Determination of haemato-biochemical profile and acid–base status is very important for monitoring the metabolism and health status of small ruminants (Carlos et al., 2015; Antunović et al., 2017a, b). The values of haemato-biochemical parameters and acid–base indicators of goats are influenced by several factors such as breed, age, physiological status, sex, nutrition or season (Piccione et al., 2010; Arfuso et al., 2016; Antunović et al., 2017a; Redlberger et al., 2017). Biochemical parameters, primarily metabolites, enzymes, proteins and acid–base parameters of the blood, indicate possible metabolic disorders and disorders caused by inadequate nutrition (Antunović et al., 2002; Rios et al., 2006). In recent years, the determination of the acid–base status of blood has included more parameters such as the calculation of the anion gap (AG), strong ion difference (SID), z values, base excess, and the determination of organic acids (lactate, keto acids) and inorganic anions (sulfates, phosphates, etc.). The values of the acid–base status have not yet been determined for goats, and the precise negative charge of albumin and globulins and their dissociation constant is still unknown (Redlberger et al., 2017). Gärtner et al. (2019) suggested using more information about the acid–base status by calculating SID, weak anions and cations (ATOTs), the strong ion gap (SIGalb), or unmeasured anions (XA) compared to traditional parameters. Besides, it is important to specify the exact values for every breed of goat of all ages, since climate zones and regions affect the haemato-biochemical profile and acid–base status of animals equally.

The Croatian spotted goat, known as a Balkan goat, is the most abundant goat breed in Croatia (estimated population is approximately 25 000 heads; CAA, 2018), developed and mostly reared in the karst, barren and inaccessible area of the Velebit, Dinara, Kamešnica and Biokovo mountains. Due to their skills and mobility, these goats are reared on the most inaccessible terrain, which is not adequate for rearing other livestock, especially cows (Mioč et al., 2008). The Croatian spotted goat is resistant and flexible with modest requirements for rearing and feeding. Production traits of an adult goat are as follows: average body weight is 44 kg, while the milk yield in 150–250 d of lactation is 100–250 L of milk (Mioč et al., 2008; Antunović et al., 2018). However, comprehensive research on the haemato-biochemical profile and the acid–base blood status of the Croatian spotted goat is missing. Therefore, the aim of the present research is to determine the haemato-biochemical profile and blood acid–base status of Croatian spotted goats in Mediterranean production systems.

Analysing Table 2 it is evident that the WBC number and MCV in the blood of goats from group I was higher compared to groups of older goats. Haemoglobin in group I was higher compared to groups II–IV, while in group IV, it was higher compared to group II. By contrast, the MCHC content was higher in older goats compared to group I. No differences were observed among groups II–IV. The distribution of leukocytes did not differ between goats of different ages.

Significantly lower concentrations of urea, Mg, Cl and lactate as well as lower activities of ALP and CK in goats' blood were observed in older goats compared to group I (Table 3). Significantly higher concentrations of Ca, Na, total cholesterol, HDL cholesterol, VLDL cholesterol and BHB as well as higher activity of ALT in the blood of older goats were observed compared to group I (Tables 3 and 4). The concentration of Na, VLDL and TGC did not differ when group III was compared with other groups. The concentration of Cl did not differ when group I was compared with group II and group IV. The cholesterol concentration was lower in group I compared to group IV, while groups II and III did not differ. The concentration of NEFA was higher in group I than in groups II and III, while the concentration of BHB was lower compared to group IV. The activity of SOD and GPx did not differ when comparing young with older goats, although a tendency of decrease with age was observed.

Analysing Table 5 in the blood of goats, lower pH, SID, AG and z values as well as the content of HCO3 and ctCO2 was determined in group I compared to older goats. By contrast, significantly higher pCO2, Cbase (B)e and Cbase (Ecf)c in the blood of older goats compared to group I was observed. The values of AG did not differ when comparing group I or group IV with groups II and III. The values of pO2, O2, ATOTtp, ATOTalb, SIGtp, SIGalb and XA did not differ between groups.

