During pre-slaughter handling, animals are exposed to considerable stress related to fasting, loading onto vehicles, duration of transportation, transportation conditions, unloading in the slaughterhouse, conditions in the slaughterhouse, and waiting time before slaughter (Kannan et al., 2003; Kadim et al., 2006; Terlouw et al., 2008; Minka and Ayo, 2010b; Miranda-de la Lama et al., 2010; Akin et al., 2018).

Numerous handling operations are performed within a short period and are a source of stress, which decreases immunity and disrupts homeostasis in animals (Kannan et al., 2003; Sowińska et al., 2006; Kadim et al., 2007; Minka et Ayo, 2007; De la Fuente et al., 2010, 2012; Adenkola and Ayo, 2010). These stressors increase susceptibility to infection, contribute to body weight loss, and compromise meat quality (Kannan et al., 2002; Kadim et al., 2006, 2007; Ferguson and Warner, 2008; Nikbin et al., 2016; Yalcintan et al., 2018).

Considerable research has been devoted to minimizing stress responses in various animal species through the use of pharmaceuticals, herbs, yeast, seaweeds, minerals, and vitamins (Ali and Al-Qarawi, 2002; Young et al., 2003; Galipalli et al., 2004; Kannan et al., 2007a,b; Minka and Ayo, 2007, 2013; Ali et al., 2006; Ferguson and Warner, 2008; Ambore et al., 2009; Sowińska et al., 2016, 2017). However, not all pharmaceuticals and preparations, in particular antidepressant drugs, can be administered to livestock due to the safety requirements imposed on animal products (Ali and Al-Qarawi, 2002; Young et al., 2003; Ali et al., 2006).

β-hydroxy-β-methylbutyrate (HMB) is an oxidation product of α-ketoisocaproic acid, which is produced primarily in muscle, liver, and fat tissue from the amino acid leucine (Brosnan and Brosnan, 2006). Leucine plays an important role for the organism, participating in the processes of protein metabolism and inhibiting the processes of their breakdown occurring during effort. Leucine activates the signaling factor of rapamycin (mTOR) in mammals to promote protein synthesis in skeletal muscle and adipose tissue. It is also the main regulator of mTOR-sensitive feed intake responses to high-protein diets. Meanwhile, leucine regulates blood glucose levels by promoting gluconeogenesis and helps maintain lean mass in a hypocaloric state. It is also beneficial for animal nutrition (Li et al., 2011).

Numerous researchers have demonstrated that dietary supplementation with β-hydroxy-β-methylbutyric acid (HMB), a leucine derivative with immunostimulatory properties, has a positive effect on defense mechanisms, improves performance in animals (Gatnau et al., 1995; Papet et al., 1997; Peterson et al., 1999; Puchajda-Skowrońska et al., 2006; Wiąz et al., 2010; Wójcik et al., 2014; Ząbek et al., 2016), and is a safe compound (Rathmacher et al., 2004). Previous reports in animals are very inconsistent with respect to HMB doses and supplementation duration. The predominant HMB doses used in pigs were 15 mg kg¹ of BW (Flummer et al., 2012) and 50 mg kg¹ of BW (Tatara et al., 2007); in cattle, 20,5 mg kg¹ of BW (Van Koevering et al., 1993); but in sheep, higher than 100 mg kg¹ of BW (Tatara, 2008).

The results of the above studies encouraged the authors to investigate whether the immunostimulant could also minimize stress responses in goat kids during pre-slaughter handling and transportation to the slaughterhouse. Therefore, the objective of this study was to determine the effect of dietary supplementation with HMB on live weight loss and selected blood parameters in goat kids after transportation to the slaughterhouse.

Research on animals was conducted according to the institutional committee on animal use (protocol No. 18/2013). The study was conducted in Olsztyn, Warmia and Mazury, Poland (53°46' North latitude, 20°30' East longitude, and altitude of 128 m).

