The impact of early and mid-pregnant Hu ewes’ dietary protein and energy levels on growth performance and serum biochemical indices

ABSTRACT This study explored the effects of different protein and energy levels on growth performance and serum biochemical indices of ewes during early and mid-pregnancy. A total of 132 ewes were assigned to 5 groups (P1, P2 = E2, P3, E1, E3) to dietary protein levels (P1: 9.22%, P2: 10.04%, P3: 10.86%) and dietary energy levels (E1: 9.04 MJ/kg, E2: 9.53 MJ/kg, E3: 10.02 MJ/kg). Blood urea nitrogen (BUN) increased with an increase in protein levels in early pregnancy (P = .008). In the P2 group, creatinine, triglyceride, and lactate levels were lowest and total protein levels were highest in mid-pregnancy (P < .05). Final weight, total weight gain, and average daily weight gain increased with an increase in energy levels of ewes (P < .05). BUN, aspartate transaminase, cholesterol, and high-density lipoprotein decreased with an increase in energy levels in mid-pregnancy (P < .05). Alanine aminotransferase, lactate dehydrogenase, and glucose increased initially and then decreased with an increase in energy levels (P < .05). To sum up, we suggest that Hu sheep be fed diets with energy and protein levels of 10.02 MJ/kg and 10.04% in early and mid-pregnancy, respectively.


Introduction
Hu sheep are a precocious and prolific indigenous Chinese sheep breed that is well adapted to high temperature and humidity conditions (Nie et al. 2015).They are among the dominant breeds for lamb meat production in China.Nutrient requirements of Hu sheep during pregnancy have not been fully established, which limits the development of effective feeding systems.Therefore, an assessment of the nutritional requirements of Hu sheep should be performed to maximize performance and feed utilization.
The relationship between maternal nutrient intake and fetal growth during pregnancy determines the success of pregnancy and lifelong health and productivity (Redmer et al. 2004).Annett and Carson (2006) reported that highlevel feeding in the early stage of pregnancy is detrimental to the success of pregnancy in ewes.Muñoz et al. (2008) aver that even in the early stages of embryonic life, the mother's dietary intake directly meets the nutritional needs of foetal growth.Previous studies on sheep and cattle breeding report that energy intake is a major factor affecting birth weight, lactation, performance and productivity of offspring (Wang et al. 2021).The energy level is the most common nutritional limiting factor in small ruminants (NRC 2006).Energy, protein, vitamin and mineral deficiencies lead to a decline in growth performance, poor carcass characteristics and low productivity of sheep (Abdullah et al. 2008;Hosseini et al. 2008).Increasing feed energy levels improve ADG and feed efficiency in sheep (Song et al. 2018).Crude protein (CP) supplementation promotes the maintenance of birth weight (BW) and body condition score in dams.Furthermore, it improves offspring performance (Larson et al. 2009).A previous study explored the effects of high dietary protein levels during late gestation in ewes on colostrum yield and lamb survival rate and reported that excess dietary CP results in low colostrum yield and lamb survival (Ocak et al. 2005) it difficult for ewes to give birth.Consequently, dietary CP levels fed to pregnant ewes during gestation and ensuing lactation have been a major research focus of livestock scientists.
Previous studies report that different dam sizes or maternal nutrition influences growth rates, carcass characteristics, and reproductive performance of offspring (Bell 2006).They report that the production characteristics of sheep, including growth and reproductive performance, are affected by nutrition, weight and physical condition (Paten et al. 2016).Although the possible effects of nutrition of ewes in late pregnancy on birth weight and other production characteristics are well known, the potential effects of nutrition in early pregnancy in ewes have not been well established (Zhang et al. 2018).To give full play to the production potential of lambs and ewes, we assume that properly reducing the dietary energy and protein levels of pregnant ewes can also meet the growing needs of ewes and their offspring.Therefore, the purpose of this experiment is to find the lowest and most appropriate dietary energy and protein level for pregnant ewes without affecting the growth and development of ewes and their offspring.

Materials and methods
The experimental design and procedures were reviewed and approved by the Animal Care and Use Committee of Hunan Normal University, Changsha, Hunan, P.R. China.

