rbz Revista Brasileira de Zootecnia R. Bras. Zootec. 1516-3598 1806-9290 Sociedade Brasileira de Zootecnia 00907 10.37496/rbz5320230106 Ruminants Effect of supplementation strategies for ewes during gestation 0000-0001-6942-3106 Silva Neyton Carlos da Conceptualization Data curation Investigation Methodology Project administration Resources Validation Writing – original draft Writing – review & editing Supervision 1 * 0000-0003-3458-9135 Cabral Sergio Lucio Salomon Filho Data curation Formal analysis Methodology Project administration Resources Supervision 2 0009-0006-2249-076X Ferreira Marília Saraiva Investigation Resources 2 0000-0001-8558-5586 Silva Cássio José da Funding acquisition Project administration Resources Supervision 2 0000-0003-2203-604X Fonseca Angela Aparecida da Investigation Resources 1 0000-0001-8926-0367 Geraseev Luciana Castro Formal analysis Resources 3 0000-0003-2206-2600 Silva Bianca Damiani Marques Formal analysis Investigation Methodology Resources 4 0000-0001-5548-4709 Ribeiro Marinaldo Divino Conceptualization Funding acquisition Methodology Project administration Resources Visualization 1 Universidade Federal de Goiás Escola de Veterinária e Zootecnia Goiânia GO Brasil Universidade Federal de Goiás, Escola de Veterinária e Zootecnia, Goiânia, GO, Brasil. Universidade de Brasília Faculdade de Agronomia e Medicina Veterinária Brasília DF Brasil Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, Brasília, DF, Brasil. Universidade Federal de Minas Gerais Instituto de Ciências Agrárias Montes Claros MG Brasil Universidade Federal de Minas Gerais, Instituto de Ciências Agrárias, Montes Claros, MG, Brasil. Empresa Brasileira de Pesquisa Agropecuária, Recursos Genéticos e Biotecnologia Brasília DF Brasil Empresa Brasileira de Pesquisa Agropecuária, Recursos Genéticos e Biotecnologia, Brasília, DF, Brasil. neytoncarlos@yahoo.com.br

Marcio de Souza Duarte

Guilherme Pugliesi

The authors declare no conflict of interest.

 18 10 2024 2024 53 e20230106 12 07 2023 20 07 2024 This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT

This study aimed to evaluate the productive and reproductive performance and the nutritional metabolic condition of ewes supplemented at different stages of gestation in a synchronized reproduction system. Fourty Santa Inês × Dorper crossbreed ewes, pregnant with an average age of ten months and two previous births, were distributed in a completely randomized design, with four treatments: mineral salt, supplementation started 20 days before the estrus synchronization (ES) protocol, supplementation from confirmation of pregnancy (60 days after ES), and supplementation in the final third of gestation (90 days after ES). The adoption of supplementation strategies during the beginning, middle, or end of pregnancy changed the intake and digestibility of dry matter (g/day), with a reduction of 10.53 and 9.14%, respectively, for the mineral salt treatment, in addition to organic matter, crude protein, neutral detergent fiber, acid detergent fiber, non-fibrous carbohydrates, ether extract, and total digestible nutrients, as well as protein, energy, and mineral profiles. There was no difference in the number of ovulations and reproductive and productive performance using supplementation alternatives. Supplementation strategies during the gestation stages influence the intake and digestibility of nutrients, as well as the animal metabolic profiles. However, it does not change reproductive and productive performance.

 nutrition performance reproduction sheep 1. Introduction

Adequate supplementation for pregnant ewes is a nutritional strategy that impacts the sheep production system, such as obtaining healthy lambs (Silva et al., 2019). However, attention must be paid to the metabolic and physiological adaptations that such a period requires, like increased nutritional requirements of females.

Oliveira et al. (2014), evaluating the nutritional metabolic condition of sheep in different stages of pregnancy supplemented with 1.2 kg of concentrate/animal/day, observed that in none of the stages of pregnancy there were severe deficiencies in the protein and energy supply of the diet, with the need for calcium adequacy and phosphorus towards the end of pregnancy. Therefore, knowing the metabolic profile and its relationship with nutritional status can be an important alternative for sheep farmers to improve performance indices.

Nutritional techniques, such as flushing, are strategies used to increase fertility. Flushing consists of the increase in nutritional intake, mainly with energy feeds, before mating, aiming to increase the ovulation rate (Macedo Junior et al., 2018).

The final third of pregnancy corresponds to the period in which 70% of the fetus is developed, and is therefore critical for ewe and lamb health. The energy demand is destined to maintain the mother and develop the fetus in a more delicate way than in other phases of pregnancy. At this time, the low availability of nutrients and energy can negatively compromise the animal’s physiology after birth, being defined as fetal programming. Klein et al. (2021) reported that the effects of prenatal restriction have an impact on the animal’s performance even when subjected to correct nutritional management after birth, resulting in lower weight gain and late age at slaughter, justifying supplementation of sheep in the final third of pregnancy.

In a study carried out by Brondani et al. (2020) ewes that received food supplementation throughout the gestational period had higher body weight, differing from the other experimental treatments. This result indicates that the nutritional supply promoted by supplementation met the physiological needs of the animals, which did not need to overload the body for its physiological maintenance.

Thus, this study aimed to evaluate the productive and reproductive performance and the nutritional metabolic condition of ewes supplemented at different stages of gestation in a synchronized reproduction system.

 2. Material and Methods

The experiment was conducted in an experimental area of animal production in Brasília, DF, Brazil (15°56ʹ to 15°59ʹ S and 47°55ʹ to 47°58ʹ W) from February to July 2019. The research on animals was conducted according to the institutional committee on animal use (protocol 10/2019).

