Effects of nano zinc oxide supplementation on metabolic parameters during the transition period in Lacaune ewes

ABSTRACT

This study was conducted to determine the influence of nano zinc oxide (N-ZnO) on metabolic parameters in transition periods of the Lacaune ewes. The animals were allocated into two equal groups: the control group (n = 10) and the experimental group (n = 10) supplemented with 20 mg/kg of N-ZnO. Blood samples were collected from all ewes on days −30, −15, 0 (parturition), + 15 and +30. A time effect was observed on aspartate aminotransferase which decreased in both groups from day −30 to day 0 (P ≤ 0.05). All ewes on day 0 had lower alanine aminotransferase than on day −30 (P ≤ 0.001) and day −15 (P ≤ 0.05). In both groups, albumin and total protein levels on day −30, blood urea nitrogen level on day +30 (P ≤ 0.001), β-hydroxybutyric acid and non-esterified fatty acid levels on day +15 (P ≤ 0.05) were higher. Triglycerides on day +30, glucose on days −15, 0, + 30 were lower (P ≤ 0.001). Blood calcium was lower (P ≤ 0.05) and zinc was higher on day +30 (P ≤ 0.001). In conclusion, there was no significant effect of N-ZnO/kg supplementation during the transition period, but changes in metabolic parameters due to a time effect were observed.

KEYWORDS:

• Nano zinc oxide
• Lacaune ewes
• transition period

1. Introduction

The high productivity of ewes depends on the nutrients consumed. Imbalances in the intake of trace minerals such as zinc (Zn) either directly affect nutritional digestibility and metabolic parameters negatively or cause metabolic diseases that indirectly affect them (Ladipo 2000). Zn is vital for nutrient utilization, many metabolic functions, reproduction, performance, immune response and antioxidant protection, especially in critical conditions such as the presence of stress factors or during the transition period (Geetha et al. 2020; Hosseini-Vardanjani et al. 2020). Zn is commonly provided in the inorganic form of zinc oxide (ZnO); however, increased Zn bioavailability may help to improve performance and health (Shafi and Kumar 2020). ZnO nanoparticles (N-ZnO) have been the subject of nutritional applications due to their biodegradability into ions that can be easily adsorbed by the metabolism and become a part of the nutritional cycle (Swain et al. 2016). The mechanisms of action of nano minerals, whose sizes vary between 10 and 100 nm compared to their organic–inorganic forms, have been associated with behavioural changes related to their ability to penetrate tissues faster, especially with a higher surface area and higher enzyme activities and bioavailability without undergoing structural changes (Pandurangan et al. 2015; Mohamed et al. 2017; Uniyal et al. 2017; Shafi and Kumar 2020).

Some researchers have indicated that N-ZnO have been shown to exhibit strong protein adsorption properties, which can be used to modulate metabolism or cellular responses. One more feature of N-ZnO is their ability to remain in circulation for a longer time and take charge as a dietary modulator for hydrolase activity relevant to controlling diabetes, cholesterol and glucose metabolism by reducing blood glucose, triglycerides and non-esterified fatty acid (NEFA). It is also reported to exhibit strong gastrointestinal protective, antibacterial and antioxidative properties (Bedi and Kaur 2015; Chikkanna et al. 2019; Geetha et al. 2020). Furthermore, it was reported that because of their high bioavailability, the use of trace minerals such as N-ZnO can decrease mineral excretion and environmental pollution (Padmavathy and Vijayaraghavan 2008; Hosseini-Vardanjani et al. 2020).

The transition period is a critical process in which physiological and metabolic changes are most intense (Salar et al. 2018). Currently, studies are quite limited regarding the effect of N-ZnO on the metabolic parameters and the blood enzymes in the pre- and postpartum periods for ewes. It was hypothesized that N-ZnO due to its high bioavailability in metabolism, could induce positive effects on metabolic parameters during this period. Hence, this study was conducted to evaluate the effect of dietary supplementation with N-ZnO on metabolic parameters during transition period of ewes.

