ologun, A.G. and Ikeobi, C.O.N., 2006. Haematological parameters in rabbit breeds and crosses in humid tropics. Pak J Biol Sci, 9: 2102-2106.?obanovi?, N., Vasilev, D., Dimitrijevi?, M., Teodorovi?, V., Parunovi?, N., Beti?, N. and Karabasil, N., 2017, September. The interactive effects of transportation and lairage time on welfare indicators, carcass and meat quality traits in slaughter pigs. In IOP Conference Series: Earth and Environmental Science, 85: 012049.Dantzer, R. and Mormède, P., 1983. Stress in farm animals: a need for reevaluation. Journal of Animal Science, 57: 6-18.Dalla Costa, F.A., Devillers, N., da Costa, M.P. and Faucitano, L., 2015. Influence of preslaughter feed withdrawal time applied at the farm or at abattoir on behaviour and meat quality in pigs. In 61st International Congress of Meat Science and Technology, Clermont-Ferrand, France.Docan, A., Grecu, I., Cre?u, M., Antache, A. and Dediu, L., 2017. Haematological And Serum Biochemical Changes Associated With Bacteriological Infection In Acipenser Gueldenstaedt II Reared In Intensive Condition. Lucr?ri ?tiin?ifice-Universitatea de ?tiin?e Agricole ?i Medicin? Veterinar?, Seria Zootehnie, 67: 104-109.Dikeman, M.E., 2017. Understanding the effects of handling, transportation, lairage and slaughter on cattle welfare and beef quality Michael S. Cockram, University of Prince Edward Island, Canada. In Ensuring safety and quality in the production of beef Volume 2: 157-202. Burleigh Dodds Science Publishing.Ekiz, B., Ekiz, E.E., Kocak, O., Yalcintan, H. and Yilmaz, A., 2012. Effect of pre-slaughter management regarding transportation and time in lairage on certain stress parameters, carcass and meat quality characteristics in Kivircik lambs. Meat Science, 90: 967-976.Etim, N.N., Williams, M.E., Akpabio, U. and Offiong, E.E., 2014. Haematological parameters and factors affecting their values. Agricultural Science, 2: 37-47.Galipalli, S., Gadiyaram, K.M., Kouakou, B., Pringle, T.D. and Kannan, G., 2004. Oxidative stability of chevon as influenced by dietary Tasco supplementation in Boer goat bucks. South African Journal of Animal Science, 34.Grant, R.J. and Albright, J.L., 2001. Effect of animal grouping on feeding behavior and intake of dairy cattle. Journal of dairy science, 84: 156-163.Gupta, S., Earley, B. and Crowe, M.A., 2007. Effect of 12-hour road transportation on physiological, immunological and haematological parameters in bulls housed at different space allowances. The Veterinary Journal, 173: 605-616.Grandin, T., 2010. Auditing animal welfare at slaughter plants. Meat Science, 86: 56-65.Grandin, T., 2017. On-farm conditions that compromise animal welfare that can be monitored at the slaughter plant. Meat science, 132: 52-58.Jarvis, A.M., Harrington, D.W.J. and Cockram, M.S., 1996. Effect of source and lairage on some behavioural and biochemical measurements of feed restriction and dehydration in cattle at a slaughterhouse. Applied Animal Behaviour Science, 50: 83-94.Knowles, T.G., Warriss, P.D. and Vogel, K., 2014. 21 Stress Physiology of Animals During Transport. Livestock handling and transport: Theories and applications, 399.Knowles, T. and Warriss, P., 2007. 19 Stress Physiology of Animals During Transport. Livestock handling and transport, 312.Knowles, T.A., Southern, L.L., Bidner, T.D., Kerr, B.J. and Friesen, K.G., 1998. Effect of dietary fiber or fat in low-crude protein, crystalline amino acid-supplemented diets for finishing pigs. Journal of animal science, 76: 2818-2832.Liu, H.W., Zhong, R.Z., Zhou, D.W., Sun, H.X. and Zhao, C.S., 2012. Effects of lairage time after road transport on some blood indicators of welfare and meat quality traits in sheep. Journal of animal physiology and animal nutrition, 96(6), pp.1127-1135.Li, X., Xia, A.Q., Chen, L.J., DU, M.T., Chen, L., Kang, N. and Zhang, D.Q., 2018. Effects of lairage after transport on post mortem muscle glycolysis, protein phosphorylation and lamb meat quality.Losada-Espinosa, N., Villarroel, M., María, G.A. and Miranda-de la Lama, G.C., 2017. Pre-slaughter cattle welfare indicators for use in commercial abattoirs with voluntary monitoring systems: A systematic review. Meat science.Llonch, P., King, E.M., Clarke, K.A., Downes, J.M. and Green, L.E., 2015. A systematic review of animal based indicators of sheep welfare on farm, at market and during transport, and qualitative appraisal of their validity and feasibility for use in UK abattoirs. The Veterinary Journal, 206: 289-297.McKenna, C., 2017. Industry Standards on Farm Animal Welfare. The Meat Crisis: Developing more Sustainable and Ethical Production and Consumption.Miranda-de la Lama, G.C., Leyva, I.G., Barreras-Serrano, A., Pérez-Linares, C., Sánchez-López, E., María, G.A. and Figueroa-Saavedra, F., 2012. Assessment of cattle welfare at a commercial slaughter plant in the northwest of Mexico. Tropical animal health and production, 44: 497-504.Pérez, M.P., Palacio, J., Santolaria, M.P., del Carmen Aceña, M., Chacón, G., Verde, M.T., Calvo, J.H., Zaragoza, M.P., Gascón, M. and García-Belenguer, S., 2002. Influence of lairage time on some welfare and meat quality parameters in pigs. Veterinary Research, 33: 239-250.Phillips, C., 2008. Cattle behaviour and welfare. John Wiley & SonsRalph, C.R. and Tilbrook, A.J., 2016. INVITED REVIEW: The usefulness of measuring glucocorticoids for assessing animal welfare. Journal of animal science, 94: 457-470.Schwartzkopf-Genswein, K.S., Faucitano, L., Dadgar, S., Shand, P., González, L.A. and Crowe, T.G., 2012. Road transport of cattle, swine and poultry in North America and its impact on animal welfare, carcass and meat quality: A review. Meat science, 92: 227-243.Schaefer, A.L., Jones, S.D.M. and Stanley, R.W., 1997. The use of electrolyte solutions for reducing transport stress. Journal of animal science, 75: 258-265.Sommavilla, R., Faucitano, L., Gonyou, H., Seddon, Y., Bergeron, R., Widowski, T., Crowe, T., Connor, L., Scheeren, M.B., Goumon, S. and Brown, J., 2017. Season, transport duration and trailer compartment effects on blood stress indicators in pigs: relationship to environmental, behavioral and other physiological factors, and pork quality traits. Animals, 7: 8.Selye, H., 1973. The Evolution of the Stress Concept: The originator of the concept traces its development from the discovery in 1936 of the alarm reaction to modern therapeutic applications of syntoxic and catatoxic hormones. American scientist, 61: 692-699.Tadich, N., Gallo, C., Bustamante, H., Schwerter, M. and Van Schaik, G., 2005. Effects of transport and lairage time on some blood constituents of Friesian-cross steers in Chile. Livestock Production Science, 93: 223-233.Vimiso, P. and Muchenje, V., 2013. A survey on the effect of transport method on bruises, pH and colour of meat from cattle slaughtered at a South African commercial abattoir. South African Journal of Animal Science, 43: 105-111.Von Borell, E.H., 2001. The biology of stress and its application to livestock housing and transportation assessment 1. Journal of Animal Science, 79: 260-267Wigham, E.E., Butterworth, A. and Wotton, S., 2018. Assessing cattle welfare at slaughter–Why is it important and what challenges are faced?. Meat Science.Chapter 3: Investigating the response of cortisol, glucose and creatine kinase on meat quality parameters of slaughtered Boran and Nguni breeds after a 16 and 24 hour lairage durationAbstract IntroductionA study by Ekiz et al. (2012) defined transport and slaughter of livestock as the last phases of an animal production system meant to produce high-quality meat. Among the most important factors in welfare and the meat production system transportation and lairage are regarded as the most significant components. However, Grandin (1998) reported that animal welfare can be linked to carcass and meat quality. Transportation is an unavoidable farming practice that animals go through hence it becomes stressful for them (Ljungberg et al., 2007; Chulayo, 2015). Moreover, there are several stressful events associated with transportation and handling of slaughter animals compromising the animal well-being (Perez et al., 2002) and welfare as well as reducing and affecting meat quality in different forms and may, therefore, cause economic loss (Adzitey et al., 2011) These activities take