Dietary energy and protein levels on lactation performance and progeny growth of Hu sheep

ABSTRACT

This experiment aimed to investigate the effects of dietary energy or protein levels on lactation performance and progeny growth of Hu sheep. Thirty early lactation ewes with twin litters were assigned into five treatments (E1, E2 = P2, E3, P1, P3; n = 6) according to dietary energy levels (E1: 9.52 MJ/kg, E2: 10.02 MJ/kg, E3: 10.52 MJ/kg) and dietary protein levels (P1: 9.82%, P2: 10.99%, P3: 12.11%). The corresponding lambs were divided into SE1, SE2 = SP2, SE3, SP1 and SP3 (n = 12). Dietary energy level significantly affected the average daily gain (ADG) of Hu sheep (P = .005). The milk yield increased significantly with the increase of dietary protein level (P = .017), and then significantly increased the ADG of lambs. The serum blood urea nitrogen (BUN) of offspring (P = .006) and ewes (P = .003) increased significantly with the increase in the dietary protein level of ewes. Interestingly, at 60 d, the body size of SE3 group was the highest, and the straight crown hip length (P = .005) with the lightest birth weight (SP2) was significantly higher than the other two groups. To sum up, we suggest that Hu sheep be fed diets with energy and protein levels of 10.52 MJ/kg and 10.99%, respectively.

KEYWORDS:

• Hu sheep
• lactation performance
• serum biochemistry
• growth performance

1. Introduction

Hu sheep, a unique Chinese breed, is recognized for its high prolificacy, early sexual maturity, and year-round oestrus (Feng et al. 2018). The experiment on the nutritional requirements of ewes during lactation is a key link in the production, but it is a weak link in local sheep breeds. The requirement of energy and protein level of Hu sheep was mostly studied in the 1990s. With the gradual improvement of feeding conditions and methods, it is no longer suitable for the current requirements (Yang et al. 2019).

The rapid development of newborn lambs depends on milk feeding. Early lactation (1–60 d) is the key stage of the whole lactation period (Wang et al. 2020). At this time, the digestion and absorption of ewes are rapid and the feed conversion rate is high. If dietary energy is insufficient, it will not only affect the recovery of ewes but also reduce the birth weight of lambs. If the feed energy is too high, it will cause a waste of resources and affect economic benefits. Insufficient dietary protein intake will affect rumen fermentation and reduce growth performance and milk yield of ewes. However, excessive dietary protein intake may lead to an increase in rumen ammonia and blood urea nitrogen concentration, resulting in greater urinary nitrogen losses (Castillo et al. 2001; Hristov et al. 2004). In ruminants, a partition of dietary crude protein (CP) is more complex than in monogastric animals (Nolan 1993). This is mainly due to the unique rumen of ruminants, which can directly convert non-protein nitrogen and low-quality protein into high-quality nutrients such as short-chain fatty acids and microbial proteins through microbial fermentation (Angelidis et al. 2019). It is well known that there is a significant correlation between early weight gain of lambs and milk yield of ewes (Torres-Hernandez and Hohenboken 1980). Passos et al. (2000) found that offspring weight significantly decreased by reducing the dietary protein level of the mother, and this negative effect would last until the offspring were 6 months old. This is consistent with the experimental results of Teixeira et al.’s (2002) study. This suggests that maternal nutrition may affect the birth weight and metabolism of offspring by regulating milk yield.

In order to give full play to the production potential of lambs and ewes, we assume that properly reducing the dietary energy and protein levels of lactating ewes can also meet the growth 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 lactating ewes without affecting the growth and development of ewes and their offspring.

2. 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.

2.1. Animals, diets and treatments

This experiment was carried out in Hubei Zhiqinghe Agriculture and Animal Husbandry Co., Ltd., Yichang, Hubei, China. Thirty ewes (43.40 ± 6.17 kg initial BW) giving birth to twin lambs were randomly divided into five experimental groups. The first three groups of ewes were fed diets with different energy levels of E1: 9.52 MJ/kg, E2: 10.02 MJ/kg and E3: 10.52 MJ/kg, respectively (n = 6), the protein levels were kept at 10.99%, and the corresponding lambs were divided into SE1, SE2 and SE3 groups (n = 12). The latter three groups were fed diets with different protein levels of P1: 9.82%, P2 (E2): 10.99% and P3: 12.11%, respectively (n = 6), the energy levels remained the same as 10.02 MJ/kg, and the corresponding lambs were divided into SP1, SP2 (SE2) and SP3 groups (n = 12). The dietary energy and protein level was set according to the nutritional requirement standard (National Research Council 2006). The feed was made into a total mixed ration. Dietary nutrients are shown in Table 1.