2. Material and method

2.1. Ethic approval

This study was approved by Animal Experiments Local Ethics Committee of Aksaray University with the date and protocol number 18.05.2022/ E-60580050-125.04-5272191. The trial was conducted at a local ovine farm in Yenikent County, Aksaray, Turkey (38°25′51″K 33°51′44″D).

2.2. Animals and experimental design

The study was performed with twenty pregnant Lacaune ewes in the 4th month of gestation. The ewes were randomly divided into two groups of 10 healthy ewes each, homogeneous according to body weight (60.0 ± 2.30 kg) and age (4–5 years). Each ewe was housed individually in the trial pens (1.5 m × 2.0 m) in a closed-sided barn. They were kept under the same environmental conditions in allocated adjacent to each use was individually housed in trial pen compartment at the barn. After parturition, record 0 was taken at the 5th± 1 day of lactation, and the ewes were kept with their single suckling lambs for 30 days. During the study, no vaccination or additional drug administration was applied to the ewes.

Animals were subjected to adaptation feeding for 15 days before starting the experiment. The trial lasted for a total of 60 days. All animals were fed a Total Mixed Ration (TMR) consisting of dry alfalfa hay, wheat straw, barley grain and concentrate feed to meet the nutritional requirements of the sheep, according to NRC (2007). Alfalfa hay and wheat straw were chopped at 10–20 mm particle length, barley grain ground and feedstuffs (salt, vitamin- mineral premix) were homogenized and provided to the ewes as two TMR. The animals in the control group (CON) were fed the basal TMRs (containing 30.56 and 31.24 mg Zn/kg DM for 30-day prepartum and 30-day postpartum periods, respectively) without any zinc supplementation (Table 1). Basal TMRs contained a Zn concentration that was lower than the level (39.34 mg/kg diet DM) suggested for 60-kg sheep with an average daily gain of 300 g/day (NRC 2007). The animals in the second group were fed basal TMR supplemented with 20 mg/kg nano zinc oxide (N-ZnO) from an inorganic zinc oxide source (Molchem NK007, size-30 nm). For each of the animals in the additive group, 20 mg/kg N-ZnO which was weighed the day before with the help of precision scales was spread on the upper surface of the feed placed in the feeders every morning. During this mixing, it was observed that N-ZnO in powder form penetrated the feed rapidly.

Table 1. Feed ingredients and chemical composition of the pre- and post-partum diets (TMRs) fed to the ewes.

Ingredient (% in a DM basis)	Prepartum diet (TMR1)	Post-partum diet (TMR2)
Alfalfa hay	18.49	21.94
Wheat straw	31.52	22.43
Barley grain, flake	24.66	27.42
Concentrated feed^a	24.38	27.12
Salt	0.54	0.54
Vitamin-Mineral Premix^b	0.41	0.54
Chemical composition
Dry matter (DM),%	89.47	89.28
Crude protein,% of DM	10.34	11.35
Crude ash,% of DM	7.35	7.39
Ether extract,% of DM	2.05	2.16
NDF,% of DM	42.08	39.86
ADF,% of DM	25.71	22.75
Ca,% of DM	0.58	0.66
P,% of DM	0.25	0.27
Zn, mg/kg DM	30.56	31.24
ME, MJ/kg DM^c	8.91	9.23

^a Containing (per 50 kg); 11.5 MJ/kg metabolizable energy, %14 crude protein.

^b Containing per kg of TMR; 40000 IU vitamin A, 10000 IU vitamin D3, 50 mg vitamin E, 90 mg Fe, 30 mg Cu, 40 mg Mn, 2 mg Se, 0.8 mg I, 0.70 mg Co.

^c ME (metabolizable energy) was calculated according to NRC (2007).

In the pre- and postpartum periods, the ewes had free access to the feed and clean freshwater. The feeders in ewes’ compartments were disposed of 0.5 m from the floor to avoid lambs consuming these feeds. Feeding was performed twice daily at 08:00 and 18:00 o’clock with a feed mixer on a drive-through feeder.

2.3. Chemical analyzes

Procedures described by AOAC (1997) were used to determine the values of crude nutritional matters, and Van Soest (1994) procedure was followed for determining the amounts of neutral detergent fibre (NDF) and acid detergent fibre (ADF) in the TMRs (Table 1).