2.1. Animals, feed, and management

Forty crossbred Santa Inês × Dorper pregnant ewes with an average age of ten months and two previous births were distributed in a randomized design, according to age, weight, and body score, with four treatments and 10 replications. The ewes were maintained on a pasture with Brachiaria brizantha cv. Marandu, in an area of 5 ha. The area was divided into seven paddocks, where the animals grazed together to remove the effect of pasture. Supplements formulated according to NRC (2007) were provided daily at 16:00 h in an amount equivalent to 1% of body weight (BW), and this value was adjusted every 15 days when the ewes were weighed. The animals were confined in stalls, where they received supplementation according to the treatment. After intake, they were kept in the same pen with water ad libitum, where they spent the night. Due to the stage of pregnancy, the needs for metabolizable energy and proteins were different (Table 1).

Centesimal and nutritional composition of concentrate and pasture (<italic>Brachiaria brizantha</italic> cv. Marandu) in terms of natural matter (%) supplied to pregnant ewes
Item (%) Treatment1
T1 T2 T3 T4
Soybean meal − 56.37 56.37 36.04
Corn − 40.24 40.24 61.53
Calcitic limestone − − − 0.59
Vitamin-mineral supplement2 + 3.39 3.39 1.84
Nutrient T2; T3 T4
Supplement Pasture Supplement Pasture
DM (% NM) 96.61 93.05 92.75 94.79
CP (% DM) 29.98 9.55 23.14 4.70
NDF (% DM) 18.33 75.56 25.75 71.11
ADF (% DM) 4.98 35.25 5.61 38.84
EE (% DM) 2.74 4.60 2.65 2.85
MM (% DM) 7.33 8.96 5.61 6.22
CHOt (% DM) 59.91 80.54 68.58 86.20
NFC (% DM) 41.58 4.98 42.83 15.09
NDIN (% DM) 13.15 3.55 21.94 2.82
ADIN (% DM) 6.68 1.26 9.37 0.96
TDN (% DM) 58.42 68.43

NM - natural matter; DM - dry matter; CP - crude protein; NDF - neutral detergent fiber; ADF - acid detergent fiber; EE - ether extract; MM - mineral matter; CHOt - total carbohydrates; NFC - non-fibrous carbohydrates; NDIN - neutral detergent insoluble nitrogen; ADIN - acid detergent insoluble nitrogen; TDN - total digestible nutrients.

1 T1 = mineral salt; T2 = supplementation started 20 days before the estrus synchronization protocol (ES); T3 = supplementation from confirmation of pregnancy (60 days after ES); T4 = supplementation in the final third of gestation (90 days after ES).

2 Composition of vitamin-mineral premix: calcium (max.), 150 g; calcium (min.), 130 g; phosphorus (min.), 65 g; sodium (min.), 130 g; fluorine (max.), 650 mg; sulfur (min.), 12 g; magnesium (min.), 10 g; iron (min.), 1000 mg; manganese (min.), 3000 mg; cobalt (min.), 80 mg; zinc (min.), 5000 mg; iodine (min.), 60 mg; selenium (min.), 10 mg; vitamin A (min.), 50,000 IU; vitamin E (min.), 312 IU.

The treatments consisted of only mineral salt specific for sheep, only supplementation started 20 days before the estrus synchronization (ES) protocol, only supplementation from confirmation of pregnancy (60 days after ES), and only supplementation in the final third of gestation (90 days after ES). Ewes were identified with paint on the lumbar region according to their treatment aiming to generate less stress. The ewes were synchronized with the presence of a Dorper sire, causing the effect of male with release of pheromones to stimulate estrus at the start of the breeding season. Oil and powder paint were used in the breast region of the sire to control which sheep were covered. The type of mounting was natural, which consists of releasing the sire into the batch of females. A ratio of one male to up to fifty females is recommended. Gestation was diagnosed about 55 days after mating using an Aloka 500 ultrasound device (Aloka Co. Ltd., Japan) coupled to a 5 MHz linear transducer positioned in the rectal region.

 2.2. Reproductive performance

Reproductive performance was evaluated using measurements of pregnancy rates ((number of pregnant ewes/number of mated ewes) × 100), parturition rates ((number of lambed ewes/number of mated ewes) × 100), and birth rates ((number of lambs born/number of mated ewes) × 100).

The ewes in estrus had the number of ovulations determined by transrectal ultrasonography, using a CHISSON 500 device with a 5 MHz linear transducer, which was attached to a rigid extension (3 cm in diameter and 16 cm in length) that allows its manipulation via the rectum. Ovulation was determined by the disappearance of the dominant follicles present on the surface of the ovaries in the final third of estrus or heat, between 24 and 30 h after the beginning of estrus, being the phase of the estrous cycle in which the female accepts mating, letting itself be mounted by the male (Saunders et al., 2012).

 2.3. Weight variation and body score condition

Performance was evaluated by weighing all dams every 15 days to obtain the average daily weight gain (ADG, g/animal/day), calculated by the difference in animal weight between weighings divided by the number of days between each weighing. Total weight gain (kg/animal) was obtained by the difference between the initial live weight and the day of birth.

Body condition score (BCS) was determined by palpations of the spine after the last rib, above the kidney region, according to the methodology described by Osório and Osório (2005), assigning values from 1 to 5, with 1 corresponding to animals excessively thin animals and 5 to excessively fat animals, also considering intermediate values in variations of 0.25.