Diets, animals, and experimental procedures
The current study was undertaken in Hubei Zhiqinghe Agriculture and Animal Husbandry Co., Ltd.Hu ewes were impregnated by artificial insemination and estrus synchronization.Successful pregnancy in ewes was confirmed by ultrasound.A total of 132 pregnant ewes (37.1 ± 1.79 kg initial weight) were selected from the successful pregnant ewes based on the parity and body weight.The pregnant ewes were assigned to 5 groups with 2 replicates in each group and 11 ewes in each replicate.The first three groups of ewes were fed diets with different energy levels of E1: 9.04 MJ/kg, E2: 9.53 MJ/kg and E3: 10.02 MJ/kg.The protein levels were kept at 10.04%.The latter three groups were fed diets with different protein levels of P1: 9.22%, P2 (E2):10.04% and P3: 10.86%.The energy levels remained the same at 9.53 MJ/kg.The dietary energy and protein level was set according to the nutritional requirement standard (National Research Council 2006 and Chinese Mutton Sheep during pregnancy NY/T816-2004).The feed was made into a total mixed ratio.Dietary nutrients are shown in Table 1.Day 0 to 39 post-mating period was considered early pregnancy, whereas day 40 to 90 was considered mid-pregnancy.Experimental ewes were randomly divided into study groups.Feeds were provided daily at 07:00 and 17:00 h and the feeding amount was adjusted in the morning to ensure approximately 5% feed refusal.Feeds were provided in the form of total mixed ration (TMR).Ewes were housed in straw-bedded individual pens (5.0 × 2.5 × 1.0 m) and had continuous access to fresh water.
From day 91 of pregnancy to parturition, all ewes were fed on the same sheep farm feed.After parturition, the sex and weight of lambs were recorded, and the number of live and dead lambs within 24 h were counted to calculate the lambing rate and survival rate.

Chemical analysis
Samples of forage were dried at 60°C for 24 h in a forced-air drying oven and ground to pass through a 1 mm screen (Yang et al. 2018).The dry matter content was measured after the samples were dried in a forced-air oven at 105°C for 24 h (method 934.01;

Growth performance
Body weights of ewes were determined before the start of the experiment and at day 90 of pregnancy obtain the initial body weights (IBW), and final body weights (FBW).Feed consumption was determined to compute average daily feed intake (ADFI).The average daily gain (ADG) and the feed conversion ratio (FCR) were calculated based on a previous method described by Li et al. (2015).a Premix provides the following per kg: Vitamin A, 120 KIU; Vitamin D3, 60 KIU; Vitamin E, 200 mg; Cu, 0.15 g; Fe, 1 g; Zn, 1 g; Mn, 0.5 g; I, 15 mg; Se, 5 mg; Co, 2.5 mg; Ca, 20 g; NaCl, 100-250 g; P, 10 g. b Except for metabolic energy was the calculated value, the rest were measured values.

Serum biochemical parameters
Blood samples were collected from the jugular vein of 5 ewes randomly selected from each group into 5 mL vacuum tubes without anti-coagulant on day 30 and day 90 of pregnancy (Changsha Yiqun Medical Equipment Co., Ltd., Hunan, China) in the morning before feeding.The samples were centrifuged (1006.2×gfor 15 min at 4°C) and stored at room temperature for 2-3 h.The serum was stored at −80°C awaiting the determination of biochemical parameters.

Statistical analysis
SPSS software (version 20.0;IBM Corp., Chicago, IL, USA) was used to analyse all data as a 2 × 3 factorial arrangement of treatments in a randomized complete block design.Pen (N = 132) was the experimental unit.The model included diet protein (9.22%, 10.04%, 10.86%) or energy level (9.04, 9.53, 10.02 MJ/ kg), pregnancy stage (early pregnancy and mid-pregnancy), and their interaction (diet × stage) as fixed effects and replicate as the random effect.Least squares means were calculated for each independent variable.A one-way analysis of variance and linear and quadratic contrasts was used to examine the effects of protein or energy levels on the lambing rate and growth performance of lambs.Significance was indicated by P < .05,and tendencies were indicated by P < .10.