Blood samples were collected from all ewes via the jugular vein into the evacuated tubes (10 mL-Becton Dickinson and Company, New Jersey, USA), without any anticoagulant. The samples were obtained 3 h after the morning feeding for a total of 5 times throughout the experiment, on the days 30 and 15 before the expected lambing date (prepartum), on day 0 (parturition) and on days 15 and 30 after lambing (postpartum). The blood samples were centrifuged at 3000 rpm for 15 min. Then, these samples were transferred to Eppendorf tubes and stored at −20°C until the analyzes were done. The serum biochemical parameters including aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, total protein, blood urea nitrogen (BUN), triglycerides, total cholesterol (T-cholesterol), β-hydroxybutyric acid (BHBA), non-esterified fatty acid (NEFA), glucose were measured using the commercial analytical kits (Randox, UK) and a spectrophotometer (Randox Daytona plus RX 4040, UK) colorimetrically. For the assay of AST and ALT described by the IFCC (1980) method, 50 µl of the sample and 500 µl of the ALT reagent were mixed in a test tube, and the absorbances at 340 nm was taken. The absorbance was read at 1, 2 and 3 min, respectively. Albumin was measured following the method of Gustafsson (1978) by a timed endpoint process using bromocresol green. Total protein was determined using the biuret method reported by Doumas et al. (1981). According to this method, the 3 ml total protein reagent, 20 µl of deionized water, protein standard, and 60 µl of the blood samples in each 1.0 ml tube was incubated for 10 min at ambient temperature and the absorbance at 540 nm of all tubes was read. The glucose, triglycerides and T-cholesterol contents of the plasma were determined enzymatically using commercial assay kits; BHBA was estimated colorimetrically using the Randox UV kinetic method by the D-3-hydroxybutyrate kit; and NEFA was estimated by the NEFA Kit (Randox Laboratories, UK). Calcium (Ca), phosphorus (P) and Zn were estimated by inductively coupled plasma mass spectrometry (ICP-MS, Thermo-X Series 2) using the method reported by Nuttall et al. (1995).

2.4. Statistics

The data were statistically analyzed by using IBM SPSS Statistics for Windows, Version 20.0. package programme. Kruskal Wallis test was used for independent group (control and supplemented with N-ZnO) comparisons, depending on the distributional properties of the data based on groups (according to results of Shapiro Wilk test). When the test statistics were statistically significant for the independent group comparisons, Dunn’s post hoc multi comparison test was used to know which group differ from which others. The chi-square test was used for proportions and its counterpart Fisher’s Exact test was used when the data were sparse. The difference between 2 groups (treatment), 5 time points and the interaction of these two main effects were tested with two-way repeated measures of ANOVA. The sphericity assumption was performed by using Mauchly’s test sphericity. As, a violation of this assumption, Wilk’s Lambda statistic was used as multivariate test results. For all statistical analyzes, any P value less than 0.05 was considered as statistically significant.

3. Results

3.1. Blood enzymes

In Table 2, the values of the blood enzymes (AST and ALT) are listed. There were no significant differences between the two groups due to the dietary treatment with N-ZnO. However, a time effect was observed on the AST level which decreased by 18.3% in the N-ZnO group, while a decrease of 0.99% was found in the CON group, from the start of the trial to parturition (P ≤ 0.05). Similarly, both the CON group and N-ZnO-fed ewes, on parturition (day 0), had lower serum ALT levels than on days −30 (P ≤ 0.001) and −15 (P ≤ 0.05). In addition, a time effect in the both CON and N-ZnO groups was observed with an increase in AST by 50.07% and 56.02%, respectively, from parturition to day +15, whereas a reduction in ALT levels by 26.3% and 18.8% in the parturition compared to prepartum day −15 (P ≤ 0.001). These parameters did not change during the postpartum period.

Table 2. The AST and ALT levels of Lacaune ewes fed with N-ZnO supplementation.