 2.4. Produced biological mass

The produced biological mass corresponds to the total weight of lambs produced by the female, originating from simple or twin dishes relative to the dam’s body weight (Geraseev et al., 2006).

 2.5. Nutrient intake and digestibility

Total dry matter (DM) intake was obtained by the sum of supplement dry matter intake (SDMI) and the estimated forage dry matter intake (FDMI), estimated through fecal excretion and the use of markers. Nutrient intake was obtained by the sum of SDMI and FDMI, multiplied by the respective nutrient concentrations. Supplement intake was determined in each gestational period, that is, beginning (50 and 60 days), middle (80 and 90 days), and end (130 and 140 days), by the ratio between the amount of DM supplied and the DM of leftovers. Consumption in metabolic weight corresponds to live weight raised to the power of 0.75 (LW0).

The estimate of fecal excretion per animal and experimental period was obtained using an external marker of titanium dioxide (TiO2), given orally administered in capsule form to the animals in daily doses of 5 g during nine days. Fecal samples were taken directly from the rectum of the animals on the 6th, 7th, 8th, and 9th day of each experimental period to determine the TiO2 concentration. The samples were placed in identified plastic bags and frozen at −10 °C for further analysis. Titanium concentration was determined following the methodology described by Detmann et al. (2012), in which the fecal samples were digested in sulfuric acid, and the reading was performed in a UV spectrophotometer at 410 nm, using a calibration curve with known titanium concentration.

Fecal dry matter excretion was calculated according to Burns et al. (1994), based on the ratio between the amount of indicator provided and its concentration in the feces: fecal excretion (g DM/day) = [Amount of indicator provided ( g / day)/Indicator concentration in the feces (%)] × 100 . Then, FDMI was estimated using the indigestible neutral detergent fiber (iNDF) as an internal indicator (Casali et al., 2008) and applying the equation proposed by Detmann et al. (2012): FDMI = [ ( FE × CIF ) − IS ] / CIFO , in which FE is the fecal excretion (kg/day), CIF is the concentration of the indicator in the feces (kg/kg), IS is the indicator present in the supplement (kg/day), and CIFO is the concentration of the indicator in the forage (kg/kg).

Nutrient digestibility (ND) was obtained by the following formula: [ND = (ingested DM × \% Nutrient) - (excreted DM × \% Nutrient) × 100] / (ingested DM × \% Nutrient) .

The total digestible nutrients (TDN) content was calculated considering the intake and fecal excretion of nutrients, using the following equation: TDN ( % ) = DCP + ( DEE × 2.25 ) + FDND + DNFC , in which DCP is the digestible crude protein, DEE is the digestible ether extract, DNDF is the digestible neutral detergent fiber, and DNFC is the digestible non-fiber carbohydrates (NRC, 2001).

 2.6. Metabolic profiles

A single blood collection was performed in the morning in each ewe and at different stages of gestation to allow environmental comfort for the animals. Blood samples were obtained through jugular venipuncture, using a vacuum tube (Vacutainer®) and specialized needles. Two types of duly identified vacuum tubes were used: one containing sodium fluoride for energy analysis and another without it for obtaining serum. The collections strictly followed the regime of intervals predetermined by the experimental treatments, the initial phase of gestation is between 50 and 60 days, the middle between 80 and 90 days, and the final between 130 and 150 days. After collection, the samples remained at rest until complete coagulation, followed by centrifugation for 10 min at 1400 rcf (g) and transference of the serum to microtubes with a capacity of 0.5 mL for storage at −20°C. Biochemical analyses were performed using diagnostic kits with enzymatic methodological principles from the companies Labtest Diagnóstica® S.A., BioClin®, and Randox®, following their respective protocols. Reading was performed in an automated COBAS MIRA PLUS (Roche®, Germany) equipment, specific for biochemical analysis. The evaluated metabolites were protein (total protein, albumin, globulin, and urea), energy (glucose and βhydroxybutyrate (BHB)), and mineral (magnesium (Mg2), calcium (Ca2), and phosphorus (P−)).

 2.7. Chemical analysis

Samples of the supplied diet, leftovers, and feces were partially dried in a forced-air ventilation oven at 55 °C and ground in a Willey mill with a 1-mm diameter opening sieve to determine the DM according to the AOAC (2005) (method 930.15), mineral matter (MM; method 942.05), crude protein (CP; method 984.13), and ether extract (EE; method 920.39). The fibrous fractions, that is, neutral detergent fiber (NDF) and acid detergent fiber (ADF), were determined using the methodology proposed by Van Soest et al. (1991). Neutral detergent insoluble nitrogen and acid detergent insoluble nitrogen were determined following the recommendations by Licitra et al. (1996). The percentage of total carbohydrates was calculated using the equation proposed by Sniffen et al. (1992), while the non-fibrous carbohydrates (NFC) were determined using the equation recommended by Weiss (1993).

 2.8. Statistical analysis

Data were subjected to analysis of variance, considering the type of diet (feeding strategy) of ewes and gestational stages as sources of variation. The statistical model used was:

Y i j k = μ + T i + B j + ε i j + D k + T D i k + ε i j k ,

in which µ = general average; Ti = effect of treatment (i = 1, 2, 3, 4); Bj = effect of the score range (j = 1, 2, 3, 4, 5); εij = experimental error associated with the plot, considered independent and identically distributed from a zero mean normal and variance σ2; Dk = effect of time (k = 20 days before the estrus synchronization (ES) protocol, supplementation from confirmation of pregnancy (60 days after ES), and supplementation in the final third of gestation (90 days after ES); TDik = interaction between treatment and time; and εijk = experimental error associated with the subplot, considered independent and identically distributed from a zero mean normal and variance σ2. The comparison between the effects of the type of feeding management and gestation was performed using Tukey’s test at 0.05 probability, using the statistical procedures PROC GLM of SAS (Statistical Analysis System, version 9.2.). The Kruskal-Wallis non-parametric test was used for the variable BCS, with slicing or not of the interaction according to the significance.

