Effects of pregnancy and post-kidding stages on haematochemical parameters in cross-bred goats

ABSTRACT The effects of pregnancy and post-kidding stages, and the number of in utero developing foetuses on haematochemical parameters were evaluated in stabled cross-bred goats. Blood serum samples were collected biweekly during the late-pregnancy (–49 days) and post-kidding stages (21 days) (n = 34 goats, including 17 Anglonubian and 17 Saanen × Anglonubian). Non-pregnant, cross-bred, adult does (n = 17) were used as controls. Blood serum glucose (GL), total protein (TP), albumin (AL), uric acid (UA), total cholesterol (TC), total bilirubin (TB), creatinine (CR), and alkaline phosphatase (AP) were measured using commercial kits. Average GL, TP, AL, and AP values were found to be different (p < .05) in pregnant, control, and post-kidding stages. Average GL level was found to be different (p < .05) in Anglonubian vs. Saanen×Anglonubian goats. Number of developing foetuses had no effect on any measured haematochemical parameters. In conclusion, both the late pregnancy and post-kidding stages of Anglonubian and Saanen × Anglonubian goats were accompanied by changes in blood serum GL, TP, AL, and AP. Breed effect was only observed on serum GL, while number of kids developing in utero did not affect the measured haematochemical parameters of goats.


Introduction
Concentrations of metabolites and enzymes in blood serum are biochemical values having strong relationships with physiological events in goats (Zulkifli et al. 2010). It has been reported that haematochemical parameters may be influenced by various factors, including temperature, reproductive status, metabolic disorders, age, sex, breed, season of the year, stress caused by management, road transportation (Sandabe & Chaudhary 2000;Žubčić 2001;Mellado et al. 2004; Barakat et al. 2007;Piccione et al. 2009Piccione et al. , 2012aPiccione et al. , 2012bPiccione et al. , 2013Sakha et al. 2009;Rumosa et al. 2010;Zulkifli et al. 2010;Arfuso et al. 2016), and infectious processes (Mahmood et al. 2015). Management practices, such as feeding, health condition and productivity, may influence the haematochemical parameters (Ismail et al. 2008;Waziri et al. 2010). Manat et al. (2016) pointed out the importance of determining whether normal reference haematochemical values are related to changes in physiological and metabolic stages (i.e. pregnancy, post-kidding, and number of developing foetuses). Particularly, blood physiological values may be influenced by genetic background and productive performance (Vallejo et al. 1991). Therefore, physiological blood serum-specific chemical reference parameters and their variation should be established in purebred and cross-bred dairy female goats under intensive farming systems (Tibbo et al. 2008;Mohammed et al. 2016). This knowledge can be used for purposes of diagnosis and prognosis of diseases, for criteria of adaptability, as well as to elucidate many other physiological mechanisms in goats (Gökdal 2013). Accordingly, the aim was to evaluate for the first time the effects of pregnancy and post-kidding stages, and the number of in utero developing foetuses on haematochemical parameters in stabled cross-bred dairy goats.

Animals and management
The study was carried out at the Sheep and Goat Research Unit of the Autonomous University of Baja California Sur. The average annual rainfall of this region is 200 mm and mean annual temperature is 14-24°C. Healthy, adult goats [n = 34; 17 Anglonubian and 17 Saanen (sire) × Anglonubian (dam)] were oestrus-synchronized and mated naturally with fertile Saanen bucks during June in order to have the kidding season during November. Adult, non-pregnant, cross-bred does (n = 17) were used as controls. Pregnancy and number of in utero developing kids were diagnosed by real-time ultrasonography (Preg-Alert ®, Sis-Pro, Inc. 6 Valley Trail Round Rock,TX). The number of kids and litter weight were recorded at birth. Body condition score measured 3-4 (scale 1-5) during the trial in both breeding and control goat groups. All the experimental animals received a daily diet of alfalfa hay (3.5% of live body weight) plus 0.3 kg/goat of concentrate with 16% crude protein. Fresh water and mineral blocks were offered ad libitum; mineral blocks included supplementary calcium, phosphorus, magnesium, manganese, sodium chloride, potassium, iron, iodine, and selenium. All goats were housed in open shaded allotments and provided access to sunny exercise areas.