Items	Experimental groups		SEM*	P values
	CON	N-ZnO		T	D	T x D
Ewes, no	10	10
AST (IU/L)
−30 d**	100.30Aa	120.80Aa	5.098
−15 d	99.00Ab	108.70Ab	4.134
0 d	100.40Ab	98.70Ab	4.573	0.577	<0.001	0.060
+15 d	150.67B	154.00B	5.827
+30 d	145.22B	139.88B	4.561
ALT (IU/L)
−30 d	21.68Aa	24.52Aa	1.103
−15 d	20.12Ab	18.68Ab	1.241
0 d	14.83Bc	15.16Bc	1.062	0.447	<0.001	0.263
+15 d	15.06Bc	15.36Bc	0.897
+30 d	15.77Bc	16.76Bc	1.059

*SEM: Standard error of mean, **d: Day.

T; fixed effect of treatment; D; fixed effect of day; T x D; interaction treatment x day.

a,b Means with different lower case within a column are different (P ≤ 0.05).

A,B,C Means with different upper case within a column are different (P ≤ 0.01).

3.2. Protein metabolites in the blood serum of ewes

In Table 3, the average values of albumin, total protein and BUN of ewes, recorded during the trial are shown. Albumin, total protein and BUN were not statistically influenced by the experimental treatment. However, a time effect was observed on these parameters. The albumin at the beginning of the trial (day −30) was greater (P ≤ 0.001) in both groups according to day −15; moreover, it also decreased at the end of the trial (P ≤ 0.001) according to day −30, with values of 2.95 and 2.90, respectively. In addition, this value decreased from day −15 to parturition (day 0) by 3.07−2.40% and from parturition to the end of the trial by 2.3–2.6% in both diets free of additional Zn and the dietary supplementation with N-ZnO, respectively (P ≤ 0.05). Total protein levels were higher (P ≤ 0.001) in both groups at the start of the experiment (day −30) compared to the other periods. Total protein in the CON and supplemented N-ZnO groups increased (P < 0.01) from day 0 to day +15, after that, it did not show significant changes. BUN levels of the Zn-supplemented ewes and the control animals increased significantly up to prepartum day −15 (P ≤ 0.05), parturition and postpartum day +30 (P ≤ 0.01). However, there was no significant difference regarding BUN in the 0–15 days period.

Table 3. Albumin, total protein and BUN of Lacaune ewes fed with N-ZnO supplementation.

Items	Experimental groups		SEM	P values
	CON	N-ZnO		T	D	T x D
Ewes, no	10	10
Albumin (g/dL)
−30 d	3.05A	3.00A	0.029
−15 d	2.93Ba	2.91Ba	0.029
0 d	3.02b	2.98b	0.031	0.219	0.003	0.158
+15 d	2.96	3.00	0.038
+30 d	2.95Ba	2.90Ba	0.030
Total protein (g/dL)
−30 d	7.00A	6.89A	0.160
−15 d	5.15BC	5.20BC	0.267
0 d	5.09B	5.22B	0.165	0.744	<0.001	0.798
+15 d	6.13C	5.92C	0.197
+30 d	5.70C	5.75C	0.141
BUN (mg/dL)
−30 d	4.15Aa	5.72Aa	0.787
−15 d	4.86Ab	6.09Ab	0.760
0 d	12.04B	14.40B	1.168	0.581	<0.001	0.635
+15 d	13.61B	14.19B	1.198
+30 d	20.80C	20.03C	0.985

T; fixed effect of treatment; D; fixed effect of day; T x D; interaction treatment x day.

a,b Means with different lower case within a column are different (P ≤ 0.05).

A,B,C Means with different upper case within a column are different (P ≤ 0.01).

3.3. Carbohydrate and lipid metabolites in the blood serum of ewes

The average values of triglycerides, T-cholesterol, BHBA, NEFA and glucose of the ewes, were recorded during the trial are listed in Table 4. These parameters were not statistically influenced by the experimental treatment and no significant changes were observed between the two groups. However, a time effect was observed on these parameters. In both groups; while triglycerides increased from days −30 to −15 prepartum, it decreased at parturition (0 day) and postpartum day +15 (P ≤ 0.001). From parturition to the end of the trial, the CON and the N-ZnO supplemented group exhibited 60.9% and 46.6% lower triglyceride levels, respectively. A time effect was observed for T-cholesterol, which decreased by 4.7% in the CON group and 15.5% in the N-ZnO supplemented group (P ≤ 0.05) from prepartum day −15 to the parturition, whereas the differences between groups in terms of T-cholesterol were found to be insignificant. Dietary supplementation with N-ZnO has not affected the serum BHBA and NEFA levels however, these parameters were higher (P ≤ 0.05) on postpartum day +15 ewes compared to the other periods in both groups. In both groups, serum glucose was lower on days −15 and +30, and was higher on days −30, 0 (P < 0.05), and +15 (P < 0.001) than in previous periods.