Table 1. Ingredients and chemical analyses of ewe diet.

Items	Dietary treatment^a
	E1	E2 (P2)	E3	P1	P3
Ingredient (air-dried basic %)
Corn silage	46.67	40.00	33.33	40.00	40.00
Peanut seedling	23.33	20.00	16.67	20.00	20.00
Corn	14.03	23.88	34.86	27.00	21.01
Wheat bran	5.33	5.50	4.29	5.50	5.28
Soybean meal	7.64	7.62	7.85	4.50	10.71
NaCl	0.30	0.30	0.30	0.30	0.30
NaHCO3	0.50	0.50	0.50	0.50	0.50
Premix^a	2.20	2.20	2.20	2.20	2.20
Total	100	100	100	100	100
Nutrient levels^b (DM)
Dry matter	89.88	90.15	90.44	90.06	90.27
Crude protein	10.80	10.99	11.16	9.82	12.11
Crude fat	2.21	2.31	2.46	2.38	2.30
Neutral detergent fibre	45.60	42.27	37.99	42.18	41.69
Acid detergent fibre	31.66	27.77	23.73	27.58	27.93
Ash	6.29	5.91	5.45	5.64	6.13
Acid insoluble ash	1.50	1.34	1.17	1.31	1.36
Gross energy, MJ/kg DM	17.67	17.73	17.80	17.73	17.74
ME, MJ/kg	9.52	10.02	10.52	10.02	10.02

^a The premix provided the following per kilogram of diet: VA 120, 000 IU, VD3 60, 000 IU, VE 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 ME, which was the predicted value, the rest were measured values.

Each ewe was separately and fed with concentrate and mixed roughage (corn straw and peanut seedlings) at 8:00 and 15:00, with the feeding offer adjusted in the morning to ensure approximately 5% feed refusal. Lambs feeding methods: Breast milk during 1–10 d, breast milk and a small amount of supplementary feeding (concentrate feed) during 11–30 d, breast milk and massive supplementary feeding (concentrate feed) during 31–60 d. The concentrated feed comes from Cargill Feed Co., Ltd., the product code is 3112. During breastfeeding, two offspring lambs were transferred from the next pen to the ewe’s pen.

2.2. Growth and lactation performance and milk composition of lactating ewes

From the beginning of the experiment, ewes and lambs were weighed every 10 d in the morning. The average daily gain (ADG) was calculated from the average of three measurements of individual daily gain (Yin et al. 2001). According to the difference of body weight of ewes before and after feeding their offspring, the milk yield was measured at 12:00, 16:00 and 20:00 every 10 d.

Milk samples were collected continuously during the 59 and 60 d of the formal trial period (8:00 and 17:00). Twenty millilitre of milk samples were collected and mixed with antibacterial preservative bromoalcohol (2-bromo-2-nitro-1-mine3-propanediol) and stored at −20°C. The concentration of somatic cell, butterfat rate, milk protein, rate of lactose, the total dry matter, and urea nitrogen was determined by NIRS (MilkoScan FT6000) and SCC using a fluorimetric method (FosSomatics) (both devices from Foss Electric A/S, Hillered, Denmark) (Koczura et al. 2019).

2.3. Serum biochemical indexes

On the 60 d of the lactation period, blood samples were randomly collected from 6 ewes and 8 lambs of each group via the jugular vein into a 5 ml vacuum tube without anti-coagulant (Changsha Yiqun Medical Equipment Co., Ltd., Hunan, China) before morning feeding. The samples were centrifuged (1006.2×g at 4°C for 15 min) after kept at room temperature for 2–3 h, the serum was extracted and stored at −80°C until analysed for the biochemical parameters (Yang et al. 2012).