The study was performed with 24 single-born male Alpine goat kids that were weaned at 30 days of age and divided into two equal groups: a control group and an experimental group. After weaning, both groups were administered identical diets, composed of the milk replacer (throughout the entire rearing period) and after 30 days of experiment, the following feed was introduced: cielak complementary feed mixture (Wipasz, Olsztyn) and grass haylage. Milk replacer was administered throughout the whole period by the following scheme: days 1-10 of experiment – 4200 mL/animal/day; days 11-20 of experiment – 2800 mL/animal/day; days 21-30 of experiment – 1900 mL/animal/day; and days 31-60 of experiment – 500 mL/animal/day. When the animals reached the 60th day of age, they began to receive extra grass haylage and complementary feed mixture, according to the NRC (2007), for goats with a daily average gain of 100 g. During the experiment, the amount of administered feed and leftovers was controlled. Chemical composition was determined with standard methods (AOAC, 2005). Based on the results, the amount of nutrients ingested by goat kids was determined for both groups throughout the experimental period (Table 1).

Table 1

Specification	Group
	Control	Experimental
Dry matter (kg)	228.58	230.00
Feed unit for meat production	0.29	0.29
Crude protein (kg)	27.01	27.17
PDIN (kg)	26.74	26.91
PDIE (kg)	27.01	27.17
Crude fiber (kg)	40.82	41.08

PDIN, PDIE - protein digestible in the small intestine when rumen fermentable nitrogen or energy, respectively.

The diets of experimental goats were additionally supplemented with HMB (Metabolic Technologies Inc. Ames, IA, USA), administered with the supplementary feed mix at 50 mg kg¹ of BW. In the available scientific studies, there are no results for goats; therefore, in our study, we applied the most frequently dosage used in other animal species. The animals were kept in boxes in accordance with the guidelines included in the animal protection act, and under the constant veterinarian supervision.

At the end of the 90-day rearing period, the animals were fasted for 12 h (with access to water) and were transported to the slaughterhouse in the following morning. The kids were transported in a standardized vehicle for animal transportation. During transportation, temperature and humidity inside the vehicle were measured with LB-520 thermo-hygrometers (Lab-EL, Poland). The duration of transportation and the conditions inside the stock crate are presented in Table 2. Goat kids were transported to the slaughterhouse on a windless day (in June) with ambient temperature of 14.3 ℃ and relative air humidity of 61% at 6 h. Vehicle speed, temperature, and humidity inside the vehicle during the 90 min of transportation were adequate. The space allowance per animal inside the vehicle was consistent with the provisions of Council Regulation (EC) No. 1/2005 of 22 December 2004 on the protection of animals during transportation and related operations.

Table 2

Parameter	Range
Duration of transportation	1.5 h
Vehicle speed	55-65 km/h
Stocking density	0.3 m2/animal
Air temperature	15.6-16.3 ℃
Relative humidity	70.2-76.8%

The animals were weighed before and after transportation to the slaughterhouse to determine live weight loss. Blood was sampled from the jugular vein before transportation (BST1) and after transportation (BST2) to determine stress responses. Hematological analyses were performed to determine red blood cell counts (RBC), white blood cell counts (WBC), hemoglobin concentration (HGB), hematocrit (HCT), and leukocyte profile (leukogram). Basic parameters were determined in the Sysmex hematology analyzer. A differential analysis of leukograms was performed by Pappenheim blood smear staining, and the results were used to calculate the neutrophil to lymphocyte ratio (N:L). Cortisol and glucose concentrations in the blood serum were determined in biochemical analyses with a Cobas Integra 800 analyzer (Roche, Rotkreuz, Switzerland). Cortisol levels were measured with a competitive enzyme immunoassay kit with an anti-cortisol polyclonal antibody (Elycsys Cortisol, Roche, Rotkreuz, Switzerland). Glucose levels were measured with an enzymatic reference method involving hexokinase. Blood parameters were determined in a specialist laboratory according to the Analytical Quality Control procedure (Chapter 2: Laboratory diagnostics).

The animals were handled during transportation, and blood samples were collected by the same team of experienced professionals. The duration of handling operations, including herding, weighing, and blood sampling in the goat farm and in the abattoir did not exceed 45 s.