 3. Results

Supplementation strategies affected DM and organic matter (OM) intake by the animals (Table 2). Dry matter intake ranged from 2,056 to 1,965 g/day and OM intake from 1,881 to 1,813 g/day, being higher (P<0.05) in the beginning and middle of gestation for animals that received supplementation from 20 days before the estrus synchronization ES protocol. This behavior was already expected since these animals had higher TDMI (forage + concentrate) than the other treatments. There was an effect (P<0.05) of supplementation on DM and OM intake in the final third of gestation, with higher averages for the group that received supplementation after pregnancy confirmation, that is, 2,126 and 1,997 g/day, respectively.

Nutrient intake as a function of supplementation strategy for ewes at different stages of gestation
Variable Beginning Middle End CV (%)
T1 T2 T3 T4 T1 T2 T3 T4 T1 T2 T3 T4
DMI    
g/day 1,584b 2,056a 1,425b 1,623b 1,947ab 1,965a 1,690b 1,728ab 1,774b 1,830b 2,126a 1,869ab 12.06
g/kg0.75 41.97b 53.91a 38.45b 42.83b 48.09a 45.32a 41.28a 41.62a 42.37ab 39.97b 49.68a 43.36ab 13.57
%BW 3.148b 4.043a 2.884b 3.212b 3.607a 3.399a 3.096a 3.121a 3.178ab 2.998b 3.726a 3.252ab 13.57
OMI  
g/day 1,442b 1,881a 1,297b 1,477b 1,794ab 1,813a 1,560b 1,592ab 1.664b 1,719b 1,997a 1,756b 11.82
g/kg0.75 38.21b 49.32a 35.00b 38.99b 44.30a 41.82a 38.11a 38.34a 39.74ab 37.56b 46.67a 40.74ab 13.18
%BW 2.865b 3.699a 2.625b 2.924b 3.323a 3.136a 2.858a 2.875a 2.980ab 2.817b 3.500a 3.056ab 13.18
CPI  
g/day 119.8b 285.5a 107.8b 122.8b 186.1c 306.2a 279.9b 165.1c 83.5b 188.7a 202.6a 190.5a 9.98
g/kg0.75 3.17b 7.47a 2.90b 3.24b 4.59b 7.07a 6.86a 3.97b 1.99b 4.14a 4.73a 4.42a 12.44
%BW 0.238b 0.560a 0.218b 0.243b 0.344b 0.530a 0.514a 0.298b 0.149b 0.311a 0.355a 0.331a 12.44
NDFI  
g/day 1,197a 1,222a 1,077a 1,226a 1,443a 1,132b 0.929c 1,280ab 1,262a 1,049b 1,259a 1,077ab 14.84
g/kg0.75 31.72a 32.08a 29.05a 32.36a 35.63a 26.07bc 22.62c 30.83ab 30.13a 22.84b 29.43a 24.98ab 15.65
%BW 2.379a 2.406a 2.179a 2.427a 2.672a 1.955bc 1.697c 2.312ab 2.260a 1.713b 2.207a 1.873ab 15.65
ADFI  
g/day 558.69a 549.62a 502.67a 572.39a 675.05a 509.10b 410.86c 599.5ab 689.26a 526.03b 640.97a 541.34b 14.83
g/kg0.75 14.80a 14.43a 13.55a 15.10a 16.66a 11.71a 9.98a 14.42a 16.46a 11.43c 14.97ab 12.55bc 15.46
%BW 1.110a 1.082a 1.016a 1.132a 1.250a 0.878b 0.749b 1.081a 1.234a 0.857c 1.122ab 0.941bc 15.46
NFCI  
g/day 52.16b 289.70a 46.93b 53.44b 97.06b 310.13a 296.42a 86.13b 267.87c 430.6b 475.35a 436.6b 8.22
g/kg0.75 1.38b 7.56a 1.26b 1.41b 2.39b 7.18a 7.28a 2.07b 6.39b 9.44a 11.11a 10.12a 15.94
%BW 0.103b 0.567a 0.094b 0.105b 0.179b 0.538a 0.546a 0.155b 0.479b 0.708a 0.833a 0.759a 15.94
EEI  
g/day 73.03b 83.99a 67.63b 74.82ab 68.12a 64.38ab 55.14b 60.45ab 50.69b 51.19b 60.81a 52.31ab 13.30
g/kg0.75 1.93ab 2.20a 1.83b 1.97ab 1.68a 1.48ab 1.34b 1.45ab 1.21a 1.11a 1.42a 1.21a 15.13
%BW 0.145ab 0.165a 0.137b 0.148ab 0.126a 0.111ab 0.100b 0.109ab 0.090a 0.083a 0.106a 0.091a 15.14
TDNI (kg) 1.001b 1.418a 0.891b 1.058b 1.244b 1.447a 1.152b 0.835c 1.146b 1.249b 1.493a 1.299b 13.21

DMI - dry matter intake; OMI - organic matter intake; CPI - crude protein intake; NDFI - neutral detergent fiber intake; ADFI - acid detergent fiber intake; NFCI - non-fibrous carbohydrates intake; EEI - ether extract intake; TDNI - total digestible nutrient intake; CV - coefficient of variation.