During analysis, serum samples were dissolved on ice, and centrifuged (1006.2×g at 4°C for 10 min) before the analysis of total protein (TP) content, albumin (ALB), alanine aminotransferase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH), blood urea nitrogen (BUN), blood glucose (GLU), triglyceride (TG), blood cholesterol (CHOL), high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDLC), lactic acid (LACT) and blood ammonia (NH3L) using commercial kits by manufacturer instructions (Roche Diagnostics Products Co., Ltd., Shanghai, China) and were identified using a Cobas C311 analyser (Roche Diagnostics, Rotkreuz, Switzerland) according to the methods described by Chen et al. (2019).

2.4. Growth and body size of lambs

The body size of lambs was measured at 0 and 60 d, respectively. The indexes and methods were as follows (Kabir et al. 2014).

• Body height: the vertical distance from the shoulder to the ground.

• Body length: the distance from the shoulder to the end of the ischial tuberosity.

• Chest depth: the vertical distance from the nail to the lower edge of the sternum.

• Chest circumference: the length of the posterior edge of the scapula around the chest.

• Abdominal circumference: the vertical circumference of the largest part of the abdomen.

• Head width: length of transverse muscle lines on both sides of the head.

• Head length: the vertical distance from the top of the head to the chin.

• Straight crown hip length: the vertical distance from the forehead to the end of the coccyx.

• Curved crown hip length: the distance between the end of the forehead along the back and the end of the tailbone.

2.5. Statistical analysis

The data were initially processed by Excel, then analysed by IBM SPSS 20.0 (IBM Corp, Armonk, New York), and multiple comparisons were carried out by DUNCAN. ‘Individual animal effects’ were included as a random term, while diet effects were used as a fixed term. Data were presented as means ± SEM, the significance was analysed by univariate analysis, and probability values P < .05 indicated that the differences were statistically significant.

3. Results

3.1. Growth and lactation performance of lactating ewes

With the increase of dietary energy level to 10.52 MJ/kg, the ADG of ewes changed from negative to positive (P = .005) (Figure 1(a)). When the dietary energy was maintained at 10.02 MJ/kg, and the protein level was more than 10.99%, the ADG of lactating ewes increased significantly (P < .001) (Figure 1(b)). Milk yield was not affected by the dietary energy levels of ewes; however, it increased at d 10, d 20 and d 30 with the increase of dietary protein level and reached a significant level at d 20 (P = .017). (Figure 1(b)). Dietary energy and protein levels had no significant effect on the milk composition of lactating ewes (P > .05) (Figure 1(c,d)).

Figure 1. Effect of dietary energy and protein level on lactation performance and average daily gain of lactating ewes.

Notes: Ewes were weighed every 10 d in the morning. The average daily gain (ADG) was calculated from the average of three measurements of individual daily gain. According to the difference in body weight of ewes before and after feeding their offspring, the milk yield was measured at 12:00, 16:00 and 20:00 every 10 d. Milk samples were collected continuously during the 59 and 60 d of the formal trial period (8:00 and 17:00). (a) Effect of dietary energy level on milk yield and ADG of ewes. (b) Effect of dietary protein level on milk yield and ADG of ewes. * a and b represent significant differences between each group. (c) Effect of dietary energy level on milk composition of ewes. (d) Effect of dietary protein level on milk composition of ewes. The energy levels of the three diets of ewes were E1 (9.52 MJ/kg), E2 (10.02 MJ/kg) and E3 (10.52 MJ/kg). The protein levels of the three diets of ewes were P1 (9.82%), P2 (10.99%), P3 (12.11%), E2 = P2 (n = 6).

3.2. Serum biochemical indexes

With the increase in dietary energy level, the serum TG content of lactating ewes decreased significantly (P = .018), reached a stable value when the energy level increased to 10.02 MJ/kg, and the glucose (GLU) content reached the highest at 10.02 MJ/kg (Table 2). The dietary energy level of ewes had no significant effect on the serum biochemical indexes of their offspring lambs, but the concentration of CHOL increased when the energy level of ewes increased from 10.02 to 10.52 MJ/kg (Table 3). With the increase in dietary protein level, the serum TG content of lactation Hu sheep decreased (P = .062), and the BUN concentration of lactation Hu sheep increased significantly (P = .003) (Table 4). It is worth noting that the change of serum BUN of lambs is consistent with that of ewes, and the difference is significant (P = .006). The changes of LDH and CHOL in lambs reached a significant level, and the concentration of HDL decreased at first and then increased, the concentration of LDH (P = .049) in SP2 group was the highest, while that of CHOL (P = .033) and HDL (P = .067) was the lowest (Table 5).