The results were processed statistically in the Statistica 13.0 PL program (StatSoft Inc., Tulsa, OK, USA). Live weight loss and blood parameters were analyzed (x⁻ ±SD). The results were validated by two-way (GK × BST) or one-way (GK or BST) ANOVA. The differences among groups of kids (GK) or blood sampling time (BST) were estimated using the following model:

in which Y_ij is the observation of dependent variable, µ is the overall mean, Y_i is fixed effect of supplementation = (Y_i = µ_i - µ), µ_i is the mean for the i group, and e_ij is the random residual error.

Fixed effect of supplementation (GK) and blood sampling time (BST) and their interaction were included in the model. The model used was:

in which Y_ij is the observation of dependent variable, µ is the overall mean, S_i is the effect of HMB supplementation (GK), T_j is the effect of blood sampling time (BST), (S × T)_ij is the interaction term of GK and BST, and e_ij is the random residual error.

During the 60 days of the study, no great differences in the feed intake of the kids were recorded (Table 1). The experimental goats consumed no more than 0.62% DM and, consequently, no more crude protein and crude fiber. It may be thus assumed that it was comparable in both groups.

No significant differences in body weights were observed between groups before transportation and after unloading in the slaughterhouse (Table 3). Body weights were somewhat higher in animals whose diets were supplemented with HMB, but the noted difference was not significant. Live weight loss after transportation (kg, %) was significantly lower in the experimental group than in the control group.

Table 3

Indices	Group of kids
	Control	Experimental
Body weight before transportation (kg)	15.78±2.30	16.64±2.76
Body weight after transportation (kg)	15.25±2.27	16.25±2.63
Body weight loss during transportation
kg	0.53±0.16x	0.39±0.14y
g kg−1	33.7±8.9x	23.6±9.7y

x,y - means within rows followed by different letters are different at P≤0.05.

In the group of hematological parameters, RBC, WBC, HBC, and HCT values did not differ significantly between GK or BST (Table 4).

Table 4

Blood parameter	Group of kids (GK)				Significance P-value
	Control (CG)		Experimental (EG)
	CG-BST1	CG-BST2	EG-BST1	EG-BST2	GK	BST	GK × BST
RBC (1012⋅L−1)	2.61±0.51	2.90±0.47	2.42±0.48	2.72±0.48	NS	NS	NS
WBC (109⋅L−1)	14.49±2.42	17.01±3.21	13.58±3.40	16.08±5.27	NS	NS	NS
HGB (mmol⋅L−1)	6.23±0.48	6.54±0.58	5.96±0.66	6.25±0.75	NS	NS	NS
HCT (L⋅L−1)	0.86±0.18	0.96±0.17	0.79±0.17	0.89±0.18	NS	NS	NS
N:L	0.62±0.11B	1.29±0.45A	0.83±0.17D	1.18±0.30C	NS	**	NS
Cortisol (mmol⋅L−1)	45.54±13.80B	136.62±36.16AY	35.88±15.18D	97.70±14.35CZ	**	**	**
Glucose (mmol⋅L−1)	3.76±0.62B	7.56±1.91A	4.03±0.53D	7.06±1.75C	NS	**	NS

BST - blood sampling time; BST1 - before transportation; BST2 - after transportation; RBC - red blood cell; WBC - white blood cell; HGB - hemoglobin; HCT - hematocrit; N:L - neutrophil to lymphocyte ratio; NS - not significant.

Cortisol levels were significantly affected by both GK and sampling dates. Transportation led to a significant increase in cortisol concentration in both groups, but it was significantly higher in the control than in the experimental group, in which the diet was supplemented with HMB (Table 4).

Serum glucose concentration did not differ significantly between groups. The above parameter increased significantly after transportation in both groups. A similar pattern of changes was observed in the N:L ratio (Table 4).

In the current experiment, live weight loss after transportation (Table 3) was significantly lower in the experimental group than the control group. The experimental kids were heavier and characterized by significantly lower weight loss.