place on the farm, during transportation, and before slaughter. This factor does not only affect the meat quality but also changes the physiology of an animal (Averos et al., 2007; Tadich et al., 2005; Jama et al., 2016). In agreement with Njisane (2016) transportation has been defined as the main element that contributes the most to the pre-slaughter stressors in farm animals leading to poor meat quality and alterations in blood constituents (Warriss et al., 1998; Tadich et al., 2005).After entry in the slaughterhouse, two important factors that may influence the level of stress in cattle, and therefore meat quality, are lairage time and handling technique immediately prior to slaughter (Tadich et al., 2005). Dokmanovic et al. (2014) reported that time spent in the slaughterhouse, lairage potentially allows cattle to replenish muscle glycogen concentrations to supply ATP with the production of lactic acid which help in reducing meat pH (Li et al., 2018), reduce dehydration of body tissues and carcase weight loss (Gupta et al., 2007) and to rest and recover from some of the effects of transport and hence improve meat quality. Despite the fact that reactions to transportation and lairage occasions are to a specific extent, quality ward factors; they can be affected among different components, by substandard transport as well as lairage durations or overcrowding including mixing of animals (Jama et al., 2005).Moreover, according to Liotta et al. (2007), lairage before slaughtering has three vital functions: it allows animal’s sanitary control antemortem, it permits recuperation from physical and nervous stress caused by previous transportation, and it ensures a constant supply to the slaughtering chain. On the other hand, Adzitey (2011) and Dokmanovik et al. (2014) reported that lairage time was an important source of variation determining meat quality.As reported earlier (Melesse et al., 2011), transportation stimulates body responses that are aimed at re-establishing homeostatic conditions. When animals experience changes in the environment such as climate, temperature, and nutrition, they become stressed and this results in physiological changes such as increases in the activity of enzymes, stress hormones, body temperature and glycolysis (Mader, 2003; Melesse et al., 2011). Examples of enzymatic secretions which are released into the serum through leakage arising from altered membrane permeability include alkaline phosphate (ALP), acid phosphate (ACP), aspartate aminotransferase (AST), creatine kinase (CK) and lactate dehydrogenase (Sattler and Fürll, 2004). Creatine kinase is one of the enzymes that are used in clinical biochemistry to perform a differential diagnosis. In some cases, lactate, glucose, and plasma CK are produced when an animal’s physiological system is formulating ways through which it can cope with the environment that might lead to poor welfare (Terlouw, 2005). The increased levels of CK in the blood are an indication of how stressful the handling facilities were before the animal was slaughtered and the extent of muscular damage during handling (Kannan et al., 2007; Edwards et al., 2010). Moreover, the presence of this enzyme in the blood is due to breed temperament, excitability, and fighting. This enzyme is mostly located in different tissues and its presence in the blood plasma serves as an indication of muscle damage (Tackett et al., 2008). On the other hand, it was reported (Warriss et al., 1998) that longer lairage reduced stress levels based on the concentrations of cortisol. Despite its variability and short life, cortisol is still one of the most used indicators of stress (Cooper et al., 1995; Muchunje et al., 2009). These plasma indicators of stress may get further significance if imagined as a helpful tool for highlighting the low attention regarding animal welfare, frequent in the slaughterhouse (Micera et al., 2007). However, in the area of transportation of cattle, lairage duration at the abattoir, animals and breed effects on the physiological response and meat quality is limited. Hence, the objective of the study was to determine the response of cortisol, glucose and creatine kinase in meat quality parameters of slaughtered Nguni and Boran breeds and their correlations with the carcass liveweight rinkage.3.2 Materials and Methods3.2.1 Ethical clearanceDuring the course of the study, routine farm to abattoir practices and conditions were maintained. Consent to carry out the study was approved and issued by the University of Fort Hare Ethical Clearance committee (Reference Number: NJ1011SGUZ01).3.2.2 Animal description and management Twenty beef steers of two genotype, Nguni (n=10) and Boran (n=10), were used in the study and were all reared on the farm (Honeydale Fort Hare farm). The age of the steers was 18 months old, the initial weight of the steers ranged from 210-350 kg. All steers were individually marked and identified alphabetically. There were all running together as a group throughout the experiment and were fed directly from the pastures, and drinking water from the near dams at the farm. After the observations and experiments in the farm, the animals were all transported to the abattoir for the final stages of the experiment.3.2.3 Experimental Site descriptionThe steers were transported from the farm (Fort Hare Farm) to the East London Abattoir (High-throughput), which is situated in Cambridge in the Eastern Cape of South Africa. Its geographical coordinated are 32.58° S and 27.53° E. Average midday temperatures in East London range between 20?C (July) to 26?C 98 (February) with an annual rainfall of about 593 mm mostly occurring during the summer months. The abattoir operates under typical commercial conditions and is equipped with modern technology to enhance production. It operates according to standard laws and regulations governing abattoirs such as “The Meat Safety Act (Act No. 40 of 2000) (SAMIC, 2006), the Animal Protection Act, 1962 and 1935 for animal welfare maintenance” to ensure public health safety. 3.2.4 Transportation detailsTwenty steers were transported to East London commercial abattoir. They were all handled and loaded from Fort Hare Honey Dale farm. Prior to loading, the group were randomly divided into two groups of 10 steers for the morning session (Group 1– Boran: b2, b3, b6, b8, b9; Nguni: n1, n2, n5, n6, n9) and another 10 steers for the afternoon session (Group 2 – Boran: b1, b4, b5, b7, b10; Nguni: n3, n4, n7, n8, n10). The breed distribution ratio between the two trips was considered and catered for to ensure that both breeds are represented in the two trips. To avoid separation stress, the steers were moved and observed in groups of tens as opposed to individual animal assessment. The first trip was off-loaded at the East London abattoir at 08:00 am after a three-hour drive and the transportation distance was 250m on gravel road within the farm and 120 km on a tarred road. The second trip arrived at 16:00 pm in the afternoon. Traveling time was within two hours in a 2 m high 2010 Mitsubishi (Model: Fuso FM 16-253) with two compartments of 4 m x 3 m size and approximately 0.15 m spacing in-between the side rails. An average speed of 80 km/h will be used throughout the distance. During transportation, the position and posture (e.g. standing or lying down and direction faced) of animals were observed before leaving the farm and on arrival at the abattoir.3.2.5 Lairage duration and Slaughter procedureThe steers were transported to the East London abattoir, transport time was ±4h; the high variability was due to the time spent for the intermediate unloading as well as to road traffic during the journeys. Instead, lairage duration was conditioned by the day of arrival of the lorry at the abattoir, considering that all the animals were slaughtered on the following day. The experiment was conducted over a one day period, within a commercial meat abattoir, with an average throughput of 5000 livestock per week (1000 per day). Cattle arrived in a truck and were taken to the restraining chute.At arrival for both groups the steers were confined into four roofed lairage pens (5.3 m × 5.3 m). For the morning group which arrived at 8 o’clock, the lairage was of 16h, whereas for those of the second group, which arrived