Table 2. Effects of different energy levels on serum biochemical indexes of lactating ewes.

Items	Groups			SEM	P-value
	E1	E2	E3
TP, g/L	73.62	69.13	69.13	1.158	.195
ALB, g/L	34.80	33.95	34.10	0.644	.863
ALT, U/L	28.47	24.95	25.22	1.348	.523
AST, U/L	135.83	112.00	118.50	5.582	.253
LDH, U/L	521.17	491.83	569.00	14.388	.079
BUN, mmol/L	1.55	2.77	2.77	0.282	.123
CREA, μmol/L	47.17	50.83	55.17	1.787	.120
GLU, mmol/L	3.42	3.78	3.48	0.075	.099
TG, mmol/L	0.32^a	0.21^b	0.21^b	0.019	.018
CHOL, mmol/L	2.43	2.45	2.31	0.086	.784
HDL, mmol/L	1.88	1.89	1.78	0.072	.796
LDL-C, mmol/L	0.52	0.57	0.51	0.029	.724
CRPL, mmol/L	4.37	4.37	4.37	0.004	.617
LACT, mmol/L	4.49	3.58	4.25	0.313	.497
NH3L, μmol/L	184.20	166.02	181.35	8.323	.459

Notes: The energy levels of the three diets of ewes were E1 (9.52 MJ/kg), E2 (10.02 MJ/kg) and E3 (10.52 MJ/kg). TP, total protein; ALB, albumin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; LDH, lactic dehydrogenase; BUN, blood urea nitrogen; CREA, creatinine; GLU, glucose; TG, triglyceride; CHOL, cholesterol; HDL, high-density lipoprotein; LDL-C, low-density lipoprotein; CRPL, C-reactive protein; LACT, lecithin-cholesterol acyltransferase; NH3L, ammonia. SEM, standard error of the mean (n = 6). In the table, the superscripts a and b represent significant differences between each group.

Table 3. Effects of lactating ewes fed diets with different energy levels on serum biochemical indexes of offspring.

Items	Groups			SEM	P-value
	SE1	SE2	SE3
TP, g/L	55.89	56.33	53.98	0.868	.522
ALB, g/L	35.39	34.43	32.61	0.528	.088
ALT, U/L	11.55	10.63	10.41	0.327	.335
AST, U/L	94.63	84.00	93.43	2.477	.165
LDH, U/L	567.75	593.38	570.88	14.096	.738
BUN, mmol/L	8.31	8.24	7.81	0.285	.751
CREA, μmol/L	40.38	38.00	44.75	1.292	.091
GLU, mmol/L	2.94	2.96	3.26	0.058	.074
TG, mmol/L	0.30	0.37	0.38	0.019	.264
CHOL, mmol/L	1.87	1.80	2.13	0.062	.055
HDL, mmol/L	1.25	1.11	1.32	0.046	.188
LDL-C, mmol/L	0.54	0.58	0.68	0.040	.321
CRPL, mmol/L	4.33	4.33	4.35	0.006	.136
LACT, mmol/L	4.85	4.85	5.41	0.140	.113
NH3L, μmol/L	288.54	302.85	322.13	7.155	.159

Notes: TP, total protein; ALB, albumin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; LDH, lactic dehydrogenase; BUN, blood urea nitrogen; CREA, creatinine; GLU, glucose; TG, triglyceride; CHOL, cholesterol; HDL, high-density lipoprotein; LDL-C, low-density lipoprotein; CRPL, C-reactive protein; LACT, lecithin-cholesterol acyltransferase; NH3L, ammonia. SEM, standard error of the mean (n = 8).

Table 4. Effects of different protein levels on serum biochemical indexes of lactating ewes.