Growth performance of ewes
With the increase in the dietary energy level to 10.02 MJ/kg (E3), the initial body weight (diet, P = .025)and final body weight (diet, P = .012) of ewes increased significantly.
Compared with early pregnancy, the ewes in mid-pregnancy showed a significantly increased in final body weight, total gain, ADG and FCR (stage, P < .001).The dietary energy level and pregnancy stage had no interactional effect on final body weight, ADG and ADFI (D × S, P > .05,Table 2), energy level and pregnancy stage had a tendency to interactional effect on total gain and FCR (D × S, .05< P < .1).
The results in Table 3 show that 10.04% (P2) protein level significantly increased the initial body weight (diet, P = .033)and final body weight of ewes (diet, P = .031).Different protein levels significantly increased body weight, total weight gain, ADG and FCR of ewes in mid-pregnancy (stage, P < .001),and had a tendency effect on ADFI (stage, P = .081).Dietary protein level and pregnancy stage had no interactional effect on final body weight, total gain, ADG, ADFI and FCR (D × S, P > .05).
The results in Tables 4 and 5 show that different energy and protein levels had no effect on lambing rate, death rate, survival rate, female birth weight and total average birth weight (P > .05).With the increase in dietary energy level, male birth weight tended to increase (P = .058).With the increase in dietary protein level, male birth weight tended to decrease (P = .078).
The results showed significant differences in final weight and ADG between P2 and E2 groups (P < .05,Table S1).Moreover, significant differences were observed in the lambing rate between E2 and P2 groups (P = .04,Table S2).However, no significant differences were observed in offspring growth performance between E2 and P2 groups (P > .05,Table S2).

Blood biochemical indices of ewes
As shown in Table 6, with the increase in dietary energy level, the serum AST (diet, P = .010)and GLU (diet, P = .011)contents of ewes decreased significantly, the serum LDH content of ewes showed a decreasing trend (diet, P = .054).In mid-pregnancy, serum LDH (stage, P = .049),BUN (stage, P < .001)and GLU (stage, P = .017)contents significantly decreased, and CREA (stage, P = .043)and HDL (stage, P = .042)contents significantly increased.The dietary energy level and pregnancy stage had no interactional effect on serum biochemical indices (D × S, P > .05).
As shown in Table 7, the BUN contents of the P2 and P3 groups were greater than those of the P1 group (diet, P = .007).The CREA contents of P1 and P3 in mid-pregnancy were greater than those in other groups (diet, P = .006).The TG content of the P1 group was greater than that in other groups (diet, P = .001).An increasing trend was observed for LACT content.As the ewe's pregnancy progresses, the contents of AST, CREA, TG, CHOL and HDL in mid-pregnancy were significantly higher than those in early pregnancy (stage, P < .05).Some significant interactions between diet and pregnancy stage (D × S, P = .008)were observed in the serum content of TP.