on the afternoon, lairage duration was of 24h respectively. Therefore, in relation to the wide variability of lairage duration, then, the subjects were collected into two groups, called Short lairage (16 h) and Long lairage (24h). At the lairages, the animals were given ad-libitum access to water, although feed was not provided to avoid carcass contamination at slaughter. When the cattle were required for slaughter, they were moved from chute through gates that allowed for a sequential release of animals into a circular crowd with minimal disruption in the lairage. They were passed on into a race. The race incorporated a standard crush where cattle were held, in line to allow normal identification procedures to be counted out before slaughter. The steers were slaughtered in accordance with approved commercial procedures of the abattoir (Meat Safety Act, 2000: Act No. 4). The live slaughter weight for the Nguni steers was approximately ± 356kg while Boran steers weighed on average ± 298kg. An electric prodder was used sparingly to move the steers from the pens to the slaughter area. The captive bolt method of stunning was used.Blood sampling Blood samples were collected at slaughter (during bleeding) for analysis. Samples were kept on ice until they were centrifuged and taken to the laboratory for routine haematological measurements. All the samples were centrifuged at 3000 rpm and 10 ºC for 15 min using Model 5403 Centrifuge (Gatenbay Eppendorf GmbH, Engelsdorp, Germany) before analysis for cortisol, glucose and lactate was carried out. The plasma samples were stored at -20 ºC until the assay to avoid loss of bioactivity and contamination. The analyses were done in University of Pretoria, Pathology lab (Cortisol and Lactate) and National Health Laboratories (glucose). As for haematological measurements the analysis was done in Victoria Hospital (Eastern Cape, Alice).Physiological ParametersHaematological, Cortisol, Glucose and Creatine kinase analysis Two blood samples (samples for haematological analysis, samples for plasma cortisol and biochemical analysis) were collected at each sampling. Immediately at the exsanguination, individual blood samples (10mL) from each steer were collected from the jugular vein into tubes; then, they were split into two aliquots: on the first one, haematological variables were determined and on the second aliquot, biochemical and hormonal variables were determined.Haematological AnalysisLeukocyte count (WBC), Erythrocyte count (RBC), Haemoglobin (Hgb), Haematocrit (Hct), Mean Corpuscular Volume (MCV), Mean Cell Haemoglobin (MCH), Mean Cell Haemoglobin Concentration (MCHC), Red Cell Distribution (RDW) and Platelet Count (Plt), were analysed by using an automated electronic particle analyser (GENIUS – VET; Celltac, MEK-6108K). Neutrophils, lymphocytes, eosinophil, basophils, monocyte and other cells the cells were counted ad identified using an optic immersion microscope and was analysed using an electronic haematological analyser fallowing the procedures described in Gupta et al. (2007) and Liotta et al. (2007). Neutrophil Lymphocyte ratio (N/L) was determined using blood smears stained with Wright stain. The neutrophil lymphocyte ratio was used as a chronic stress indicator. Hormonal (Cortisol) AnalysisThe blood samples were centrifuged, and the plasma was removed and stored at -20 ºC until analysis. Plasma cortisol concentrations were determined by enzymatic immunoassay (EIA) kit using an automatic analyser (DPC immulite and a Corti-cote kit). The enzyme activity which was bound into the solid phase was inversely proportional to the cortisol concentration in the standards and samples. Colorimetric readings were carried out using a spectrophotometer at 450 and 540 nm. The assay was performed in ng/ml and then the concentrations of cortisol were determined as described (ASPA, 1999).Biochemical (Glucose and Creatine Kinase) AnalysisIn the stored serum samples, the levels of creatine kinase were determined using a commercial colorimetric diagnostic kit (CK; IL Test kit, No. 181,605–90) on the Monarch 2000 Chemistry system (Monarch Chemistry System, Instrumentation Laboratories, Zaventem, Belgium). Concentrations of CK in serum were expressed in units per litre (U/L).Glucose was determined by taking 50 µl of blood serum and the samples were added to the measuring cells for dilution. Glucose concentrations in blood serum samples were determined by using the calibration curve in nmol/L. On individual plasma sample Glucose and Creatine Kinase (CK) contents were determined by using a biochemical automatic analyser (BM HITACHI 911 – Roche).Meat samples and quality testsAfter carcasses dressing, 2.5 kg of meat samples were harvested from the Loin muscle (Longissimuss thoracic et. Lumborum LTL) from each steer. Smaller sub-sections of the LTL muscle (approximately 100 mm thick) from the left side of each carcass were from the 10th rib in the direction of the rump for meat quality analysis. Samples were sealed in plastics before they were stored in a cooler box filled with ice cubes for 3 hours during transportation. When the samples arrived at the University of Fort Hare Meat Science Lab, they were weighed unfrozen, and frozen at -20º C refrigerator temperature until analysis of meat quality attributes including pH, meat colour (lightness; L*, redness; a*; and yellowness; b*) and thawing loss (TL%) were analysed. Before meat quality experiment was performed, the samples were thawed at 4ºC for 24 hours. After 7 days of refrigeration, measurements for cooking loss (CL %) and Warner Bratzler Shear Force (WBSF) were performed. For WBSF measurements, 100mm thick sub-sections were processed into the 20mm steak for CIE (Commission on Illumination lab color) measurements and 30mm steaks for WBSF measurements.22.214.171.124 Determination of meat pHA portable pH meter with a sharp metal sheath (A Testo 205 model) was used to measure the pH of the Loin muscles 24 hours after slaughter. The pH meter was calibrated before use. 126.96.36.199 Determination of meat color (L?, a?, and b?)The color of meat coordinates L* (lightness), a* (redness) and b * (yellowness) was determined using a Minolta colour-guide 45/0 BYK-Gardener GmbH machine (illuminant: D65; Visual angle or standard observer: 10º; diameter measurement: 20 mm). The machine was calibrated using the white standards before taking measurements. The results were taken after 3 readings achieved by rotating the device by 90º on the sample surface 3 times so as to obtain the average value for the colour. Chroma (Cr) was calculated as (a*2+ b*2) ½ and the Hue angle (HA) was also calculated as tan-1(b/a). The colour values were then record accordingly. 188.8.131.52 Thawing and cooking loss measurementsAfter determination of pH and colour, the samples were vacuum packed. The samples were weighed before freezing using a LBK weighing scale and then were kept at -20 ºC. The samples were subsequently weighed 7 days after refrigeration, then thawed for 24 hours at 4ºC and weighed again. The recorded weight differences were expressed as the thawing loss percentage which was calculated as follows:Thawing Loss (TL %) = Weight before thawing-Weight after thawing Weight before thawing x 100The same meat samples were then placed into thin-walled plastic bags and placed in water bath until an internal temperature of 71ºC (recorded by a portable thermometer) was reached. The meat samples were cooked for 45 minutes at 71ºC. After cooking, the samples were cooled to room temperature for 4 hours and weighed again so as to calculate the amount of water lost after thawing and during cooking. The results were expressed as the cooking loss percentage which was calculated as follows:Cooking loss %=Weight before cooking -Weight after cooking Weight before cooking x 1003.2.8.4 TendernessFollowing the thawing and cooking loss measurements, for measuring texture of the cooked meat, the samples were sheared perpendicular to the fibre direction using a Warner- Bratzler Shear Force (WBSF) device mounted on an Instron 3344 Universal Testing apparatus. Cross head speed was at 20 mm/min, one shear in the centre of each core. From each sample, three sub samples of proximately 11cm core diameter were extracted parallel to the long axis of the muscle