Items	Groups			SEM	P-value
	P1	P2	P3
TP, g/L	67.05	69.13	69.67	0.971	.537
ALB, g/L	32.72	33.95	32.63	0.606	.638
ALT, U/L	24.75	24.95	29.42	1.125	.161
AST, U/L	110.17	112.00	112.50	3.867	.971
LDH, U/L	520.00	491.83	527.00	9.867	.324
BUN, mmol/L	2.12^b	2.77^b	4.83^a	0.386	.003
CREA, μmol/L	47.83	50.83	46.00	1.259	.303
GLU, mmol/L	3.30	3.78	3.44	0.111	.192
TG, mmol/L	0.24	0.21	0.19	0.009	.062
CHOL, mmol/L	2.37	2.45	2.20	0.101	.624
HDL, mmol/L	1.82	1.89	1.68	0.074	.500
LDL-C, mmol/L	0.53	0.57	0.49	0.046	.830
CRPL, mmol/L	4.38	4.37	4.37	0.005	.182
LACT, mmol/L	4.46	3.58	3.87	0.264	.408
NH3L, μmol/L	193.43	166.02	187.20	9.625	.504

Notes: The protein levels of the three diets of ewes were P1 (9.82%), P2 (10.99%) and P3 (12.11%). TP, total protein; ALB, albumin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; LDH, lactic dehydrogenase; BUN, blood urea nitrogen; CREA, creatinine; GLU, glucose; TG, triglyceride; CHOL, cholesterol; HDL, high-density lipoprotein; LDL-C, low-density lipoprotein; CRPL, C-reactive protein; LACT, lecithin-cholesterol acyltransferase; NH3L, ammonia. SEM, standard error of the mean (n = 6). In the table, the superscripts a and b represent significant differences between each group.

Table 5. Effects of lactating ewes fed diets with different protein levels on serum biochemical indexes of offspring.

Items	Groups			SEM	P-value
	SP1	SP2	SP3
TP, g/L	53.29	56.33	51.10	1.021	.107
ALB, g/L	34.35	34.43	32.81	0.607	.493
ALT, U/L	11.20	10.63	11.43	0.323	.600
AST, U/L	89.14	84.00	79.63	2.227	.219
LDH, U/L	562.50^ab	593.38^a	527.00^b	11.171	.049
BUN, mmol/L	6.66^b	8.24^ab	8.60^a	0.287	.006
CREA, μmol/L	40.88	38.00	37.13	1.526	.597
GLU, mmol/L	3.11	2.96	3.14	0.062	.468
TG, mmol/L	0.48	0.37	0.47	0.033	.322
CHOL, mmol/L	1.94^ab	1.80^b	2.21^a	0.067	.033
HDL, mmol/L	1.34	1.11	1.41	0.055	.067
LDL-C, mmol/L	0.50	0.58	0.68	0.037	.105
CRPL, mmol/L	4.34	4.33	4.33	0.005	.806
LACT, mmol/L	4.77	4.85	4.75	0.107	.927
NH3L, μmol/L	297.34	302.85	279.79	5.864	.254

Notes: TP, total protein; ALB, albumin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; LDH, lactic dehydrogenase; BUN, blood urea nitrogen; CREA, creatinine; GLU, glucose; TG, triglyceride; CHOL, cholesterol; HDL, high-density lipoprotein; LDL-C, low-density lipoprotein; CRPL, C-reactive protein; LACT, lecithin-cholesterol acyltransferase; NH3L, ammonia. SEM, standard error of the mean (n = 8). In the table, the superscripts a and b represent significant differences between each group.

3.3. Growth and body size of lambs

On the 11 d of the experiment, the lambs were fed with concentrate supplements based on breast milk. Although the birth weight of lambs in the three groups increased with the increase of dietary energy level of ewes, the difference was not significant (Table 6). However, there was a significant difference in ADG at 51–60 days (P = .011), SE3 > SE1 > SE2 group (Figure 2(a)). After 60 d of experiment, the oblique body length (P = .048), head width (P = .020), head length (P = .033) and curved crown hip length (P = .009) of lambs in SE3 and SE2 groups were significantly higher than SE1 group (Table 6).