Discussion
Current research results show that the growth rate of the foetus in early pregnancy was low and the nutritional requirements were low.Generally, only extreme feeding levels (very low or very high) during the first month of pregnancy may impair the subsequent productive performance of ewe-lamb pair (Álvarez-Rodríguez et al. 2012).Changes in body weights of ewes during pregnancy are an important index to assess nutritional effects during pregnancy.Insufficient nutrition commits nutrients obtained by ewes only to the promotion of growth and development of embryos, which leads to reduced body weight of ewes.In this study, in early pregnancy, 10.04% dietary protein significantly increased the ADG of ewes.In mid-pregnancy, a 10.04% protein level significantly increased the ewes' initial body weight, final body weight and ADG.High energy levels caused an increase in the initial body weight and final body weight of ewes, and the result was consistent with Song et al. (2018), which avers that the increase in dietary energy levels can improve the growth performance of sheep.The birth weight of lambs is an important index to determine the role of the nutritional level of pregnant ewes on foetal development.The findings of the current study showed no difference in the birth weights of lambs between different protein and energy groups.This shows that the ewes had adequate nutrition during pregnancy.Furthermore, ewe feeding during early gestation did not affect lamb birth weight, which is consistent with the findings reported by Ford et al. (2007).Studies in pigs (Bee 2004) found that neither nutrient restriction nor increased energy intake during the first 50 d of gestation affected the birth weights of resulting piglets.Similarly, birth weight was not affected by maternal nutrition in these studies or in our previous studies where ewes were restricted during early-mid gestation (d 30 to 70; Fahey et al. 2005).In the current study, male lambs were heavier at birth than females, which was consistent with results reported by Daniel et al. (2007).Effects of diet on pre-and post-partum BW have been attributed to the mobilization of energy reserves by dams, either to support foetal development or meet requirements for lactation (Olafadehan and Adewumi 2009).The current study showed the same between protein and energy levels in the E2 and P2 groups of ewes.However, the growth performance of ewes in the two groups was different.This can be explained by the possibility that ewes in the P2 group had more litters and increased uterine weight, which resulted in a higher weight gain.In addition, the current study showed that the feeding of ewes in early pregnancy had no effect on the birth weight of lambs and the survival rate of lambs.The findings of the current study were consistent with those reported by Kenyon et al. (2011). Furthermore, McGovern et al. (2015) reported that altering the nutrition (80%, 100% or 120% of AFRC (1993) ME requirements) of twin-bearing ewes in late gestation did not affect the birth weight of newborn lambs.The current study showed that maternal nutrition did not affect lamb weight.However, previous studies report that the uterine environment has long-term effects on offspring (Gluckman et al. 2010).Therefore, the potential long-term effects of these nutritional treatments in animals need to be explored further.
The growth and health status can be reflected indirectly by the determination of biochemical indexes (Lou et al. 2022).The concentration of BUN in the serum of early and mid-pregnancy ewes increased with the increase in protein levels.Low BUN content indicates high nitrogen metabolic efficiency.High BUN content indicates a high protein intake or excessive muscle mobilization (Wang et al., 2021).However, excess protein intake can place a burden on the growth and metabolism of the ewe.The part which cannot be used will be deaminated and the excess N is excreted from the body, causing environmental pollution (Nuno et al. 2004).The results of the study showed that the serum BUN concentration in mid-pregnancy was significantly lower than that in early pregnancy.This can be explained by the possibility that with the progress of pregnancy, the energy requirement of ewes is increased, which promotes microbial protein synthesis.As a result, the rate of protein utilization was increased, and serum BUN levels were reduced.Previous studies reported that BUN concentration decreases with an increase in energy level, and that weight gain of ewe increases with an increase in energy level.This shows that an increase in energy level can promote protein absorption (D'Mello 2003).An increase in serum creatinine levels has been reported in cases of injury, surgical trauma and rumenotomy (Obidike et al. 2009), where severe haemorrhage and electrolyte loss are prominent.Parturition is characterized by a moderate to a high degree of blood and electrolyte loss.Therefore, CREA levels in the midpregnancy were higher following parturition than in early pregnancy.However, the lowest CREA levels were observed in the P2 group.As a result, ewes in the P2 group were in a relatively good physiological state, which improves the physiological status of ewes during pregnancy.According to the relationship between the growth performance of ewes and the indices of serum BUN, CREA and TG, it can be inferred that P2 (10.04% protein level) was the optimum protein level of ewes in early and mid-pregnancy.
Levels of TP in serum partly represent a nutritional level of protein in the diet and the degree of protein digestion and absorption by animals (Hussain et al. 1996).Serum TP levels increased upon enhancement of protein synthesis and nitrogen deposition in the body.In the current study, the TP content of the P1 and P2 groups in mid-pregnancy was lower than that in early pregnancy, while the TP content of the P3 group in mid-pregnancy was higher than that in early pregnancy.This was probably because the high dietary protein level (10.86%) exceeded the nutritional needs of ewes, which resulted in an amino acid imbalance in the body.Therefore, ewes were probably unable to effectively digest and absorb proteins, which led to a low total protein deposition rate.
AST is a liver enzyme that catalyses the transamination of Aspartate to form Oxaloacetate, the main metabolite in the tricarboxylic acid cycle.AST can pass through the hepatocyte membrane.Therefore, an increase in serum AST activity indicates an overload of hepatocyte activity.Compared with early pregnancy, the AST activity of the different protein and energy level groups increased in mid-pregnancy, indicating that the burden on the body will increase with pregnancy, but AST levels in the E3 group of ewes were the lowest in mid-pregnancy.The results showed that the active load of sheep hepatocytes in the E3 (10.02 MJ/kg) group decreased with the progress of the pregnancy.Previous studies reported that neuroendocrine requirements in late pregnancy and the lambing period upset the homeostatic regulation mechanism, which is complicated by high metabolic energy requirements (Greenfield et al. 2000).An increase in AST level in Hu ewes during pregnancy signifies an increase in liver metabolism.Changes in AST activity may be associated with reduced intake of dry matter before and after delivery and may lead to liver lipidosis, which alters the normal function of the liver (Aliasghar et al. 2019).
The dietary protein and energy levels affected the lipid metabolism of ewes.The serum TG content of ewes fed with a dietary protein level of 9.22% was significantly higher than that of the other two groups in mid-pregnancy.The serum TG content of ewes fed with protein levels of 10.04% and 10.86% was the same, indicating that the dietary protein level of 9.22% might not meet the metabolic requirements of Hu sheep.This may be explained by the fact that triglycerides, being the storage forms of lipids in animals, were mainly used as fuel.Ewes decompose and consume lipids upon an increase in energy demand (Haffaf and Benallou 2016), which explains why the P1 group had higher serum levels of TG.In this experiment, fat utilization increased with the increase of protein level until the protein level reached 10.04%.The subsequent continuous increase in protein levels did not affect the fat metabolism of ewes.The TG concentration of ewes decreased, indicating that the intake of a high protein diet increased the fat utilization rate of ewes.Cholesterol, an indicator of lipid metabolism, was within the reference limits for an adult sheep (1.5-3.9 mmol/L) (Wittwer 2012).Fat mobilization in adipose tissue during pregnancy and lactation is an alternative energy source in ruminants (Mohammadi et al. 2016).In this experiment, TG and HDL levels were higher in mid-pregnancy than the levels in early pregnancy (different protein levels).This is possible because as the pregnancy progresses, the embryo matures and more energy is needed.The HDL content decreased with an increase in energy level.This finding was consistent with the findings by Ban-Tokuda et al. (2007) that CHOL was positively correlated with HDL and slightly positively correlated with TG.
The activity of serum LDH can reflect the stress sensitivity of the body, which can catalyse the formation of lactic acid from pyruvate.In mid-pregnancy, the concentration of LDH in the serum of ewes in the E3 group was significantly lower than that in E1 and E2 groups, indicating that the increase in energy intake of ewes may significantly inhibit the production of lactic acid from pyruvate and reduce the sensitivity to stress.Furthermore, the current study showed that GLU levels in sheep in energy groups decreased in mid-pregnancy compared with early pregnancy.Notably, serum GLU levels increased at first and decreased with an increase in energy levels, this shows that E3 (10.02 MJ/kg) has met the growth and development needs of ewes, which was consistent with the results reported by Wang et al. (2020).
In conclusion, in early and mid-pregnancy, 10.04% dietary protein level decreased the levels of CREA and LACT, and increased the final body weight in mid-pregnancy, thus reducing the body burden and improving the growth performance of ewes.10.02 MJ/kg dietary energy level improved the growth performance, and fat and glucose utilization efficiency of ewes.We recommend that Hu sheep be fed diets with energy and protein levels of 10.02 MJ/kg and 10.04%, respectively, to ensure the growth and pregnancy needs of ewes.