fibre. The mean maximum load recorded for the three cores represents the average of the peak force in Newtons (N) for each sample.Statistical AnalysisGeneral linear model was used to determine the association between stress indicators and independent variables. The independent variables were breed type (Nguni and Boran), and lairage period (slaughter group). The values of haematic, hormonal and biochemical parameters, and those of meat quality were analysed for normal distribution. Blood analysis data were processed using a repeated measure analysis of variance by GLM procedure of SAS (2001). This model included time at slaughter. Meat quality data were also subjected to analysis of variance with the same procedure of SAS using general linear model. The following model was used Yijk = µ + ?i + ?j + ?k +?ijk. Where Yijk is the response variable (Meat quality, Haematological parameters, plasma, weight loss, plasma cortisol, glucose and creatine lactate levels); µ is the mean; ?i is the breed effect; ?j is the lairage period effect; ?k is the slaughter group effect; and ?ijk is the standard error. Data for haematological were also analysed by ANOVA using GLM.Results Table 2Mean and standard error for plasma cortisol, glucose, and Ck as affected by pre-slaughter conditions after a16 hour lairage duration ParameterBreed type (Mean±S.E.)Significance levelsBoran Nguni Cort 104,6a±18,136 159,68b±18,136 ?Gluc 6,06a±0,567 5,71a±0,567 NSCk 383,50a±64,567 335,6b±64,567 NSpHu 5,62a±0,025 5,61a±0,025 NSL* 34,71a±0,923 34,86a±0,923 NSa* 17,25a±0,368 17,20a±0,368 NSb* 11,62a±0,605 12,14a±0,605 NSHue 33,78a±1,269 35,22a±1,269 NSChr 20,89a±0,531 21,09a±0,531 NSTL 7,90a±0,569 7,93a±0,569 NSCL 22,24b±1,295 16,87a±1,295 ?Tenderness 19,13a±1,387 23,96b±1,387 ?a,b —Means with different superscripts within the same row are signi?cantly different. CORT — cortisol, GLUC — glucose, pHu — ultimate pH, L* — lightness, a* — redness, b* — yellowness, HUE — Hue angle, Chr — Chroma, TL— thawing loss, CL — cooking loss, SE — standard error, NS — not significant (P ; 0.05), ? (P ; 0.05).Table 3Mean and standard error for plasma cortisol, glucose, and Ck as affected by pre-slaughter conditions after 24-hour lairage duration ParameterBreed type (Mean±S.E.)Significance levelsBoran Nguni Cort 115,92±18,136 148,45±18,136 NSGluc 5,27±0,567 6,50±0,567 NSCk 378,90±64,567 340,20±64,567 NSpHu 5,62±0,025 5,61±0,025 NSL* 33,17±0,923 36,41±0,923 ?a* 17,56±0,369 16,89±0,369 NSb* 11,29±0,605 12,47±0,605 NSHue 32,71±1,269 36,28±1,269 ?Chr 20,95±0,531 21,03±0,531 NSTL 7,63±0,569 8,21±0,569 NSCL 18,68±1,295 20,44±1,295 ?Tenderness 20,91±1,388 22,17±1,388 NSa,b —Means with different superscripts within the same row are signi?cantly different. CORT — cortisol, GLUC — glucose, pHu — ultimate pH, L* — lightness, a* — redness, b* — yellowness, HUE — Hue angle, Chr — Chroma, TL— thawing loss, CL — cooking loss, SE — standard error, NS — not significant (P ; 0.05), ? (P ; 0.05).Plasma cortisolThe above tables consist of the predicted mean values of the stress indicators for the different breeds and lairage time. Table 1 contains the parameter estimates for the 16 hours of lairage time. The capture of the model is based on the abattoir measurements (at slaughter) after lairage. Table 1 shows that cortisol concentrations between Boran and Nguni steers were statistically significantly (p; 0.05) after a 16h of lairage. The steers transported for 3 h with a lairage time of 16h, had significantly lower cortisol levels at slaughter. However, for the second group (24h of lairage time) concentrations in steers at slaughter were not different. This could be due to the correspondences in their behaviour during handling. Even though, boran steers slaughtered after 16h of lairage time had a lower cortisol level compared to those who were kept for 24 hours before slaughter. Meanwhile, Nguni steers at slaughter with a lairage time of 16 h had higher cortisol concentrations than the steers with a lairage time of 24h. This shows that breed type has a slight influence on the physiology of animals. The responses of stress indicators on meat quality for different lairage time are provided in Table 2 and 3. However, Table 2 shows that the influence of lairage time on both breeds on instrumental meat quality traits was not significant except for cooking loss and tenderness. CL mean values in Nguni steers were significantly lower compared to Boran steers and its meat was tenderer. After 24 h of lairage for both breeds, lairage duration on meat quality traits only affected colour (lightness), hue and also cooking loss. Ultimate pH, Chroma, thawing loss, and tenderness were not significant (Table 3).Glucose and CkGlucose concentrations were similarly increased fallowing lairage duration on both breeds. Table 2, and 3 shows that glucose levels at slaughter for both breeds were not statistically significant. The breed type and lairage time had no effect on glucose concentrations. Furthermore, the plasma ck also did not show any changes during and after lairage time. Lairage did not have a significant influence on ck after 16 h or 24 h of lairage duration. Table 4Effect of lairage duration on live weight loss (Mean±S.E.) of a Boran and Nguni breed Variable Breed Mean± SE Significance levelLD16hr. LD- 24hr. Liveweight loss Boran 4,85a±0,591 4,89a±0,591 NSNguni 4,46a±0,591 4,42a±0,591 NSMeans with different superscripts within the same row are signi?cantly different at p ; 0, 05; SE (Standard Error), NS — not significantWeight lossThe changes in the mean (SE) live weights of the animals at slaughter after the different periods in lairage are shown in Table 4. In the recent study, the overall loss of the initial live weight of Boran steers was 8.38% and the overall mean loss was 10.08% after 16 h of lairage time. Increasing their time in lairage led to further loss of live weight. The lower percentage in the current study may have been due to several factors, including breed, their behaviour, feed deprivation, transport and environmental conditions (Teke et al., 2014). Table 4 shows that the effect of lairage time on live weight loss was not significant (p; 0.05). Therefore, weight was not significantly different among the two groups, but it showed a decreasing trend with lairage time. DiscussionThe experiment was designed to study pre-slaughter stressors and meat quality implications of lairage duration, and breed type into account individual physiological characteristics. The animals were all kept on the same farm and the transport conditions were the same for all the groups. During transportation in our study, there was no skin damage, and no deaths were registered. Since during the pre-slaughter period, farm animals become exposed to a number of stressful events such as grouping and moving animals, loading and unloading, and noise and physical discomfort (Muchenje et al., 2009; Chulayo et al., 2016).In agreement with Ekiz et al. (2012), in their study, they reported that the loading and unloading process are the most stressful events of pre-slaughter handling. Therefore, lairage period may give a chance to animals for a recovery of the road stress before taken to slaughter (Dikeman, 2017). Ekiz et al. (2012) opposed according to their findings, they said lairage may cause an additional stress in farm animals due to the exposure of new environment and food withdrawal. The results show biochemical measurements (cortisol, glucose, and ck) and meat quality parameters as affected by breed type and in relation to the 16 h (short) and 24 h (long) lairage duration. Cortisol is an index of stress experienced by an animal. Based on the results in Table 2, steers given a resting period of 16 h had a higher cortisol level, Nguni had high plasmatic cortisol concentrations at slaughter than those slaughtered after a 24 h resting time. Therefore at 16 h of lairage, cortisol was significantly affected by lairage time. Meanwhile, Table 3 shows no significant effect (p ; 0.05) of breed and lairage period on plasma cortisol. But the decrease of cortisol concentrations was higher in 24 h of lairage time (results not shown). This result agrees with Liotta et al. (2007), who stated that a decrease in plasma cortisol levels at longer lairage was due to a decrease in stress-inducing the