T1 = mineral salt; T2 = supplementation started 20 days before the estrus synchronization protocol (ES); T3 = supplementation from confirmation of pregnancy (60 days after ES); T4 = supplementation in the final third of gestation (90 days after ES).

Means followed by different letters in the row differ from each other by Tukey’s test (P<0.05).

There was an increase (P<0.05) in CP intake (g/day, g/kg0, and % BW) with the supplementation due to the effect on DM and OM intake (Table 2) and the difference in the isonitrogenous profile of the diets offered (Table 1).

A significant increase (P<0.05) in the average NDF intake was observed for the control group (Table 2), which can be explained by the pasture quality during the experiment (Table 1) and DM intake (Table 2). However, there were no differences among treatments in early gestation. The higher intake of roughage in the treatment that received mineral supplementation during gestation increased (P<0.05) ADF intake among periods (Table 2), with an average of 641.0 g/day. However, no differences in the early stage of gestation were observed.

The average NFC intake was influenced (P<0.05) by diet, with increasing effect. This result can be justified by the increase in NFC concentration in diets with the supplement (Table 1), as roughage results in an increase in the fiber content of the diet and a reduction in non-fiber carbohydrates.

There was an effect (P<0.05) on EE intake (g/day, g/kg0, and % BW) with the supplementation strategy due to the presence of an effect on DM intake (Table 2) and the heterogeneous profile of the offered diets (Table 1).

Total digestible nutrients intake varied (P<0.05) along the gestation stages (Table 2), as verified for DM and OM intake.

Dry matter (DMD) and OM digestibility (OMD) was influenced (P<0.05) by the treatments in the gestational stages (Table 3). However, ewes supplemented 20 days before the ES protocol showed higher coefficients in the initial period. In contrast, the middle of gestation showed lower DMD for the group supplemented 90 days after ES, with no effect on OMD. Ewes from the control treatment in the final third had a lower OMD (66.73%) but with no effect on DMD (67.96%).

Digestibility coefficient of nutrients as a function of the supplementation strategy for ewes at different stages of gestation
Variable Beginning Middle End CV (%)
T1 T2 T3 T4 T1 T2 T3 T4 T1 T2 T3 T4
DMD (%) 63.32b 71.85a 61.71b 64.93b 64.01a 67.00a 66.53a 59.14b 63.99a 67.92a 70.23a 69.73a 4.77
OMD (%) 64.73b 72.82a 62.97b 65.89b 65.92a 68.84a 68.38a 61.12a 66.73b 70.27ab 72.60a 72.23a 4.45
CPD (%) 47.19b 77.96a 44.77b 49.85b 56.76b 74.15a 77.00a 50.90c 23.29b 70.66a 68.98a 68.24a 6.48
NDFD (%) 63.09a 60.88a 60.66a 61.69a 70.85a 67.93b 67.06b 40.47c 67.47a 65.31a 68.61a 66.86a 7.05
ADFD (%) 69.23a 68.98a 68.56a 70.00a 69.24a 64.71ab 63.88b 63.88b 69.16a 64.44a 69.42a 67.20a 6.72
NFCD (%) 61.74b 85.91a 61.82b 60.58b 78.24a 85.07a 80.12a 69.10b 79.04b 82.11ab 86.62ab 89.71a 8.04
EED (%) 43.62b 48.35a 39.97b 48.54a 26.83b 27.74ab 24.06b 37.63a 54.96b 70.39a 57.58b 49.74b 15.19
TDND (%) 63.55a 69.09a 62.55a 65.68a 63.84b 74.57a 69.06ab 52.39c 64.54a 68.48a 70.33a 69.61a 12.06

DMD - dry matter digestibility; OMD - organic matter digestibility; CPD - crude protein digestibility; NDFD - neutral detergent fiber digestibility; ADFD - acid detergent fiber digestibility; NFCD - non-fibrous carbohydrates digestibility; EED - ether extract digestibility; TDND - total digestible nutrient digestibility; CV - coefficient of variation.

T1 = mineral salt; T2 = supplementation started 20 days before the estrus synchronization protocol (ES); T3 = supplementation from confirmation of pregnancy (60 days after ES); T4 = supplementation in the final third of gestation (90 days after ES).

Means followed by different letters in the row differ from each other by Tukey’s test (P<0.05).

A higher effect (P<0.05) of the supplementation was observed on the CP digestibility coefficient. Possibly, it reflects the lack of similarity in the CP contents of the diets (Table 1).

There was an effect (P<0.05) on NDF digestibility among treatments (Table 3), with an average of 67.13%. Despite this, no significant difference was observed among treatments in the initial and final stages of gestation, possibly reflecting the quality of roughage fiber during the study. However, in the middle of gestation, ewes supplemented in the final third had lower NDFD (40.47%).

No significant difference (P>0.05) was observed in the initial and final stages of gestation. In the middle of gestation, ewes supplemented with mineral salt had a higher ADF digestibility (69.24%).

The NFC digestibility was influenced (P<0.05) by supplementation strategies during gestation (Table 3). However, animals supplemented 20 days before the ES protocol had higher coefficients at the initial stage (85.91%). Despite this, groups supplemented in the final third of gestation had lower and higher NFC digestibility in the middle and end of gestation, with averages of 69.10 and 89.71%, respectively.