Figure 2. Effects of lactating ewes fed diets with different energy and protein levels on average daily gain of offspring.

Notes: Lambs were weighed every 10 d in the morning. The average daily gain (ADG) was calculated from the average of three measurements of individual daily gain. (a) Effects of lactating ewes fed diets with different energy levels on ADG of offspring. (b) Effects of lactating ewes fed diets with different protein levels on ADG of offspring. SE2 = SP2. * a and b represent significant differences between each group (n = 12).

Table 6. Effects of lactating ewes fed diets with different energy levels on body size of offspring.

Items	Groups			SEM	P-value
	SE1	SE2	SE3
Born body measurements, cm (within 24 h)
Weight	2.97	3.13	3.23	0.092	.502
Body height	28.58	28.92	29.02	0.212	.563
Body oblique length	30.67	31.71	31.33	0.311	.301
Chest depth	13.58	13.46	13.25	0.104	.408
Chest girth	33.46	32.96	33.58	0.310	.696
Abdominal circumference	30.92	30.88	30.58	0.334	.911
Head width	6.38	6.39	6.58	0.048	.168
Head length	10.46	10.56	10.38	0.066	.708
Straight crown hip length	43.00	43.25	42.42	0.425	.725
Curved crown hip length	49.83	49.17	48.50	0.540	.615
Weaning body measurements, cm (60 d)
Weight	15.43	15.55	15.98	0.314	.767
Body height	49.27	50.75	51.09	0.412	.168
Body oblique length	52.18^b	53.67^ab	54.27^a	0.368	.048
Chest depth	24.45	24.92	25.40	0.171	.051
Chest girth	56.55	57.17	57.09	0.420	.816
Abdominal circumference	57.91	59.58	58.45	0.466	.328
Head width	8.46^b	8.83^ab	9.07^a	0.091	.020
Head length	16.14^b	16.36^ab	16.68^a	0.080	.033
Straight crown hip length	68.60	71.33	70.09	0.604	.160
Curved crown hip length	83.00^b	87.50^a	88.36^a	0.781	.009

Notes: SEM, standard error of the mean (n = 12). In the table, the superscripts a and b represent significant differences between each group.

The concentrate feed intake of lambs in the SP2 group was significantly higher than other two groups (Figure 3). There was no significant difference in total weight gain among the three groups, but the ADG of lambs in SP2 group reached the maximum at 0–10 d (P = .034). At 11–20 days, the daily gain was SP3 > SP2 > SP1 (P = .021) (Figure 2(b)). When the lambs were 60 d, the straight crown hip length of SP2 group was the highest (P = .005) (Table 7).

Figure 3. Feed intake of concentrated feed for lambs (air-dried basic, kg).

Notes: On the 11 d of the experiment, the lambs were fed with concentrate supplements based on breast milk. SE2 = SP2. ADFI, average daily feed intake (n = 12).

Table 7. Effects of lactating ewes fed diets with different protein levels on body size of offspring.

Items	Groups			SEM	P-value
	SP1	SP2	SP3
Born body measurements, cm (within 24 h)
Weight	3.23	3.13	3.28	0.094	.827
Body height	29.41	28.92	29.17	0.193	.597
Body oblique length	31.79	31.71	31.79	0.274	.990
Chest depth	13.43	13.46	13.61	0.103	.767
Chest girth	33.33	32.96	33.46	0.320	.812
Abdominal circumference	31.25	30.88	31.63	0.290	.587
Head width	6.51	6.39	6.42	0.040	.459
Head length	10.46	10.56	10.73	0.068	.282
Straight crown hip length	43.25	43.25	44.67	0.443	.330
Curved crown hip length	48.75	49.17	50.67	0.532	.311
Weaning body measurements, cm (60 d)
Weight	14.41	15.55	15.65	0.416	.412
Body height	48.67	50.75	49.00	0.428	.100
Body oblique length	52.09	53.67	52.73	0.401	.272
Chest depth	25.14	24.92	24.75	0.205	.758
Chest girth	56.82	57.17	56.58	0.544	.910
Abdominal circumference	57.82	59.58	58.67	0.552	.460
Head width	8.78	8.83	8.88	0.074	.927
Head length	16.38	16.36	16.48	0.085	.797
Straight crown hip length	66.58^b	71.33^a	68.17^ab	0.632	.005
Curved crown hip length	83.92	87.50	85.64	0.780	.165

Notes: SEM, standard error of the mean (n = 12). In the table, the superscripts a and b represent significant differences between each group.