Table 4. Triglycerides, T-cholesterol, BHBA, NEFA and glucose of Lacaune ewes fed with N-ZnO supplementation.

Items	Experimental groups		SEM	Significance
	CON	N-ZnO		T	D	T x D
Ewes, no	10	10
Triglycerides (mg/dL)
−30 d	34.84A	29.93A	2.226
−15 d	41.24B	37.06B	2.840
0 d	23.94C	26.35C	3.361	0.491	<0.001	0.649
+15 d	9.61D	13.00D	2.587
+30 d	9.34D	14.06D	2.800
T-cholesterol (mg/dL)
−30 d	44.47	56.96	2.859
−15 d	46.91a	56.96a	2.739
0 d	44.69b	48.12b	3.043	0.200	0.024	0.218
+15 d	49.82	46.15	3.267
+30 d	50.69	52.80	2.649
BHBA (mmol/L)
−30 d	0.45a	0.38a	0.035
−15 d	0.39a	0.38a	0.042
0 d	0.43a	0.43a	0.040	0.0852	0.019	0.430
+15 d	0.52b	0.59b	0.064
+30 d	0.38a	0.33a	0.041
NEFA (mmol/L)
−30 d	0.57a	0.63a	0.137
−15 d	0.63a	0.92a	0.152
0 d	0.73a	0.76a	0.151	0.664	0.006	0.740
+15 d	1.42b	1.51b	0.265
+30 d	0.68a	0.67a	0.158
Glucose (mg/dL)
−30 d	28.66Aa	26.00Aa	1.451
−15 d	13.22B	12.99B	1.327
0 d	19.69C	16.72C	1.693	0.081	<0.001	0.252
+15 d	22.06Db	23.58Db	1.538
+30 d	16.52C	14.53C	1.199

T; fixed effect of treatment; D; fixed effect of day; T x D; interaction treatment x day.

a,b Means with different lower case within a column are different (P ≤ 0.05).

A,B,C Means with different upper case within a column are different (P ≤ 0.01).

3.4. Minerals in the blood serum of ewes

According to Table 5, the Ca, P and Zn concentrations in the pre- and postpartum blood serum of the ewes were not statistically influenced by the experimental treatment. However, a time effect was observed on Ca and Zn levels. The dietary N-ZnO supplementation; from an inorganic nano-oxide source, did not affect on serum Ca and P levels of ewes (P ≥ 0.05). However, blood Ca concentration decreased by 3,7% and %1.9 (P ≤ 0.05) in the CON and N-ZnO supplemented group, respectively, from parturition to the end of the trial. Compared to the prepartum (−15 day), serum Ca values resulted in higher (P ≤ 0.001) levels on days 0 and +15, feeding with or without N-ZnO-supplemented diets. The dietary N-ZnO supplementation did not affect the blood serum P levels and no changes were observed between the groups. The Zn levels in the blood serum at the end of the trial (day +30) were greater (P ≤ 0.001) in the groups compared to −15 day; it increased during the trial from −15 to +30 days, with values of 10.30 and 10.57, respectively.

Table 5. Ca, P and Zn concentrations of Lacaune ewes fed with N-ZnO supplementation.