An effect (P<0.05) of the supplementation addition on the EE digestibility coefficient was observed (Table 3), with averages of 45.12, 29.02, and 58.16%, respectively, in the gestational periods. Possibly, it reflects the lack of similarity in the EE contents of diets. Furthermore, ewes supplemented 20 days before and 90 days after ES had higher digestibility in the initial stage and in the middle of the period; however, only the second treatment had the greatest value at the end of gestation.

Supplementation did not affect (P>0.05) %TDN in the initial and final thirds of gestation, with averages of 62.25 and 68.24%, respectively. However, there was an increase (P<0.05) in the middle of gestation, with an average of 71.81% TDN (Table 3) as a function of supplementation.

The number of ovulations between the ovaries and the total corpus luteum did not differ (P>0.05) between ewes subjected or not to supplementation strategies (Table 4), with average counts of 0.7, 0.8, and 1.65, respectively.

Number of ovulations and reproductive performance according to supplementation strategies
Item Treatment1 P-value
T1 T2 T3 T4
Left ovary 0.9a 0.6a 0.9a 0.7a 0.8633
Right ovary 0.8a 0.8a 1.2a 0.7a 0.0988
Total corpus luteum 1.7a 1.4a 2.1a 1.4a 0.2708
Pregnancy (%) 100 100 100 100 -
Parturition (%) 100 100 100 100 -
Birth (%) 1.20a 1.20a 1.20a 1.4a 0.4949

1 T1 = mineral salt; T2 = supplementation started 20 days before the estrus synchronization protocol (ES); T3 = supplementation from confirmation of pregnancy (60 days after ES); T4 = supplementation in the final third of gestation (90 days after ES).

Means followed by different letters in the row differ from each other by Tukey’s test (P<0.05).

Pregnancy, parturition, and birth variables (Table 4) were not influenced (P>0.05) by supplementation in pre-breeding, with means of 100, 100, and 1.25%, respectively. The absence of the effect of supplementation, offered from 20 days before the estrus synchronization ES protocol, indicates that the nutritional condition of ewes was adequate for all treatments to guarantee good reproductive performance.

The supplementation strategy did not affect (P>0.05) the final live body weight, daily weight gain, and total weight gain of ewes despite the observed changes in nutrient intake (Table 5).

Live weight gain, produced biological mass, and body condition score (BCS) according to supplementation strategies
Item Treatment1 CV (%) P-value
T1 T2 T3 T4
FLW (kg) 65.51a 71.65a 69.72a 66.70a 13.52 0.1735
ADG (g/animal/day) 98.71a 130.42a 134.29a 102.75a 28.10 0.0899
TLWG (kg/animal) 14.81a 19.57a 20.14a 15.41a 28.09 0.0892
BM (kg) 0.072a 0.069a 0.076a 0.078a 20.61 0.0017
Final BCS 2.5a 3.50a 3.0a 2.75a 10.47 0.1276

FLW - final live weight; ADG - average daily gain; TLWG - total live weight gain; BM - biological mass; CV - coefficient of variation (CV).

1 T1 = mineral salt; T2 = supplementation started 20 days before the estrus synchronization protocol (ES); T3 = supplementation from confirmation of pregnancy (60 days after ES); T4 = supplementation in the final third of gestation (90 days after ES).

Means followed by different letters in the row differ from each other by Tukey’s test (P<0.05).

No effect (P>0.05) of treatments was observed on the produced biological mass (Table 5), with an average of 0.073 kg. Body condition score (Table 5) was not affected by supplementation (P>0.05), with an average of 2.93. Thus, the similarities in the final BCS observed in this study occurred because the supplementation did not reduce the daily, total, and final weight gains (Table 5).

An increase was observed among the evaluated groups, with a difference (P<0.05) for protein metabolites (Table 6), which remained within the reference limits, except for urea in the final third of gestation, which showed increased levels for the group that received supplementation started 20 days before the ES protocol.

Metabolic profile of ewes at different stages of gestation according to supplementation strategy
Variable Beginning Middle End RV1 CV (%)
T1 T2 T3 T4 T1 T2 T3 T4 T1 T2 T3 T4
Protein metabolism
Total proteins (g/L) 60.92b 69.89a 61.74b 63.12b 62.40b 72.54a 70.77a 64.10b 66.55c 75.89a 72.22b 66.39c 60-79 3.18
Albumin (g/L) 27.78b 31.87a 28.15b 28.78b 27.42b 31.88a 31.10a 28.17b 30.77c 35.10a 33.40b 30.71c 26-42 3.18
Globulin (g/L) 33.14b 38.02a 33.59b 34.34b 34.97b 40.65a 39.66a 35.92b 35.76c 40.78a 38.81b 35.68c 35-57 3.35
Urea (mg/100 mL) 24.65b 36.12a 26.53b 26.14b 25.86c 38.46a 31.89b 27.30c 29.25c 46.00a 36.41b 32.03c 17-43 8.10
Energy metabolism
Glucose (mg/100 mL) 33.87c 45.13a 37.85b 35.19bc 36.19c 48.28a 40.15b 38.04bc 40.98c 55.42a 47.87b 32.27d 50-80 4.99
Β-hydroxybutyrate (mmol/L) 0.13d 0.22c 0.76a 0.58b 0.12d 0.22c 0.74a 0.58b 0.16d 0.26c 0.78a 0.62b <0.6 8.34
Mineral metabolism
Calcium (mg/100 mL) 11.69a 10.58b 9.68c 8.71d 11.79a 10.70b 9.80c 8.81d 11.40a 10.30b 9.40c 8.45d 11.5-12.8 6.15
Phosphorus (mg/100 mL) 6.21b 7.04a 5.82b 5.08c 6.40b 7.20a 6.50b 5.59c 5.10c 6.30a 5.70b 5.04c 5.0-7.3 6.09
Magnesium (mg/100 mL) 2.21b 2.55a 2.19bc 2.11c 2.21b 2.55a 2.20bc 2.11c 2.22b 2.57a 2.21b 2.12c 2.2-2.8 5.35

CV - coefficient of variation.