activation of the hypothalamic-adrenal axis. Moreover, Tadich et al. (2005) reported that cortisol levels increase during the lairage starting from 3 to 24 hours. According to the current study, this could be due to the fact that both breeds (Boran and Nguni steers) has a potential to respond to various handling and has similarities in terms of adaptation. Therefore, in order to recuperate from stress sufficient lairage time should be provided to farm animals. The other possible reason could be the fact that, those animals who are subjected to long lairage has a chance to relax and recover from stress, and also the group gets the opportunity to drink enough water to recover from dehydration that probably took place during transportation time (Liotta et al., 2007). No significant difference was observed for glucose and Ck for 16 h and or 24 h of lairage period (shown in Table 2 and 3). However, both breeds did not differ in terms of glucose and Ck concentrations. This can be a result of small circulations of glycolysis caused by catecholamine (Marencis et al., 2012).As for instrumental meat quality traits, there are many factors associated with pre-slaughter conditions that affect meat quality including transportation and lairage duration (Liotta et al., 2007). Dokmanovic et al. (2014) carried out a study on effects of different lairage times on meat quality; they found that the effect of lairage times on meat quality was significant (p ; 0.05). However, Perez et al. (2012), Teke et al. (2014), also discovered that cattle which had shorter lairage were more stressed at slaughter, than those of the longer lairage time. They concluded that lairage time had a significant effect on meat quality such tenderness. The result of the current study is therefore similar to those of the other studies mentioned. On the other hand, the discoveries (results) of some studies are in disagreement with ours. For an example, Liste et al. (2009) in rabbits and they found that lairage had no significant effect on meat tenderness.The discoveries of meat colour results are inconsistency in many studies. In the current study at slaughter after 16h of resting period, lairage had no significant effects on colour mean values (p ;0.05) (Table 2), but on the other side lairage had an effect on one of the meat colour value after 24 h of resting period (lairage time affected L? significantly) except for a?, and b? values. The L* value is a measurement for brightness, ranging from black to 100 white. Hence in meat classification, they explain that the higher the L*value, the paler the meat (Kerry, 2009). However, Kadim et al. (2009) reported no significant effect of lairage duration on meat colour coordinates (L?, a?, and b?). In agreement with Ekiz et al. (2012), they said lairage duration does not have an influence on meat colour values either during short or longer lairage times given to animals. Conversely, Muchenje (2009) found that there was a significant relationship between stress responsiveness hormonal concentrations and L? as a meat quality trait. Also, Zhong et al. (2011) stated that in their study lairage period had a significant effect on a? during 24 h of resting period.There was no significant difference of pH found between the two groups at slaughter. This indicates that breed type had no effect on muscle characteristics. These findings are in disagreement with Gallo et al. (2003) study, as they found colour highly affected by 24 h lairage. Nevertheless, the cooking loss was significantly affected by both resting periods. The cooking loss is usually associated with firmness and water holding capacity as it works in relation to drip loss (Chikuni et al., 2010). However, Sikes and Warner (2016) revealed that meat contains approximately 75% water and the water is dominatingly held by capillary force in the myofibrils, inside the muscle cell. Hence, loss of water from meat during boiling, which results in higher cooking loss and decreased yield, is caused by loss of water from myofibrillar structures.The steers were discovered of most common productivity. Stress disturbs the homeostasis of farm animals resulting in increased activity of enzymes and hormones (Yalcintan et al., 2018). Hence, plasma cortisol, glucose, and ck were used because they are the most reliable stress responses and have been widely used. Even though, glucose and ck were not significantly affected by lairage times. There are still several arguments between the authors regarding the preferred lairage time needed for animals on abattoir arrival. Other recommends short lairage ranging from 3 to 8 hours beneficial, while approve longer lairage ranging from 12- 48 hours of lairage time. For an example, Tadich et al. (2005) explained that in their study glucose levels increased after 3 h and 6 h of lairage but further decreased after 12 h and 24 h of resting period. They further reported that there is no valuable effect on animal welfare by a long lairage time at the slaughterhouse. In contrary Gallo et al. (2003), stated that loss of live weight and increases in the pH of meat prove that longer transportation and longer resting periods had an adverse influence on the animal welfare.ConclusionThe results shows that from an economic point of view and for animal corner, times in lairage should be long, and the should be a firm and legit laws that will be against the case slaughtering animal as soon as they arrive to slaughter plants.ReferenceChikuni, K., Oe, M., Sasaki, K., Shibata, M., Nakajima, I., Ojima, K. and Muroya, S., 2010. Effects of muscle type on beef taste?traits assessed by an electric sensing system. Animal science journal, 81: 600-605.Dokmanovi?, M., Velarde, A., Tomovi?, V., Glamo?lija, N., Markovi?, R., Janji?, J. and Balti?, M.Ž., 2014. The effects of lairage time and handling procedure prior to slaughter on stress and meat quality parameters in pigs. Meat science, 98: 220-226.Dikeman, M.E., 2017. Understanding the effects of handling, transportation, lairage and slaughter on cattle welfare and beef quality Michael S. Cockram, University of Prince Edward Island, Canada. In Ensuring safety and quality in the production of beef, 2: 157-202. Gallo, C., Lizondo, G. and Knowles, T.G., 2003. Effects of journey and lairage time on steers transported to slaughter in Chile. Veterinary Record, 152: 361-364.Liotta, L., Costa, L.N., Chiofalo, B., Ravarotto, L. and Chiofalo, V., 2007. Effect of lairage duration on some blood constituents and beef quality in bulls after long journey. Italian Journal of Animal Science, 6: 375-384.Liste, G., Villarroel, M., Chacón, G., Sañudo, C., Olleta, J.L., García-Belenguer, S., Alierta, S. and María, G.A., 2009. Effect of lairage duration on rabbit welfare and meat quality. Meat science, 82: 71-76.Maren?i?, D., Ivankovi?, A., Pinti?, V., Kelava, N. and Jakopovi?, T., 2012. Effect of the transport duration time and season on some physicochemical properties of beef meat. Archives Animal Breeding, 55: 123-131.Tadich, N., Gallo, C., Bustamante, H., Schwerter, M. and Van Schaik, G., 2005. Effects of transport and lairage time on some blood constituents of Friesian-cross steers in Chile. Livestock Production Science, 93: 223-233.Teke, B., Akdag, F., Ekiz, B. and Ugurlu, M., 2014. Effects of different lairage times after long distance transportation on carcass and meat quality characteristics of Hungarian Simmental bulls. Meat Science, 96: 224-229.Kerry, J. ed., 2009. Improving the sensory and nutritional quality of fresh meat. Elsevier.Chapter 4: Investigating the effects of a 16 and 24- hour lairage period on haematological parameters of Nguni and Boran breedIntroductionTransported animals are frequently exposed to a range of physical and psychological stimuli that disturb their homeostasis and metabolism and therefore increase the activity of enzymes and hormones (Giannetto et al., 2011; Adenkola and Ayo, 2010). On the other hand, stressful lairage conditions resulted from dehydration and food deprivation during lairage can result to more stress in farm animals (Bornez et al., 2009). These stressors may lead to drastically changes in blood constitutes such as plasma concentration of creatine kinase (CK), lactate, glucose, cortisol and packed cell volume (Grandin, 2007). Therefore, these parameters have