Items	Experimental groups		SEM	Significance
	CON	N-ZnO		T	D	T x D
Ewes, no	10	10
Ca (mg/dL)
−30 d	9.94a	9.99a	0.129
−15 d	9.98A	9.89A	0.109
0 d	10.31Bb	10.20Bb	0.115	0.439	<0.001	0.980
+15 d	10.39Bb	10.48Bb	0.118
+30 d	9.92a	10.00a	0.111
P (mg/dL)
−30 d	4.53	4.05	0.975
−15 d	4.88	5.77	1.055
0 d	3.98	4.67	1.105	0.242	0.197	0.554
+15 d	3.79	3.89	1.205
+30 d	3.89	4.80	1.095
Zn (mg/dL)
−30 d	9.37	9.42	0.660
−15 d	9.41A	9.56A	2.201
0 d	9.75	9.91	1.285	0.416	<0.001	0.656
+15 d	9.83B	10.09B	1.483
+30 d	10.30B	10.57B	1.317

T; fixed effect of treatment; D; fixed effect of day; T x D; interaction treatment x day.

a,b Means with different lower case within a column are different (P ≤ 0.05).

A,B,C Means with different upper case within a column are different (P ≤ 0.01).

4. Discussion

All parameters did not show statistically significant differences between groups. However, there was a significant time effect on levels of metabolic parameters parallel to the changing physiology of Lacaune ewes during the transition period. The nutrient requirements of ewes increase during the prepartum period due to the growth of the fetus. If ewes do not receive required nutrient of the required during the transition period, nutrient depots are mobilized in large quantities (Gürgöze et al. 2009).

There was a gradual decrease in serum AST and ALT levels in ewes fed with N-ZnO in the prepartum period. In contrast, the AST level increased up to day +15 after parturition and did not change between the next periods. However, a similar effect was observed in the CON group. At the end of the trial, AST increased and ALT decreased in both groups compared to the prepartum period. Despite this fluctuation, there was no significant difference between the two groups during the trial. This finding can be interpreted as the absence of adverse effects and liver damage due to the supplementation of Zn nanoparticles. Hosseini-Vardanjani et al. (2020) reported that fluctuation in blood enzymes may be an indicator of possible toxicity of N-ZnO and may elevate liver function due to hepatocellular damage. However, the researchers emphasized that this effect was achieved by adding 30 or 40 mg N-ZnO/kg DM/sheep in the pre- and postpartum periods. The results regarding AST and ALT levels in this study were consistent with the previous findings on sheep fed with the dietary supplementation N-ZnO (Mohamed et al. 2017; Alijani et al. 2020). In addition, Wang et al. (2017) showed negative effects of feeding N-ZnO (basal diet + 1200 mg supplemental nano-ZnO/kg for piglets), leading to increased blood serum ALT and AST activities. According to Bedi and Kaur (2015), as a result of systemic absorption of ZnO nanoparticles, ALT and AST activities increase significantly in a dose-dependent manner due to the elevation of zinc levels in the liver, adipose tissue and pancreas. On the other hand, Najafzadeh et al. (2013) reported mild liver degeneration and severe renal damage in lambs because of nano Zn feeding at a dose of 20 mg/kg body weight for a period of 25 days. The mechanism of high dose N-ZnO toxicity may be, as the N-ZnO can be rapidly activated and transformed into metal ions in the gastrointestinal canal. These large amounts of metal ions are subsequently brought to the liver and kidney for metabolism and excretion, which cause damage to hepatic and renal tissues (Chen et al. 2007). Liver enzymes such as ALT and AST content in serum were increased by feeding N-ZnO at a dose of 300 mg/kg to mice for 14 days (Sharma et al. 2012). These results, according to the authors, cytotoxic effects of feeding N-ZnO include induced oxidative stress increase in lipid peroxidation and accumulation of N-ZnO in the liver. It is clear from these results in the present study, the dose of 20 mg/kg DM N-ZnO in lambs did not cause any toxic effect and did not tend to accumulate in the liver tissues.