T1 = mineral salt; T2 = supplementation started 20 days before the estrus synchronization protocol (ES); T3 = supplementation from confirmation of pregnancy (60 days after ES); T4 = supplementation in the final third of gestation (90 days after ES).

1 Reference values for sheep (Kaneko et al., 1997).

Means followed by different letters on the row differ from each other by Tukey’s test (P<0.05).

Mean globulins were different (P<0.05) between times and treatments, averaging 34.77, 37.80, and 37.75 g/L (Table 6). The values are within the normality range for sheep (from 35 to 57 g/L). However, a higher serum concentration of globulin was observed in the initial and final stages of gestation for animals supplemented from 20 days before the ES protocol (Table 6).

Glucose means were different among treatments (P<0.05), showing a numerically increasing concentration with the advancement of gestation, especially in the final third, in which animals supplemented 20 days before the ES protocol had an average of 55.42 mg/100 mL, which is within the minimum normal value for sheep, that is, 50 to 80 mg/dL (Table 6).

The variation of BHB was significant (P<0.05) in the three stages of gestation (Table 6), with increasing levels. It also remained close to the reference standards for sheep (<0.6 mmol/L).

A significant difference (P<0.05) was observed with an increase and decrease in serum calcium levels (Table 6), but animals supplemented with mineral salt were within the reference values, with an average of 11.62 mg/100 mL, showing that the balance of this mineral was adequate for the gestational period.

The serological concentration of phosphorus increased during the beginning and middle of gestation, with an average of 6.23 mg/100 mL, but a significant decrease (P<0.05) was observed in the final third of pregnancy (5.53 mg/100 mL) (Table 6).

Serum magnesium levels remained within the reference standards (Table 6), with an increasing effect. However, they were statistically different (P<0.05) among groups, with the highest concentrations observed for animals that received supplementation from 20 days before the estrus synchronization ES protocol.

 4. Discussion

The supplement increased the supply of nutrients to the animals, which increases the CP and NFC intake in the different periods of gestation, thus leading to higher DM and OM intake for the supplemented groups.

Sousa et al. (2018), evaluating the impact of supplementation levels of 0.5 to 1.5% concentrated based on body weight in the reproductive phases (gestation and lactation) of Santa Inês hair sheep, observed that sheep fed 1.5% of BW had higher dry matter intake (DMI). This behavior was already expected since these animals had higher TDMI (forage + concentrate) than the other treatments.

According to Silva et al. (2019), intake becomes limited by the physiological demand for energy if energy density is high or fiber concentration is low compared with the requirements. This finding is probably related to the energy density of the diet, which is lower based on roughage, with a consequent lower amount of corn, starch, and energy supply, which justifies the increase in intake to meet energy requirements (Table 2).

The fact that the ewe is in the final third of gestation, a stage in which the gravid uterus is compressing the gastrointestinal tract, may have significantly affected its intake. In addition, supplements increase energy density, which, in turn, may also affect intake. Geraseev et al. (2023) stated that satiety is due to the caloric density of the diet. This fact was not observed for animals supplemented in the final third of gestation, which had higher DMI and OM intake, even when large amounts of soluble carbohydrates were used in the diets.

According to NRC (1985), DMI for ewes with 60 kg of live weight in the last four weeks of gestation should be 1.6 kg or 2.8% of live weight. In the study, the average DMI was 1.89 kg, or 3.28% of live weight, showing that the results observed in the present study are within the recommended by this committee.

Similarly, Macedo Junior et al. (2012) observed an NDFI of 34.37 g/kg0 when evaluating the influence of different roughage and concentrate ratios (10, 20, 30, and 40% of roughage) in the diet of sheep on intake, glycemia, and apparent digestibility in pregnant females. Intake in the present study was 28.97 g/kg0, showing that the results are within the range recommended for animals on pasture.

The DMD and OMD of animals that received supplementation 20 days before ES may have occurred due to the higher energy intake provided to animals in this treatment, which possibly resulted in an adequate balance of carbohydrates and nitrogenous compounds available in the rumen, which contributed to the development of cellulolytic microorganisms, responsible for the degradation of cell wall components, thus resulting in increased digestibility. Sousa et al. (2018) verified that an increase in the energy value of diets improved the digestibility coefficients of DM, OM, total carbohydrates, and NFC of Santa Inês ewes supplemented (0.5 and 1.5% live weight) on pasture pre-and postpartum.

The reduction of NFC affects the development of the rumen microbiota (Zhang et al., 2020). Geraseev et al. (2023), when evaluating the effects of including sunflower bran on performance, nutrient digestibility, and respirometric parameters in Dorper × Santa Inês sheep, observed a decrease in the in vivo digestibility of DM, NFC, and NDF with the inclusion of bran, which may be associated with the reduction of NFC in diets and roughage quality.

Flushing is one of the nutritional management tools with an impact on reproduction and is perhaps the most widespread among technicians and producers. This tool provides supplementation with a high nutritional level during a short period, which includes before and during the breeding season (Macedo Junior et al., 2018). However, significant increases in the ovulation rate through flushing will only be obtained if ewes are subjected to a period of supplementation equivalent to one estrous cycle before the beginning of the breeding season, with no effect when ewes present satisfactory body condition (Yıldırır et al., 2022). Thus, ewes could have better reproductive performance throughout the experimental period by receiving supplementation starting 20 days before the ES protocol until lambing, however, without effect because the sheep have satisfactory nutritional and body conditions.