been used in order to assess stress regarding to pre-slaughter handling in farm animals. Dalla et al. (2015) reported that, the measurement of plasma constituents in a blood sample can give information about the status of stress of the animal during handling and management procedures. Physiological indicators of animal welfare may differ on blood sampling time, breed, gender and or the age of the animals (Warris, 1998).The measurement of plasma constituents in a blood sample can provide information about the stress of the animal during the various management operations (Shaw and Tume, 1992Shaw and Tume, 1992). Moreover, a combination of several measurements is better to assess the effects of a particular handling practice (Haresign et al., 1995Haresign et al., 1995). However, it is probable that not all parameters give the same information about the possible stress that animals can suffer in several situations such as after transport or after lairage. Physical and mental efforts occurring during road transportation of livestock and during the lairage time disrupt their homeostasis and metabolism and therefore, increase activity of enzymes and hormones (Adenkola and Ayo, 2010). Additionally, Knowles et al. (2014) showed that numerous stressors instigated the activation of the autonomic nervous system as prior revealed by (Bornez et al., 2009) with the pituitary-adreno-cortical system likewise assuming a vital part in the inflection of stress reaction (Fazio et al., 2018). Adenkola and Ayo (2010) reported a rise in adrenal cortical movement in pigs transported by s road by demonstrating an expansion in blood level of 11hydroxycorticosteroids (11-oxy) 30 min after the beginning of transport. Furthermore, in an ongoing report, Fazio et al. (2015) observed the initiation of the hypothalamic-pituitary-adrenal axis and the catecholaminergic system in long transportation of calves. Observations of an increase in blood hormone levels, lymphocyte, glucocorticoid and adrenergic receptor, cortisol and catecholamine concentrations were noted (Yalcintan et al., 2018). However, the increased qualities came back to typical at 24 h and 7 days after arrival. Also Gupta et al. (2007) proven that transportation induced leucocytosis, neutrophilia, lymphocytosis, eosinophilia in bulls.Haematological studies are of ecological and physiological enthusiasm for understanding the relationship of blood qualities to the environment (Etim et al., 2014a) thus could be helpful in the determination of animals that are genetically impervious to specific sicknesses and environmental conditions (Addass et al., 2012; Isaac et al., 2013). Hematological parameters are great indicators of the physiological status of animals (Kubkomawa et al.,2016). Haematological parameters are those parameters that are associated with the blood and blood framing organs (Addass et al., 2012; Bamishaiye et al., 2009). Blood go about as a neurotic reflector of the status of presented animals to toxicant and different conditions (Etim et al., 2014a). As reported by Parvez et al. (2017) animals with good blood sythesis are probably going to demonstrate good execution. Research facility tests on the blood are imperative apparatuses that assist distinguish and detect any deviation from ordinary in the animal. The examination of blood gives the chance to explore the presence of a few metabolites and different constituents in the body of animals and it assumes an imperative part in the physiological, nutrition and neurotic status of either an animal or human (Onasanya et al., 2015). According to Zakari et al. (2016) inspecting blood for their constituents can give essential data to the analysis and visualization of infections in animals. Blood constituents change in connection to the physiological states of wellbeing (Etim et al., 2014b). These changes are of incentive in assessing reaction of animals to different physiological circumstances (Oramari et al., 2014). As indicated by Kana et al. (2014), changes in haematological parameters are frequently used to decide different status of the body and to determine stresses because of environmental, nutrition (Etim et al., 2014b) or potentially obsessive variables.The commonly used haematological parameters are erythrocytes (red blood cells, RBC), leucocytes (white blood cells, WBC), hemoglobin concentration (HBC), packed cell volume (PCV) and values which include Mean Corpuscular Volume or cell (MCV), Mean Corpuscular Hemoglobin (MCH) and Mean Corpuscular Hemoglobin Concentration (MCHC) (Carlson, 1996). Specific causes of erythrocytic abnormalities, which might manifest in chronic blood loss, include bloody diarrhoea, ulcers, bleeding, neoplasm and blood sucking parasites (Johnston and Morris, 1996).Materials and Methods4.1.1 Ethical clearanceDuring the course of the study, routine farm to abattoir practices and conditions were maintained. Consent to carry out the study was approved and issued by the University of Fort Hare Ethical Clearance committee (Reference Number : NJ1011SGUZ01).4.1.2 Animal description and management Twenty beef steers of two genotype, Nguni (n=10) and Boran (n=10), were used in the study and were all reared on the farm (Honeydale Fort Hare farm). The age of the steers was 18 months old, the initial weight of the steers ranged from 210-350 kg. All steers were individually marked and identified alphabetically. There were all running together as a group throughout the experiment and were fed directly from the pastures, and drinking water from the near dams at the farm. After the observations and experiments in the farm, the animals were all transported to the abattoir for the final stages of the experiment.4.1.3 Experimental Site descriptionThe steers were transported from the farm (Fort Hare Farm) to the East London Abattoir (High-throughput), which is situated in Cambridge in the Eastern Cape of South Africa. Its geographical coordinated are 32.58° S and 27.53° E. Average midday temperatures in East London range between 20?C (July) to 26?C 98 (February) with an annual rainfall of about 593 mm mostly occurring during the summer months. The abattoir operates under typical commercial conditions and is equipped with modern technology to enhance production. It operates according to standard laws and regulations governing abattoirs such as “The Meat Safety Act (Act No. 40 of 2000) (SAMIC, 2006), the Animal Protection Act, 1962 and 1935 for animal welfare maintenance” to ensure public health safety.4.1.4 Transportation detailsTwenty steers were transported to East London commercial abattoir. They were all handled and loaded from Fort Hare Honey Dale farm. Prior to loading, the group were randomly divided into two groups of 10 steers for the morning session (Group 1– Boran: b2, b3, b6, b8, b9; Nguni: n1, n2, n5, n6, n9) and another 10 steers for the afternoon session (Group 2 – Boran: b1, b4, b5, b7, b10; Nguni: n3, n4, n7, n8, n10). The breed distribution ratio between the two trips was considered and catered for to ensure that both breeds are represented in the two trips. To avoid separation stress, the steers were moved and observed in groups of tens as opposed to individual animal assessment. The first trip was off-loaded at the East London abattoir at 08:00 am after a three-hour drive and the transportation distance was 250m on gravel road within the farm and 120 km on a tarred road. The second trip arrived at 16:00 pm in the afternoon. Traveling time was within two hours in a 2 m high 2010 Mitsubishi (Model: Fuso FM 16-253) with two compartments of 4 m x 3 m size and approximately 0.15 m spacing in-between the side rails. An average speed of 80 km/h will be used throughout the distance. During transportation, the position and posture (e.g. standing or lying down and direction faced) of animals were observed before leaving the farm and on arrival at the abattoir.4.1.5 Lairage duration and Slaughter procedureThe steers were transported to the East London abattoir, transport time was ±4h; the high variability was due to the time spent for the intermediate unloading as well as to road traffic during the journeys. Instead, lairage duration was conditioned by the day of arrival of the lorry at the abattoir, considering that all the animals were slaughtered on the following day. The