Blood sampling
Blood samples were taken from the goats during the latepregnancy and post-kidding stages by jugular venipuncture using Vacutainer tubes without additives (BD, 1 Becton Drive, Franklin Lakes, 07417 NJ, USA) and sterile needles. Pregnant goats were bled weekly from day -49 (n = 7 sampling dates) prior to kidding to day +21 (n = 3 sampling dates) postkidding (day of kidding = 0). During this period, it was supposed that goat physiology adjusts in order to fulfil the foetuses' demand for nutrients and, later, to reach the lactation peak production (Celi et al. 2008;Magistrelli & Rosi 2014;Radin et al. 2015). Control goats were sampled the same as pregnant/ post-kidding goats. In all cases, withdrawal of feed and water was carried out 12 hours prior to the time of bleeding (07:00 am), since fasting has been recommended prior to the measurement of the haematochemical parameters (Rezaei et al. 2013;Yatoo et al. 2015).

Statistical analysis
Overall descriptive statistics (mean, standard deviation, and ± 95% confidence intervals of mean) for each blood constituent were calculated. The data were tested for normality (Shapiro-Wilk W normality test) and homogeneous variance (Levene test). The effects of the factors evaluated were tested by separate analyses of variance for a complete random design (Steel & Torrie 1960). For evaluating each factor, the model applied was as follows: where Y is the blood constituent response (blood serum concentrations of GL, TP, AL, UA, TC, TB, CR, or AP), µ is the general mean, τ is the factor tested (physiological stage, breed group, or number of in utero developing foetuses), and ε is a random element of variation. Interactions (physiological stage × breed group × number of in utero foetuses) were formerly included in a factorial arrangement model, but were deleted later on due to a lack of significance demonstrated in an a priori analysis. When the effect of a factor was found to be significant, post hoc Tukey's test was used to determine statistically significant differences (p < .05). All statistical procedures were performed using the statistical software Statistica (StatSoft 1998).

Results
Overall means and confidence intervals of the haematochemical parameters of all goats are summarized in Table 1. Table 2 shows that mean serum concentrations of GL, TP, and AL were significantly different between pregnancy and postkidding stages, and between experimental and control groups. Particularly, the GL level in pregnant goats was significantly lower than that in post-kidding and non-pregnant control goats. The mean TP level was lower (p < .05) in postkidding goats, but similar in pregnant and control goats. AL values were significantly (p < .05) lower in pregnant compared to post-kidding and control goats. Average blood serum level of AP was higher (p < .05) in control, followed by pregnant and post-kidding groups.
The haematochemical parameters of pregnant goats according to breed group are shown in Table 2. The mean blood serum GL level was higher (p < .05) in Anglonubian in comparison to Saanen × Anglonubian goats.
Number of developing foetuses did not have a significant effect on the haematochemical parameters in pregnant goats ( Table 2). The average number of kids at birth was 1.89 ± 0.57 (range = 1-3) and litter weight at kidding was 6.0 ± 1.75 kg (range = 2.3-9.82 kg).