4. Discussion

For lactating livestock, dietary energy and protein requirements are extremely important. Lack of energy is an important source of stress (Sordillo and Aitken 2009; Celi, 2010; Singh et al. 2011). This negative energy balance produces reactive oxygen metabolites, which leads to oxidative stress and, in severe cases, diseases such as mastitis, retained and placental retention (Sordillo and Aitken 2009; Celi, 2010). Protein, as the main nitrogen donor of the three major nutrients, is the basic component of tissues and cells. If the intake of protein is insufficient, it will lead to insufficient nutrition and weakness of lactating ewes, and it is easy to lead to lamb disease, dysplasia of body size and lack of immunity. 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 survival rate of newborn lambs depends on the milk yield and milk composition of ewes (Linden et al. 2010; Allah et al. 2011). By monitoring the milk yield of ewes, it was found that the milk yield of the high protein level group was significantly higher than that of the low protein level group. Colmenero and Broderick’s (2006) results were similar to this experiment. However, Gonzalez et al. (1985), giving lactating ewes rations containing 13, 16, and 19% CP at ME levels of 19, 23, and 27 MJ/d, observed a milk yield response to protein supplementation only at relatively high-energy intakes. Cowan et al. (1980) reported that where ewes received a higher level of dietary protein during lactation (116 vs. 143 g/kg DM CP), body reserves lost during this time were used more efficiently, leading to increases in milk yield. Jaime and Purroy (1995), who kept ME intake constant at 12.5 MJ/kg, found an increase in ewe milk yield when passing from 13 to 16% CP, irrespective of the protein source (fava beans, soybean cake, and fish meal), which is the same as the results of this experiment.

Lashein et al. (2019) reported that during pregnancy and lactation period and feeding the ewes at high ME can improve the performance of growth and milk production. In this experiment, feeding 10.52 MJ/kg high-energy level diet to lactating ewes could increase the body weight of ewes from negative to positive. However, it does not affect milk production, which may be due to the fact that the energy gradient designed in this experiment is lower than the 20% ratio set by Lashein et al. Both O’Doherty and Crosby (1997) and Mcgovern et al. (2015) suggested that excess protein intake can compensate for inadequate energy nutrition, and this needs to be considered when interpreting the results of this experiment. In this experiment, the dietary protein levels did not affect milk composition, which was similar to the results of Bach et al. (2000) and Bequette et al. (1996). Neither the fat nor the protein content of milk was changed by dietary CP level (Sevi et al. 2006).

Corner-Thomas et al. (2015) reported that lambs live birth weights were unaffected by ewes’ nutritional regimens if the lack of differences in ewes’ parameters was measured in late pregnancy. It is worth noting that the ADG of the ewes fed with the P2 group was negative, but the ADG of lambs was significantly higher than that of the other two groups at 0–10 d. During early lactation, the lamb is reliant on milk to meet its nutritional demands (Campion et al. 2016), a significant correlation between the growth rate of twin lambs and ewe milk production up to Day 42 of lactation (Snowder and Glimp 1991), so we speculate that the weight loss of ewes may be due to the intake of more nutrients by their offspring.

Early supplementary feeding has become a common practice in lamb breeding (Chai et al. 2016). With the feed intake of lambs increased, breast milk quantity did not play a decisive role in the growth and development. This may be due to the fact that the rumen of lambs fed concentrate supplementation is more mature than that of breast milk alone, which was consistent with the results of Wang et al. (2016). At 60 d, there was no significant difference in total weight gain among the three groups, but it was worth noting that the growth rate of lambs in the SP2 group was increased, and the length of the straight crown hip length and body height were significantly higher than those of the other two groups, which may be due to the fact that lambs increased their growth rate by increasing feed intake and were in a state of compensatory growth (Lippens et al. 2002). In this experiment, the birth weight of lambs in high-energy group was heavier, and the difference became more obvious with the growth and development of lambs. From 51 to 60 d, the trend of ADG of lambs was consistent with the data on concentrate intake. Snowder and Glimp (1991) reported that after 42 d of lactation, the correlation between milk yield and lamb growth rate was insignificant, signifying the lamb’s decreasing dependence on milk as its main nutrient source. It was also found that the oblique body length, head length, head width and curved crown hip length of lambs with higher birth weight were significantly higher than those of the other two groups, so it was speculated that the birth weight would affect the development rate of body size.