According to Saunders et al. (2012), the minimum body condition for Santa Inês sheep to manifest their reproductive capacity is 2.5. Thus, this could probably have been the reason for the lack of difference in the number of ovulations, as ewes already started the experiment with a BCS of 3.7, above the established threshold of 2.5.

Khaiseb et al. (2022) evaluated the interactions between nutrition and the ram effect on the control of ovarian function in Merino sheep. They observed that supplementation before the introduction of rams did not affect the ovulation rate in ram-induced ovulation, with an average of 1.66, that is, close to that work with average counts of 1.65 follicles.

Yıldırır et al. (2022) found that ewes with body scores higher than or equal to 2.5 had better reproductive performance than animals with a lower body condition. Thus, the importance of using BCS as a herd nutritional monitoring tool is evident (González-Maldonado et al., 2023).

According to NRC (2007), ewes at the end of gestation have a CP requirement of 193 g/day. In the present study, ewes had an average protein intake of 186.60 g/day, which is close to the recommended intake for the three stages of gestation. Furthermore, the supplementation strategy increased CP intake (g/day, g/kg0, and % BW) (Table 2) and CP digestibility coefficient (Table 3). It is possibly a reflection of the lack of similarity in CP contents of the diets (Table 1) and, consequently, changed the serum levels of total protein (Table 6). The concentration of total proteins in the blood reflects the protein nutritional status reliably (Castillo et al., 2016).

When evaluating the nutritional metabolic condition of sheep at different stages of pregnancy, Oliveira et al. (2014) observed that the concentrations of total proteins, albumin, and globulins did not differ from each other. In our study, the protein concentration was close to the lower limit reported by Kaneko et al. (1997).

The highest urea concentrations, with averages ranging from 38.46 to 46 mg/100 mL in the middle and final third of gestation (Table 6) for animals supplemented from 20 days before the ES protocol, are related to the higher protein intake. The higher the dietary protein intake, the higher the blood urea level, and, on the other hand, blood urea levels reach low values when protein intake is insufficient (Barbosa et al., 2023).

Serological concentrations of glucose differed statistically, showing a numerically increasing concentration with the advancement of gestation and below the minimum normal value for sheep, which is 50 to 80 mg/dL. Nutrient requirements, especially energy, increase in the last six weeks of gestation due to fetal growth, in which the fetus reaches up to 80% of its final size and weight (McCarty et al., 2020). Oliveira et al. (2014) found glucose values between 38.58 and 44.29 mg/100 mL at different gestational stages of pregnant Santa Inês ewes reared in a semi-intensive system. These results are below the reference values defined by Kaneko et al. (2008) but are within the range defined in this study, with an average of 40.93 mg/mL. Song et al. (2018) found lower values of plasma glucose in sheep subjected to a diet with reduced energy intake.

The variation in BHB can be attributed to the genetic and adaptive characteristics of these animals, related to the rearing system and feed. The ewes had good BCS (3 to 3.5), thus allowing body energy reserves. These characteristics are extremely important to control metabolic disorders such as toxemia (Mendes et al., 2021).

Serum calcium levels were always below the reference values for ewes that received supplementation. Oliveira et al. (2014), working with sheep in different thirds of gestation (beginning, middle, and end), found calcium values of 9.39, 9.22, and 8.86, respectively, and observed a decrease in this mineral in the final third of gestation, which was attributed to the fetal growth and milk synthesis. In this research, the lowest value of this mineral was observed at the end of pregnancy (9.88 mg/100 mL).

Phosphorus serum concentration decreased during the final third of gestation, being different from the concentration of animals in early gestation. It may indicate a phosphorus deficiency in the soil and, consequently, the forage in the region, considering the phosphorus requirement of ewes in the final third of gestation. Oliveira et al. (2014) worked with ewes at different stages of gestation (initial, medium, and final) and found phosphorus values of 7.15, 7.42, and 5.88 mg/100 mL, respectively. The low values were attributed to phosphorus deficiency in the soil and, consequently, in the pasture ingested by the animals. Feijó et al. (2014) observed mean values of 5.33 mg/dL for non-pregnant females. Santos et al. (2014) observed that phosphorus levels decreased seven days before parturition, maintaining this behavior up to 15 days after parturition. Therefore, the balance of this mineral in the present study was adequate for the gestational period, being within the reference standards (Table 6).

Hypomagnesemia, a nutritional disease, usually caused by low magnesium intake in the diet, can lead to tetany, hyperexcitability, retained placenta, abnormalities of ruminal digestion, and decreased milk production (Moreira et al., 2019). Normally, serological alterations are due to a deficiency of this mineral in the soil and, therefore, in the pasture. However, the magnesium balance in the present study is within the reference standards (Table 6).

Nutritional restriction in the prenatal phase has negative effects during the postnatal life of the animal, causing an increase in the age at slaughter of lambs, a reduction in the quality of the final product, a delay in the age at puberty of lambs, and low reproductive efficiency of the herd (Siqueira et al., 2020). Therefore, it is essential to meet the female’s nutritional requirements during the different phases of the gestational period, due to the impact on the productive and reproductive performance of her offspring.

 5. Conclusions

Supplementation strategies during the gestation stages influence the intake and digestibility of nutrients, as well as the animal metabolic profiles. However, they do not change reproductive and productive performance.

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