Numerous authors have demonstrated that transportation to the slaughterhouse and the accompanying handling operations are considerable stressors that decrease live body weight. During transportation, animals lose weight due to dehydration and loss of energy required for the maintenance of homeostasis (Knowles, 1995). Many researchers have evaluated body weight loss in young ruminants in view of the animals’ age and breed, duration of transportation, and space allowances inside the vehicle (Kannan et al., 2000, 2007a,b; Kadim et al., 2006; Kadim et al., 2007; Nikbin et al., 2016; Akin et al., 2018; Yalcintan et al., 2018), but their findings were inconclusive. In a study by Kannan et al. (2000), live weight loss in 15-month-old goats was not affected by stocking density and reached 9.8-10.2% after 2.5 h of transportation. In contrast, Nikbin et al. (2016) reported higher live weight loss (1.36%) in 12-month-old goats after 3.5 h of transportation at higher stocking density compared with lower stocking density (0.44%). In the work of Akin et al. (2018), differences in stocking density did not influence live weight loss during short transportation (45 min), whereas significantly higher weight loss was noted after longer transportation (3 h) at higher stocking density.

Live weight loss during transportation to the slaughterhouse should be minimized to increase profits, and many researchers have analyzed the efficacy of various supplements that enhance defense mechanisms and decrease weight loss in animals (Kannan et al., 2007b; Minka and Ayo, 2007, 2013; Minka et al., 2009; Sowińska et al., 2017). The results of their studies were also ambiguous. The addition of seaweed extract to the diets of goats of two breeds did not affect live weight loss after 6 h of transportation, but significant differences in the evaluated parameter were noted between breeds (Kannan et al., 2007b). Sowińska et al. (2017) found no significant differences in the body weights of young rams fed beta-glucan, determined after 80 min of transportation. According to other authors, the administration of ascorbic acid (vitamin C) minimized live weight loss in goats. Minka and Ayo (2007) observed significantly lower weight loss (1.04%) in the group of goats supplemented with vitamin C than in the control group (11.9%) after 8 h of transportation. Lower live weight loss was also reported in goats that received ascorbic acid (1.6 and 5.7%) before a 12-h-long transportation (Minka et al., 2009). Ambore et al. (2009) demonstrated that herbal preparations (Restobal liquid, Stresomix premix) were effective in reducing transportation-related live weight loss in goats. In the present study, the experimental kids were characterized by significantly lower weight loss after transportation, which suggests that dietary supplementation with HMB results in positive effects.

Transportation is one of the greatest stressors in livestock rearing. The severity of stress is determined not only by handling operations before transportation and slaughter (Knowles et al., 1998; Fisher et al., 2005), but also by other factors that cause physiological changes in animals (Hartung, 2003; Minka and Ayo 2007). The values of RBC, WBC, HGB, and HCT did not differ significantly between blood sampling times (Table 4), which is consistent with the results of many studies. Ekiz et al. (2012) did not observe significant differences in the hematological parameters of lambs after 75 min of transportation in comparison with base levels. Bórnez et al. (2009) also reported an absence of significant variations in RBC, HCT, and HGB values in 30-day-old and 70-day-old lambs after 30 min of transportation. Ali et al. (2006) did not observe significant differences in HCT, HGB, or WBC in lambs after 2 h of transportation, and Miranda-de la Lama et al. (2011) found similar values of RBC, WBC, and HCT in lambs after 3 h of transportation.

Stress stimulates the hypothalamic-pituitary-adrenal axis to release cortisol from adrenal glands, which is why cortisol concentration in the blood is regarded as a reliable indicator of stress (Ferguson and Warner, 2008; Minka and Ayo, 2010b; Zimerman et al., 2013). Numerous authors have reported an increase in the serum cortisol levels of transported animals (Knowles, 1995; Nwe et al., 1996; Kannan et al., 2000, 2003; Kadim et al., 2006; Sowińska et al., 2006; Miranda-de la Lama, 2010; Yalcintan et al., 2018).

Kannan et al. (2000) noted a significant increase in goat cortisone levels after 2.5 h of transportation, regardless of stocking density. In another study, cortisol concentration increased after 2 h of transportation in Alpine goats from all age groups (Kannan et al., 2003). A considerable increase in the cortisol levels of male goats, regardless of breed, was also reported after 2 h of transportation by Kadim et al. (2006). In the current study, cortisol concentration increased significantly in 90-day-old male Alpine goats after 1.5 h of transportation. The above findings confirm that even short transportation is a source of considerable stress for animals.