The majority of the results in the blood of haemato-biochemical parameters and acid–base indicators were within reference values (Kaneko et al., 2008; Antunović et al., 2013, 2017a). The haematological blood parameters, such as RBC, WBC and HGB, can reflect physiological occurrences in an animal's body. In the present study the WBC number, HGB concentration and MCV in the blood of goats was lower and MCHC was higher in older goats compared to group I. Arfuso et al. (2016) in a study with Messinese goats determined more RBC and a higher content of HGB in younger goats compared with older goats, as well as similar difference in MCV and the opposite trend in MCHC in the blood of Argentata dell'Etna goats. Similar results with RBC and HGB concentration in the same goat breed determined Piccione et al. (2010). The differences in RBC could be related to the oxygen-carrying capacity of the blood, which is higher in young goats compared with old ones (Daramola et al., 2005), while changes in WBC were connected with suitable protection systems, due to immune variations in animals (Piccione et al., 2014; Arfuso et al., 2016). The MCV and MCHC in goat fluctuated mostly depending upon RBC, HGB and HCT values. This is in accordance with conclusions by Egbe-Nwiyi et al. (2000). Generally, most of the haematological parameters determined in our study were within the normal range for goats reported by other authors (Zumbo et al., 2011; Habibu et al., 2017).

In the current study significantly lower urea, Mg, Cl and NEFA concentrations in goats' blood were observed as well as lower activities of ALP and CK, while significantly higher Ca, Na, total cholesterol, HDL cholesterol, VLDL cholesterol and BHB concentrations as well as higher activity of ALT was observed in older goats compared to group I. The results from the present study (4.46–9.23 mmol L-1) were within reference values for urea (2.9–10.9 mmol L-1,; McDougall et al., 1991) which may be related to the good content of proteins in goats' diet (Table 1). In fact, Kohn et al. (2005) showed that the concentration of urea can be considered as a good indicator of the amount of nitrogen consumed through feed. Piccione et al. (2010) obtained a decreased urea concentration in the blood of Girgentana goats from 1–2 to 3–4 years and an increased concentration from 5 to 6 years. An increase in urea concentrations in young non-pregnant goats was observed compared with adult Danish landrace dairy goats (Mbassa and Poulsen, 1991a).

In the present study, plasma Ca levels in goats younger than 1 year were at the limit of the reference values (Kaneko et al., 2008; 2.3–2.9 mmol L-1). Table 1 presents adequate concentrations of Ca in feedstuffs, which was offered to goats of all ages. A higher sodium concentration in the blood of older compared to younger sheep was determined Antunović et al. (2004). Gwaze et al. (2012) determined significantly lower concentrations of P as well as lower activity of ALP in older compared to young Nguni goats from South Africa. A lowered concentration of P in the blood could be the cause of a decreased capacity to assimilate phosphorus from the diet with increasing age of animals (Blood and Radostis, 1993). Kaneko et al. (2008) reported that serum phosphate may be higher in younger animals because the growth hormone increases renal phosphate resorption. In the present study the concentration of Mg was lower in older groups compared to group I. A similar trend for Mg concentration in the blood of Angora goats (1.66–1.44 mmol L-1) was determined by Van Niekerk and Cloete (1990) and in the research by Pandey et al. (2006) in Marwari goats (1.06–1.027 mmol L-1). As reported by Bhattacharyya et al. (1994) that was associated with an increased mobilisation of minerals from the bone because of an increased demand for growth at this stage or due to an increased rate of resorption from the gastrointestinal tract.