The growth and health status can be reflected indirectly by the determination of biochemical indexes (Joshi et al. 2002). The concentration of BUN in the serum of lactating ewes and lambs increased with the increase of protein level, which may be due to the low energy of supplementary feed and the negative balance of energy and nitrogen in the rumen, which led to the inhibition of rumen microbial reproduction and the excess of ammonia nitrogen. Indeed, the values were normal even in diets with higher concentrations, a large number of studies have shown that there is a strict correlation between BUN concentration and dietary CP levels (Jaime and Purroy 1995; Cannas et al. 1998; Frank and Swensson 2002), especially when the ME to CP ratio is relatively low, it can result in lower availability of fermentable energy in the rumen, which in turn leads to high ruminal ammonia concentrations and, consequently, to a higher urea production by the liver (Cannas et al. 1998). Surprisingly, the changing trend of BUN concentration in lamb serum is the same as that of ewe, but there is no difference between concentrate composition and breast milk composition of lamb. It is well known that sheep milk is rich in protein (Park et al. 2007), whether different milk quantities of ewes will lead to different feed intake of lambs and thus affect the change of serum BUN.

The dietary energy level of ewes did not affect the serum biochemical indexes of their offspring lambs, but affected the lipid metabolism of ewes. The serum TG content of ewes fed with a dietary energy level of 9.52 MJ/kg was significantly higher than that of the other two groups. The serum TG content of ewes fed with energy levels of 10.02 and 10.52 MJ/kg was the same, indicating that the dietary energy level of 9.52 MJ/kg might not meet the metabolic requirements of Hu sheep. Fat utilization efficiency was low, and fat utilization increased with the increase of energy level to 10.02 MJ/kg. The continued increase in energy 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, which may be the reason why the ADG of ewes decreased when the protein level increased from 9.82% to 10.99%. When the protein level increased to 12.11%, the fat utilization rate continued to increase, and the ADG of ewes increased, indicating that 12.11% of the protein level exceeded the nutritional needs of ewes and reached an amount of fat deposition.

There is a glycolytic enzyme-lactate dehydrogenase (LDH), that can catalyse the formation of lactic acid from pyruvate. The activity of serum LDH can reflect the stress sensitivity of the body. The concentration of LDH in the serum of lambs in the SP3 group was significantly lower than that in SP1 and SP2 groups, which was consistent with the difference in daily feed intake of lambs, indicating that the decrease in feed intake of lambs may significantly inhibit the production of lactic acid from pyruvate and reduce the sensitivity to stress. And the concentration of CHOL in lamb serum decreased at first and then increased. All these indicate that the lipid metabolism of offspring has been changed.

In conclusion, in the early stage of lactation, dietary protein levels can affect the ADG lambs by affecting milk yield. When the dietary energy level of ewes was properly increased, the fat utilization efficiency of ewes could be improved, and ewes fed 10.52 MJ/kg diet could better meet their growth and lactation needs. We recommend that Hu sheep be fed diets with energy and protein levels of 10.52 MJ/kg and 10.99% respectively to ensure the growth and development needs of ewes and lambs.

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.

Acknowledgements

We thank the staff at the Laboratory of Animal Nutrition and Human Health, College of Life Sciences, Hunan Normal University, for support. Thank Academician Yin Yulong for his strong support of our work.

Disclosure statement

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

Data availability statement

The authors confirm that the data supporting the findings of this experiment are available within the article [and/or] its supplementary materials.

Additional information

Funding

This work was supported by the Scientific Research Project of Hunan Education Department [grant number No. 20B369] and the National Key Research and Development Program Project of China [grant numbers 2021YFD1300400, 2021YFD1300402].