experiment was conducted over a one day period, within a commercial meat abattoir, with an average throughput of 5000 livestock per week (1000 per day). Cattle arrived in a truck and were taken to the restraining chuteAt arrival for both groups the steers were confined into four roofed lairage pens (5.3 m × 5.3 m). For the morning group which arrived at 8 o’clock, the lairage was of 16h, whereas for those of the second group, which arrived on the afternoon, lairage duration was of 24h respectively. Therefore, in relation to the wide variability of lairage duration, then, the subjects were collected into two groups, called Short lairage (16 h) and Long lairage (24h). At the lairages, the animals were given ad-libitum access to water, although feed was not provided to avoid carcass contamination at slaughter. When the cattle were required for slaughter, they were moved from chute through gates that allowed for a sequential release of animals into a circular crowd with minimal disruption in the lairage. They were passed on into a race. The race incorporated a standard crush where cattle were held, in line to allow normal identification procedures to be counted out before slaughter. The steers were slaughtered in accordance with approved commercial procedures of the abattoir (Meat Safety Act, 2000: Act No. 4). The live slaughter weight for the Nguni steers was approximately ± 356kg while Boran steers weighed on average ± 298kg. An electric prodder was used sparingly to move the steers from the pens to the slaughter area. The captive bolt method of stunning was used.Blood sampling Blood samples were collected at slaughter (during bleeding) for analysis. Samples were kept on ice until they were centrifuged and taken to the laboratory for routine haematological measurements. All the samples were centrifuged at 3000 rpm and 10 ºC for 15 min using Model 5403 Centrifuge (Gatenbay Eppendorf GmbH, Engelsdorp, Germany) before analysis for cortisol, glucose and lactate was carried out. The plasma samples were stored at -20 ºC until the assay to avoid loss of bioactivity and contamination. The analyses were done in University of Pretoria, Pathology lab (Cortisol and Lactate) and National Health Laboratories (glucose). As for haematological measurements the analysis was done in Victoria Hospital (Eastern Cape, Alice).Haematological AnalysisLeukocyte count (WBC), Erythrocyte count (RBC), Haemoglobin (Hgb), Haematocrit (Hct), Mean Corpuscular Volume (MCV), Mean Cell Haemoglobin (MCH), Mean Cell Haemoglobin Concentration (MCHC), Red Cell Distribution (RDW) and Platelet Count (Plt), were analysed by using an automated electronic particle analyser (GENIUS – VET; Celltac, MEK-6108K). Neutrophils, lymphocytes, eosinophil, basophils, monocyte and other cells the cells were counted ad identified using an optic immersion microscope and was analysed using an electronic haematological analyser fallowing the procedures described in Gupta et al. (2007) and Liotta et al. (2007). Neutrophil Lymphocyte ratio (N/L) was determined using blood smears stained with Wright stain. The neutrophil lymphocyte ratio was used as a chronic stress indicator. Results Table 5: Haematological parameters (Mean±S.E.) in the meat of Boran and Nguni steer at different lairage period Characteristics Slaughter groupLD- 16hr. Significancelevels Slaughter groupLD- 24hr. SignificancelevelsB N B N WBC 14,30a±1,451 14,96a±1,451 NS14,69a±1,451 14,58a±1,451 NSRBC 7,98b±0,315 6,86a±0,315 ? 7,56a±0, 315 7,27a±0,315 NSHaemoglobin 11,15b±0,291 10,49a±0,291 NS 11,03a±0,291 10,61a±0,291 NSHaematocrit 0,33b±0,009 0,29a±0,009 ? 0,34a±0,009 0,31a±0,009 NSMCV 42,15a±1,191 43,51b±1,191 NS 43,37a±1,191 42,29a±1,191NSMCH 14,15a±0,562 15,43b±0,562 NS 14,79a±0,562 14,79a±0,562 NSMCHC 33,48a±0,498 35,41b±0,498 ? 34,03a±0,49834,86a±0,498 NSRDW 15,47a±0,469 16,95b±0,469 ? 16,19a±0,469 16,23a±0,469 NSPlatelet count 145a±24,519 145,4a±24,519 NS 140,10a±24,519 150,30a±24,519 NSMPV 8,94a±0,261 8,50a±0,261 NS 8,68a±0,261 8,76a±0,261 NSNeutrophils 8,84a±1,157 9,73b±1,157 NS 8,44a±1,157 10,13b±1,157 NSLymphocytes 63,54a±4,550 64,01a±4,550 NS 63,99a±4,551 63,56a±4,551 NSMonocytes 9,09a±3,242 11,22b±3,242 NS 10,27a±3,242 10,04a±3,242 NSEosiniphils 0,47a±0,101 0,42a±0,101 NS 0,44a±0,101 0,45a±0,101 NSBasophils 2,54a±0,425 2,71a±0,425NS2,56a±0,4252,69a±0,425 NSNTR 0,16a±0,032 0,17a±0,032NS 0,14a±0,032 0,18a±0,032 NSa,b-Means with different superscripts within the same row are signi?cantly different at p < 0, 05; SE (Standard Error), LD— lairage duration, NS — not significant, ? (p < 0.05).Discussion Our study was performed under controlled commercial abattoir using the same type of animal (breed and age). Even under those normal conditions, numerous potential stressors were identified as critical points along the pre-slaughter handling that can affect the welfare and meat quality such as season (weather changes) even though there were no results recorded. Physiological studies have discussed that stressful condition partaken by animals can disturb immune system, endocrine and haematological characteristics (Romero et al., 2014; Zhen et al., 2014). Hence, blood constituents have been used as complex indicators of the physiological responses of animals to stress (Perez et al., 2002; Bornez et al., 2009). As shown in Table 2 after 24 h of lairage duration, lairage had no influence on haematological characteristics among both breeds. This could be due to the fact that haematological parameters are not affected by the nervous system which usually release stress hormones during draining and stressful situations. Similar studies were achieved by Minka et al. (2009).On the other side, lairage duration after 16 h of lairage significantly affected RBC, haem, MCHC, and RDW on both steers. These findings could be caused by nerve system activation or by circulating catecholamine, because catecholamine keeps cattle in the place of alert. However, Tadich et al. (2005); Liota et al. (2007); and also found significant effect of lairage duration on haematocrit values and other constituents, they further describe that animals that were kept for short lairage (less than 12 hours) had an increased significant effect on the parameters than the longer lairaged animals (starting from 20 hours). In agreement with mentioned studies, Ekiz et al. (2012) reported that lairage had high significant effect on PCV of animals that were kept for 18 h of lairage duration. Furthemore, Early et al. (2006) found decreased haematocrit values in steers that were subjected to long lairage (24 hours). Liotta et al. (2012) supported Early results by further explaining that, this could be caused by dehydration or splenic concentration releasing catecholamine that might increase HCT and PCV (Tadich et al., 2005 ), thereby increasing the number of red blood cells. However, Yalcintan et al. (2018) noted that blood cell constituents retain the physiological balance in response to environmental situations by reinstating normal homeostasis. Another sensitive haematological parameter that is affected by lairage duration is neutrophil ratio (Yalcintan et al., 2018). In agreement with Gupta et al. (2007), described that neutrophil ratio has been used as a balancing measurement, it is also helpful in assessing stress in transported animals during transportation handling (loading and unloading). In addition, Miranda de-la Lama et al. (2012) and Romero et al (2014) observed that transportation handlings and blood sampling time lead to immune suppression and therefore resulting in lapsing leukocyte and increased neutrophil ratio. Therefore, many studies disagree with the current results obtained after the 24 h of lairage duration (Table 5). Some researchers consider 12 h pre–slaughter lairage for cattle being too long because stressful conditions (noise, fear, novelty, thirst, hunger) may increase with time spent at the slaughterhouse, which may lead to a decrease in meat qualityConclusionLittle is known about the effect of haematological parameters on welfare and meat quality traits in beef cattle. Handling, transport, lairage and slaughter are important components of beef production that can affect both animal welfare and meat quality. Each stage of pre-slaughter management can potentially present multiple negative welfare