Higher concentrations of total cholesterol, HDL cholesterol and VLDL cholesterol in the blood of older goats were observed compared to group I. A similar trend for blood cholesterol concentrations was determined in Marwari goats from India (Pandey and Sareen, 2007) and in Hair goats from Turkey (Karaşahin et al., 2018). Very few studies are available regarding NEFA concentrations within goats' blood. Higher NEFA in yearlings compared to older goats indicates a certain energy deficit, since, according to Dunshea and Bell (1989), a concentration of 0.21 mmol/L is suggested for lactating goat at zero energy balance. Mondal and Prakash (2004) explained that the concentration of the growth hormone in the plasma of buffaloes decreases along with lipolysis resulting in less NEFA in the plasma as they become older. An increase in BHB in goat blood determined with increased age was within the physiological range, since Doré et al. (2015) reported a hypoketonemia definition based on BHB concentrations from ≥0.4 to ≥0.9 mmol L-1. According to Herdt (2000) BHB is more likely a reflection of nutritional status and less constrained physiologically.

In the present study, higher activities of ALT and AST in the blood of older goats may be used as indicators of physical stress (Mbassa and Poulsen, 1991b). The activities of ALT and AST in the blood of Girgentana goats increased with their age, which was determined by Piccione et al. (2010). Perez et al. (2003) determined that levels of ALP are the highest in young Spanish ibex compared to adult ones; Antunović et al. (2004) also determined levels in Merinolandschaf sheep which may have been correlated with high osteoblastic activity in young animals (Durak et al., 2015). The highest CK activity in the blood of young goats could be an indicator of specific markers of damaged muscle. Similar results were obtained in Merino landschaf ewes by Antunović et al. (2004). The increasing CK activity in calves could be attributed to their growth and gaining muscle mass (Klinkon and Ježek, 2012). Besides, Mpkama et al. (2014) determined higher CK activity in 18-month-old cattle compared to 16- or 24-month-old cattle. A tendency of SOD and GPx activity decreased with age was found in serum. Dubreuil et al. (2005) observed a decrease in GPx in the serum of ewes with age when comparing yearlings and ≥4-year-old ewes.

In a general sense, metabolic activity modifies the acid–base balance, although it is difficult to estimate the degree of this contribution to the metabolic component of the acid–base balance (Castillo et al., 2000). According to Kaneko et al. (2008), the recommended value for the anion gap in goats is 10–20 mmol L-1, but for SID values, it is 40.00 mmol L-1 (Castillo et al., 2000). In the present study an increase in pH values with increased age is due to a decrease in pCO2 (12.9–9.68 kPa) and ctCO2 concentrations. A similar situation was observed by Redlberger et al. (2017) in study with goats aged 6–56 weeks. Significantly lower pH in the blood of the youngest compared to older goats in this study is related to significantly lower HCO3, Cbase (B) and Cbase (Ecf). Afterwards, pH values as well as HCO3, ctCO2, AG, SID and z values were significantly higher in older goats. The anion gap is an indicator used to investigate the presence of unmeasured anions and may differ with pCO2 and HCO3 (Fencl and Leith, 1993; Castillo et al., 1998). However, the value of the anion gap is limited for an explanation of the acid–base balance. The ketone bodies, classified as unmeasured anions, were detected by increased XA and/or decreased SIG(Alb) and SIG(Prt) in the study by Gärtner et al. (2019), although these parameters did not differ between groups of goats aged differently in the current study. Therefore, in the correct interpretation of the acid–base status of goats, during different ages, SID, base excess and z values need to be incorporated.

The haemato-biochemical profile and acid–base status of Croatian spotted goats from the present study are the first published reference values and can be useful for understanding the metabolic profile of this breed. Meanwhile, in preparing the reference values for the haemato-biochemical profile of Croatian spotted goats, the inclusion of the age effect in the model is mandatory. Goats from 2 to 7 years or older mostly do not have a changed metabolic profile. When comparing the youngest goats with older ones, it is important to include certain biochemical parameters, enzyme activities and parameters of the acid–base status. The results obtained may help in monitoring the health and nutritional status of goats, while the implementation of these parameters provides more in-depth information regarding the metabolic profile of differently aged goats.