Discussion
The majority of blood constituent levels were consistent with the general normal ranges reported for goats (Ismail et al. 2008). The results of this study agree with other authors suggesting that GL and AL are critical molecules for meeting goat's nutritional requirements during the late-pregnancy stage (Mellado et al. 2004;Ismail et al. 2008). Such nutrients are likely directed towards metabolic purposes related to either foetal growth or colostrum production in the udder, as observed in cattle (Funston et al. 2010;Bach 2012). The blood serum TP level, which was comparable to previous results (Deljou et al. 2014), did not change in pregnant or control groups. These results coincide with previous findings reported for Indian goats, as well as for Sahelian and Canarian breeds (Patra et al. 2003;Waziri et al. 2010;Zabaleta et al. 2010). Celi et al. (2008) found that blood serum TP was significantly lower after parturition when compared with that in pregnant goats. It has been demonstrated in Nubian goats that the decrease in blood serum protein, particularly γ-globulin, is due to its removal from the blood stream in order to support mammary secretion after parturition (Chen et al. 1998). Such proteins were later found in the blood serum of neonatal kids to support trophic and absorption processes (Chen et al. 1999). AL level in blood serum was also lower in pregnant goats compared to that in post-kidding stage and non-pregnant (control) goats. Similar results were obtained in pregnant and non-pregnant goats from Croatian, Red Syrian, Saanen, and Oberhasli breeds (Žubčić 2001;Celi et al. 2008;Castagnino et al. 2015). When these results are put together, it appears that blood AL was directed to foetal growth tissue, while globulin may be used mainly for milk synthesis, as occurs in ewes (Balıkcı et al. 2007;Karapehlivan et al. 2007;Obidike et al. 2009).
In the present study, control (non-pregnant) goats showed increased levels of AP activity. When pregnancy and postkidding stages were compared, AP activity was found to be higher during the pregnancy, suggesting that the physiological removal of this enzyme in goats is related to foetal growth and early milk production processes. This physiological pattern of AP has been previously observed for bone-specific AP in pregnant and kidding goats, in which AP decreased slowly from the second month of pregnancy until the first week post-kidding to support osteoblastic activity (Liesegang et al. 2006). Moreover, these results coincided with higher levels of AP in women recorded during the last third of pregnancy which decreased after parturition (Valenzuela et al. 1987). The key mechanisms of AP activity during pregnancy and post-partum are incompletely understood, and further study is needed to elucidate the role(s) of this enzyme during the peri-kidding period in goats.
CR, UA, urea, ammonia, allantoin, hippuric acid, and small quantities of amino acids are constituents of urine nitrogen losses (NRC 1985). In this regard, increased levels of CR and UA indicate kidney damage, while high levels of bilirubin and cholesterol are related to hepatic failure (Cornelius, 1989). In contrast, low concentrations of those parameters are related to dietary energy and protein levels (Mohamed et al. 2016). In this study, values of CR, UA, cholesterol, and bilirubin were not affected by the physiological status or the genetic cross and remained in the range that is considered normal. These results indicate that nutritional management of the goats was appropriate, according to their physiological stages, and unaffected by genetic background (  Blood samplings were carried out weekly from -49 to 21 days post-kidding (day of kidding = 0). Mean values were calculated from the number of serum samples collected from the goats included in each group. 2 Number of foetuses were detected by real-time ultrasound and confirmed at birth. Goats gave birth to 1 (n = 7), 2 (n = 21), or 3 (n = 6) kids. No significant statistical differences were found (p > .05). 3 Values in columns with different superscripts indicate statistical differences; n = number of valid processed blood serum samples.
In the present study, the haematochemical parameters of Anglonubian and Saanen × Anglonubian cross-bred goats were found to be in the normal range for healthy goats of various breeds and production systems (i.e. Žubčić 2001; Mellado et al. 2006;Ríos et al. 2006;Barakat et al. 2007;Ismail et al. 2008;Cook et al. 2008;Sakha et al. 2009;Okonkwo et al. 2010;Rumosa et al. 2010;El-Khodery et al. 2011;Oni et al. 2012). Values of the haematochemical parameters were similar in both breed groups, with the exception of GL levels. Similar to the results obtained here, GL levels decreased during the pregnancy stage in Red Syrian, Sahel, Alpine, and Saanen breeds (Celi et al. 2008;Waziri et al. 2010;Laporte-Broux et al. 2011). In contrast, lower GL levels were recorded in cross-bred goats, suggesting that Saanen goats have a lower normal level of GL in blood than Nubians (Mellado et al. 2004(Mellado et al. , 2006Laporte-Broux et al. 2011).
Conversely to what would be expected, the number of foetuses in utero did not cause significant differences in the haematochemical parameters. This matches well with the finding that maternal metabolism throughout the pregnancy of dairy goats did not vary regardless of the breed or number of foetuses (single or twin pregnancies) (Castagnino et al. 2015). In contrast, ovine pregnancies with more than one foetus are often accompanied by changes in the metabolic profile, compared with single pregnancies (Hefnawy et al. 2011). Similar observations have been reported in bovine pregnancies (Bach, 2012;Alberghina et al. 2015). Overall, the peri-partum period is accompanied by marked changes in the haematochemical parameters, but female goats appeared to adapt during these physiological stages by different physiological mechanisms (Azab & Abdel-Maksoud 1999;Iriadam 2007). Therefore, physiological adaptation mechanisms in Anglonubian and Saanen × Anglonubian cross-bred goats merit further research using a greater number of goats.

Conclusion
The late-pregnancy and post-kidding physiological stages of Anglonubian and Saanen × Anglonubian goats are characterized by changes in some haematochemical parameters (GL, protein, and AL). A breed effect was only observed on serum GL parameter, while number of kids developing in utero did not affect the haematochemical parameters of goats.