issues. It is difficult to repeat this difficulty in a research environment to investigate factors affecting animal welfare and beef quality. Variations in each stage of pre-slaughter management including lairage durations, variations between breeds and differences in how humans interact with the animals can have significant effects on welfare and haematological parameters, which can also be used to investigate stress levels in animal. ReferenceAdenkola, A.Y. and Ayo, J.O., 2010. Physiological and behavioural responses of livestock to road transportation stress: A review. African Journal of Biotechnology, 9: 4845-4856.Addass, P.A., David, D.L., Edward, A., Zira, K.E. and Midau, A., 2012. Effect of age, sex and management system on some haematological parameters of intensively and semi-intensively kept chicken in Mubi, Adamawa State, Nigeria. Iranian Journal of Applied Animal Science, 2: 277-282.Bamishaiye, E.I., Muhammad, N.O. and Bamishaiye, O.M., 2010. Haematological parameters of albino rats fed on tiger nuts (Cyperus esculentus) tuber oil meal-based diet. Internet Journal of Nutrition and Wellness, 10.Bórnez, R., Linares, M.B. and Vergara, H., 2009. Haematological, hormonal and biochemical blood parameters in lamb: effect of age and blood sampling time. Livestock Science, 121: 200-206.Chineke, C.A., Ologun, A.G. and Ikeobi, C.O.N., 2006. Haematological parameters in rabbit breeds and crosses in humid tropics. Pak J Biol Sci, 9: 2102-2106.Carlson, J.D., Catanzarite, D.M. and St. Clair, K.A., 1996. Commercial magneto-rheological fluid devices. International Journal of Modern Physics B, 10: 2857-2865.Etim, N.N., Offiong, E.E., Williams, M.E. and Asuquo, L.E., 2014. Influence of nutrition on blood parameters of pigs. American Journal of Biology and Life Sciences, 2: 46-52.Etim, N.N., Williams, M.E., Akpabio, U. and Offiong, E.E., 2014. Haematological parameters and factors affecting their values. Agricultural Science, 2: 37-47.Earley, B. and O’Riordan, E.G., 2006. Effects of transporting bulls at different space allowances on physiological, haematological and immunological responses to a 12-h journey by road. Irish Journal of Agricultural and Food Research, 39-50.Ekiz, B., Ekiz, E.E., Kocak, O., Yalcintan, H. and Yilmaz, A., 2012. Effect of pre-slaughter management regarding transportation and time in lairage on certain stress parameters, carcass and meat quality characteristics in Kivircik lambs. Meat Science, 90: 967-976.Fazio, F., Arfuso, F., Rizzo, M., Giannetto, C., Giudice, E., Zanghì, E. and Piccione, G., 2018. Livestock handling and road transport influence some oxidative stress parameters in ewes. Journal of Veterinary Behavior, 26: 5-10.Giannetto, C., Fazio, F., Casella, S., Marafioti, S., Giudice, E. and Piccione, G., 2011. Acute phase protein response during road transportation and lairage at a slaughterhouse in feedlot beef cattle. Journal of Veterinary Medical Science, 73: 1531-1534.Grandin, T. 2007. Livestock Handling and Transport (Third Edition), CAB International Publishing Colorado State University, United States of AmericaGrandin, T., 2010. Auditing animal welfare at slaughter plants. Meat Science, 86: 56-65.Gupta, S., Earley, B. and Crowe, M.A., 2007. Effect of 12-hour road transportation on physiological, immunological and haematological parameters in bulls housed at different space allowances. The Veterinary Journal, 173: 605-616.Isaac, L.J., Abah, G., Akpan, B. and Ekaette, I.U., 2013, September. Haematological properties of different breeds and sexes of rabbits. In Proceedings of the 18th Annual Conference of Animal Science Association of Nigeria, 24-27Kubkomawa, I.H., Ogundu, M.A., Okoli, I.C. and Udedibie, A.B.I., 2016. Seasonal haematological characterization of pastoral cattle in the Guinea Savannah zone of North-Eastern Nigeria.Knowles, T.G., Warriss, P.D. and Vogel, K., 2014. 21 Stress Physiology of Animals During Transport. Livestock handling and transport: Theories and applications, 399.Kana, J.R., Ngoula, F., Tchoffo, H., Tadondjou, C.D., Sadjo, Y.R., Teguia, A. and Gbemenou, J.G., 2014. Effect of biocharcoals on hematological, serum biochemical and histological parameters in broiler chickens fed aflatoxin B1-contaminated diets. Journal of Animal Science Advances, 4: 939-948.Liotta, L., Costa, L.N., Chiofalo, B., Ravarotto, L. and Chiofalo, V., 2007. Effect of lairage duration on some blood constituents and beef quality in bulls after long journey. Italian Journal of Animal Science, 6: 375-384.Miranda-de la Lama, G.C., Leyva, I.G., Barreras-Serrano, A., Pérez-Linares, C., Sánchez-López, E., María, G.A. and Figueroa-Saavedra, F., 2012. Assessment of cattle welfare at a commercial slaughter plant in the northwest of Mexico. Tropical animal health and production, 44: 497-504.Minka, N.S. and Ayo, J.O., 2009. Physiological responses of food animals to road transportation stress. African Journal of Biotechnology, 8.Oramari, R.A., Bamerny, A.O. and Zebari, H.M., 2014. Factors affecting some hematology and serum biochemical parameters in three indigenous sheep breeds. Advances in Life Science and Technology, 21: 56-62.Onasanya, G.O., Oke, F.O., Sanni, T.M. and Muhammad, A.I., 2015. Parameters influencing haematological, serum and bio-chemical references in livestock animals under different management systems. Open Journal of Veterinary Medicine, 5: 181.Parvez, F., Medina, S., Santella, R.M., Islam, T., Lauer, F.T., Alam, N., Eunus, M., Rahman, M., Factor-Litvak, P., Ahsan, H. and Graziano, J.H., 2017. Arsenic exposures alter clinical indicators of anemia in a male population of smokers and non-smokers in Bangladesh. Toxicology and applied pharmacology, 331: 62-68.Pérez, M.P., Palacio, J., Santolaria, M.P., Aceña, M.C., Chacón, G., Gascón, M., Calvo, J.H., Zaragoza, P., Beltran, J.A. and Garc?a-Belenguer, S., 2002. Effect of transport time on welfare and meat quality in pigs. Meat science, 61: 425-433.Romero, M.H., Uribe-Velásquez, L.F. and Sánchez, J.A., 2014. Physiological profiles of Zebu steers during transport and pre-slaughter. Revista Colombiana de Ciencias Pecuarias, 27: 282-289.Shaw, F.D. and Tume, R.K., 1992. The assessment of pre-slaughter and slaughter treatments of livestock by measurement of plasma constituents—a review of recent work. Meat science, 32: 311-329.Tadich, N., Gallo, C., Bustamante, H., Schwerter, M. and Van Schaik, G., 2005. Effects of transport and lairage time on some blood constituents of Friesian-cross steers in Chile. Livestock Production Science, 93: 223-233.Warriss, P.D., Brown, S.N., Edwards, J.E. and Knowles, T.G., 1998. Effect of lairage time on levels of stress and meat quality in pigs. Animal science, 66: 255-261.Yalcintan, H., Akin, P.D., Ozturk, N., Avanus, K., Muratoglu, K., Kocak, O., Yilmaz, A. and Ekiz, B., 2018. Effect of lairage time after 2 h transport on stress parameters and meat quality characteristics in Kivircik ewe lambs. Small Ruminant Research.Zakari, F.O., Ayo, J.O., Rekwot, P.I. and Kawu, M.U., 2016. Effect of age, sex, physical activity and meteorological factors on haematological parameters of donkeys (Equus asinus). Comparative clinical pathology, 25:1265-1272.Zhen, S., Liu, Y., Li, X., Ge, K., Chen, H., Li, C. and Ren, F., 2013. Effects of lairage time on welfare indicators, energy metabolism and meat quality of pigs in Beijing. Meat science, 93: 287-291.ConclusionThis study assessed many plasma constituents as possible indicators of stress. The main findings were that, breed type did not affect physiological indicators (haematological, hormonal and biochemical parameters). In addition, our data showed that pre-slaughter conditions after a short lairage (16h of lairage duration) may influence, although slightly, the haematological parameters as well as the meat quality, and there were no indications that increased lairage at the abattoir had beneficial influence on the welfare of animal. Though, both 16 h and 24 h of lairage had a significant influence on different hormonal, biochemical parameters, and meat quality traits. During long time lairage, a restoration of energy was observed in steers, evidence by an increase in glucose and a decrease in cortisol concentrations. On the whole, the increase in lairage time can have a beneficial effect on animal welfare and can lead to good quality meat.