Animal welfare statement
The experimental protocol was approved by the Animal Care and Use Committee of Hunan Normal University, Changsha, Hunan, China.The authors confirm that they have followed EU standards for the protection of animals used for scientific purposes and feed legislation.
. Dietary CP causes birth weight gain in lambs, which makes CONTACT Qiye Wang wangqiye@hunnu.edu.cnHunan International Joint Laboratory of Animal Intestinal Ecology and Health, Laboratory of Animal Nutrition and Human Health, College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, People's Republic of China; Hubei Zhiqinghe Agriculture and Animal Husbandry Co., Ltd., Yichang, Hubei 443106, People's Republic of China; Huansheng Yang yhs@hunnu.edu.cnHunan International Joint Laboratory of Animal Intestinal Ecology and Health, Laboratory of Animal Nutrition and Human Health, College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, People's Republic of China; Key Laboratory of Agro-ecological Processes in Subtropical Region, Hunan Provincial Engineering Research Center of Healthy Livestock, Scientific Observing and Experimental Station of Animal Nutrition and Feed Science in South-Central, Ministry of Agriculture, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, Hunan 410125, People's Republic of China *Xin Wang and Yancan Wang should be considered joint first author.Supplemental data for this article can be accessed online at https://doi.org/10.1080/09712119.2023.2170385.

Table 1 .
Dietary composition of ewes with different nutritional levels.

Table 2 .
Effects of different energy levels on the growth performance of ewes.
Notes: Abbreviation: IBW, initial body weights.FBW, final body weights.ADG, average daily gain.ADFI, average daily feed intake.FCR, feed conversion ratio.SEM, standard error of the mean.N = 22.Values within a row with different superscripts (a, b) differ significantly at P < .05.

Table 3 .
Effects of different protein levels on the growth performance of ewes.IBW, initial body weights.FBW, final body weights.ADG, average daily gain.ADFI, average daily feed intake.FCR, feed conversion ratio.SEM, standard error of the mean.N = 22.Values within a row with different superscripts (a, b) differ significantly at P < .05.

Table 5 .
Effects of different protein levels on the growth performance of offspring.

Table 6 .
Effect of different energy levels on serum biochemical indices of ewes.