The treatment with N-ZnO did not induce significant differences in values of serum concentrations of albumin, total protein and BUN. Data were within the reference range of Lacaune sheep, as reported in previous experiments by other researchers (Brito et al. 2006; Alba et al. 2021). The results in this study of albumin levels revealed that it was significantly higher on prepartum day −30; compared to days −15 and +30 and was in the reference range for sheep. Mohamed et al. (2017) recorded the highest serum albumin and total protein values in the N-ZnO group (supplemented with 5 mg N-ZnO/kg feed) for ewes over the post-lambing period. According to the authors, this superiority in the N-ZnO group may be attributed to the increase the AST enzyme activity. Gürgöze et al. (2009) reported the albumin levels on day 145 of gestation in ewes to be significantly high. On the other hand, the lower serum total protein levels on days 0 and −15 may be related to the fact that the fetus synthesizes all its proteins from the amino acids derived from the mother, and the growth of the fetus is rapid during late pregnancy. However, the higher total protein levels on postpartum day +15 can be related to colostrum production in the postpartum period. Passage of serum proteins and immunoglobulins from blood to colostrum occurs with the onset of colostrum production in prepartum period. These findings were similar to the results of Sadjadian et al. (2013) and Salar et al. (2018) obtained from Saanen goats.

In contrast to the present work, Alijani et al. (2020) observed lower blood urea-N (BUN) concentration after the supplementation of the sheep diet with N-ZnO (28 mg/kg DM) instead of Zn and ZnO. According to the researchers, the lower BUN concentrations in sheep receiving the Zn-supplemented diets were due to the higher digestibility of N. The N evaluated as ammonia is transformed to urea and excreted, whereas N absorbed as an amino acid is retained and used in protein metabolism, thus, curing N retention (McDonald et al. 2011). The mean level of BUN concentration is a significant indicator of dietary protein supply in small ruminants and plasma urea levels increase during early pregnancy and decrease in late pregnancy (Ramin et al. 2007). In this study, the highest BUN levels were recorded on day +30 compared to −30- and −15-days late pregnancy and, 0- and +15-day postpartum. The reason for high BUN levels in postpartum ewes could be related to either nutritional management or high protein metabolism during lactation.

No study has been carried out examining the effect of N-ZnO use on triglycerides, T-cholesterol, BHBA, NEFA and glucose in ewes in the transition period. In this study, the supplementation of N-ZnO did not result in significant differences in these parameters of ewes. However, it was observed a general improvement in blood lipid contents in ewes of both groups. There were significant decreases in triglycerides and T-cholesterol particularly from day +15 to parturition and this continued postpartum in triglycerides. The decrease in triglycerides and T-cholesterol may have been due to the prevention of fat accumulation in the liver and its use in lipid metabolism in order to meet the increased energy need during these periods (Sadjadian et al. 2013). In this study, BHBA levels prominently increased on day +15 postpartum all ewes. According to Salar et al. (2018), high the BHBA levels in the postpartum period can be related to the effect of the hyperketonemia on the fatty liver and the glucose need is met by lipolysis by using body fat stores. Researchers specified that also the maximum levels of triglycerides in the liver on day 30 postpartum were associated with these results. The CON and N-ZnO groups showed a greater NEFA such as BHBA from day 0 to +15.

The average serum BHBA concentration for this study was 0.43 mmol/L in the transition period and the normal values reported for BHBA were between 0.86 mmol/l (Lacetera et al. 2001), and 0.67 mmol/l (Ramin et al. 2005) in that was lower than recorded for ewes except that transition period. Ramin et al. (2005), emphasized that might observe no correlation between BHBA and glucose concentrations while negative correlation between glucose and urea and a positive correlation between BHBA and urea due to ketone bodies resulting from lipid metabolism when glucose does not meet the energy requirements. According to some researchers, in the absence of glucose, BHBA increases in serum and results in the presence of ketone bodies and is indicative of pregnancy toxaemia (Lacetera et al. 2001; Sakha 2016). The reason for hyperketonemia also could be mainly related to lactation as lower serum glucose (Ramin et al. 2005). On the contrary, in this study, while glucose increased after birth (day +15), BHBA also increased. The results of this study were in accordance with the reference values for BHBA and glucose concentrations of early lactation Lacaune sheep by Antunović et al. (2022). In pregnancy toxaemia, the BHBA value is usually quite above 3 mmol/L (Ramin et al. 2007). According to the studies, the present study results show that the animals were not exposed to pregnancy toxaemia. High ВНВА concentrations after parturition are a reaction for compensation of occurring carbohydrate deficiency and the ketone bodies formation is parallel with the lipolysis and oxidation of fatty acids. Lacaune ewes were not subjected to ketosis as BHBA levels were not over 0.8 mmol/l (Marutsova and Marutsov 2018). The data from this study are compatible with those of Antunović et al. (2022) and Balikci et al. (2007) and could be accepted as indicating a good transition from prepartum to postpartum period. In addition, the increased NEFA concentration is to meet both the energy that the animal will spend for birth and the energy needed for milk production after parturition is formed (Cheng et al. 2007). The gluconeogenesis and mobilization of adipose tissue, which occurs in the postpartum period to meet the energy needed for the development of the fetus and udder, can be associated with the concentrations of prepartum hormones. According to Herdt (2000), increases in plasma NEFA at parturition and early lactation can be interpreted as initiating the infiltration of triglycerides in the liver. The authors reported that increases in plasma lipolytic hormones may contribute to increased plasma NEFA concentrations. Obtained NEFA levels were increased up to 1.42 and 1.51 mmol/l on day +15 in ewes. These results revealed that the lipolysis in this period of postpartum, occurred at a higher rate so the NEFA levels were higher than the quantity that could be converted in the liver (Marutsova and Marutsov 2018).