Glucose is a source of energy that is rapidly depleted under exposure to stress (Broom et al., 1996), which is why serum glucose concentration is also regarded as an indicator of transportation stress in animals (Kannan et al., 2000, 2003; Minka and Ayo, 2010a). Research has demonstrated that an increase in blood glucose levels is preceded by an increase in cortisol concentration (Sanhouri et al. 1992). Cortisol plays a very important role in gluconeogenesis because it stimulates the liver to convert fat and protein to indirect metabolites. These metabolites are ultimately converted to glucose as a source of energy (Saeb et al., 2010). Numerous researchers have demonstrated that glucose is a reliable indicator of stress in animals. Glucose levels increased in response to the stress induced by 1.5-2.5 h (Kannan et al., 2000, 2003; Rajion et al., 2001; Ali et al., 2006; Sowińska et al., 2016, 2017), 6 h (Nwe et al., 1996; Galipalli et al., 2004; Kannan et al., 2007b), as well as 12 h of transportation (Minka and Ayo, 2010a). In the present experiment, a significant increase in the serum glucose levels of goat kids transported to the slaughterhouse also indicates that glucose is a useful parameter for evaluating stress responses in animals.

Many studies have also evaluated the impact of transportation stress on the N:L ratio in livestock. Stress triggers the release of corticoids, which increase neutrophil counts and decrease lymphocyte counts in the leukogram (Stanger et al., 2005). A significant increase in the N:L ratio was observed regardless of the duration of transportation (1.5-12 h) (Nwe et al., 1996; Kannan et al., 2000, 2007a; Rajion et al., 2001; Galipalli et al., 2004; Minka and Ayo, 2007; Minka et al., 2009; Sowińska et al., 2016, 2017). In the current study, the N:L ratio in goat kids also increased significantly after transportation to the slaughterhouse.

Various methods for minimizing stress and its negative effects on animal health have been evaluated in the literature. The efficacy of various preparations, including seaweed (Ascophyllum nodosum) extract (Galipalli et al., 2004; Kannan et al., 2007a,b), xylazine (Ali et al., 2006), ascorbic acid (Minka and Ayo, 2007, 2010a; Minka et al., 2009), and dried yeast (Saccharomyces cerevisiae) (Sowińska et al., 2016, 2017), has been evaluated in animals. In the works of Galipalli et al. (2004) and Kannan et al. (2007a), seaweed (Ascophyllum nodosum) extract did not lower cortisol concentration, glucose levels, or the N:L ratio in goats after transportation. Dietary supplementation with dried brewer’s yeast (Sowińska et al., 2016) and beta-glucan (Sowińska et al., 2017) did not cause significant differences in cortisol levels between control and experimental lambs, but it significantly lowered blood glucose and the N:L ratio after transportation.

Ali et al. (2006) showed a significantly higher HCT and lymphocyte counts and significantly lower cortisol and glucose levels in the group of goats which received xylazine before the 2-h transportation compared with the control group. The efficacy of ascorbic acid supplementation before transportation, in particular under exposure to high temperature and high humidity, was demonstrated in a series of experiments by Minka and Ayo (2007, 2013) and Minka et al. (2009). In the present study, goat kids fed diets supplemented with HMB were characterized by significantly lower cortisol concentration after transportation. The evaluated supplement had no significant effect on glucose levels or the N:L ratio, but glucose concentration after transportation increased 1.7-fold in the experimental group and 2-fold in the control group. The N:L ratio increased 1.4- and 2.1-fold in the respective groups, which indicates that HMB is a promising feed additive.

The results of this study demonstrated that the supplementation of goat kid diets with β-hydroxy-β-methylbutyrate (at 50 mg kg¹ of BW) can alleviate stress and its consequences on the animals’ health. The analyzed immunostimulant, a leucine derivative, has a beneficial influence on immune function and reduces stress responses evaluated based on blood parameters and live weight loss.