Several authors found that plasma glucose levels were higher during postpartum period than prepartum period in sheep. In contrast, some researchers reported greater plasma glucose concentrations in prepartum ewes (Takarkhede et al. 1999; Moghaddam and Hassanpour 2008; Gürgöze et al. 2009). Gürgöze et al. (2009) reported lower serum glucose concentrations on days 21, 120 and 145 of pregnancy than on days 7 and 14 postpartum. Balikci et al. (2007) recorded this value to be lower on day 100- and 150-days prepartum compared to 45 days postpartum period. In this study, lower serum glucose levels were recorded on days −15 and +30 prepartum compared to −30, 0 and +15 days postpartum. Glucose concentrations were lower than all period’s value on day −15 the prepartum period. Serum glucose level is known as the metabolic profile test; thus, it has distinguishable value in pregnancy toxaemia and reproduction defects (Ramin et al. 2005; Gürgöze et al. 2009). The variables in glucose concentration during the transition period show the consumption of glucose by the development of the fetus and milk yield in lactation, so glucose evaluation in metabolism prepartum and postpartum period results in improvement in hypoglycaemia and pregnancy toxaemia (Ramin et al. 2007).

In the present study, no effect of the supplementary N-ZnO displaying better absorption and bioavailability in the cellular level on the blood Ca and P levels were probably due to its not leading to the antagonistic interaction with these minerals (Patra and Lalhriatpuii 2019). However, it could be attributable to the amount (not very high) of supplemental N-ZnO (20 mg/kg). This is because Zn affects the other minerals’ status in livestock if dietary Zn is several times higher than the recommended concentration (Hosseini-Vardanjani et al. 2020). Unaffected the blood serum level of Ca and P with the Zn supplementation was consistent with the results reported for goat kids (Zaboli et al. 2013).

It should be noted that the basal diet in this study was formulated with high levels of grains such as barley containing phytate, which usually binds Zn in plants (Goff 2018). Although the feed ingredients of the present diets were flaked, the escape of undegraded phytate from the rumen may have acted as a Zn antagonist in the small intestine and increased the Zn requirement of the sheep above the recommended level (Alijani et al. 2020). In addition, high concentrations of Zn in the diet may be competed with the uptake and absorption of Ca and P as an antagonist by forming insoluble phosphates, thereby reducing the amount of these elements in blood plasma (Phiri et al. 2009). However, in this study, the levels of N-ZnO intake were not so high as to affect the levels of Zn in the blood of ewes.

5. Conclusion

Based on results in the present study, supplementation of the basal diet containing 30.56 or 31.24 mg Zn/kg DM with 20 mg N-ZnO/kg DM had no significant effect on metabolic parameters in the transition period of Lacaune ewes. In addition, clear changes in levels of metabolic parameters depending on a time effect were observed during the transition period in Lacaune ewes which must be taken into consideration for the correct exposition of serum chemistry, metabolic profile and mineral status. Studies using higher doses of N-ZnO are necessary to understand the probable effects of feeding with N-ZnO on livestock metabolism.

Disclosure statement

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

Data availability statement

All data and materials are available in the manuscript.