The Impact of Fetal Programming in Ewe Nutrition with Chromium Propionate or Calcium Salts of Palm Oil on the Meat Quality and Bone of the Progeny

This study aimed to evaluate the inclusion of chromium propionate or calcium salts of palm oil in ewes’ diet during the final third of gestation and lactation on progeny performance, carcass characteristics, non-carcass components, and bone density. Forty-three ewe, Santa Inês and Dorper breed, three ± one-year-old, and body weight 57 ± 10 kg were used. The experimental design was in casual blocks in three treatments, CTL treatment (n = 15) with starch from corn; CR (n = 15) diet CTL plus chromium propionate; PF (n = 13) diet CTL plus calcium salts of palm oil. After weaning, 23 male lambs from these ewes were confined in individual stalls, with the same diet for 60 days, slaughtered. The data were analyzed using the SAS program, PROC GLM, and compared the means using Tukey’s test at 5% probability. The maternal diet did not alter the dry matter intake, feeding efficiency, and average daily weight gain. Therefore, weights (weaning and slaughter) and carcass yield were higher for CR and PF groups than for CTL (P < 0.05). The treatment did not influence the loin eye area and fat thickness (P > 0.05). The spleen and the respiratory tract were smaller for PF and larger for CTL (P < 0.05). Leg weight was higher for CR. The perimeter and depth of the shank for the CR and PF lambs were higher, indicating an effect of maternal nutrition in this commercial cut. The CR group had a smaller epiphysis measurement and femur length than the CTL group. We concluded that the fetal programming effect in ewes fed with Cr propionate and Ca salts of palm oil benefited the progeny by increasing their body weight, better carcass yield, and a higher proportion of prime cuts.


Introduction
Ewes in gestation and lactating have an increased nutritional requirement due to fetal growth, colostrum, and milk synthesis [1], and poor nutrition can result in metabolic diseases such as pregnancy toxemia [2]. However, maternal nutrition may affect lamb's body weight [3], development of the liver, lung, and pancreas [4], meat quality, weight of premium cuts [5], and changes in bone tissue [6]. The fetal programming study is investigating the effect of maternal nutrition on offspring. The correct energetic support to the ewe at the end of gestation can result in more significant muscle fiber growth [7] and better quality meat for the progeny. Therefore, it is crucial to study feeds that promote better fetal growth and post-birth development.
Corn starch is ruminants' most common feed for energy supplementation [8]. However, using rumen-protected fat in nutrition should increase the diet's energetic support and milk production [9]. In addition, the calcium salts of palm oil reduce the risk of ruminal acidosis because of excess starch and reduce the negative energy balance at the end of pregnancy and early lactation, avoiding metabolic problems such as toxemia [10].
Nutritionists have used chromium in animal nutrition as an insulin receptor enhancer to increase the use of blood sugar in cells [11]. In ruminants, the use of chromium in an energy-rich diet aims to increase the energy utilization of this diet and, thus, promote greater productive and reproductive performance [12]. Therefore, Cr supplementation affects glucose and lipid metabolism, with changes in blood profiles such as cortisol, glucagon, insulin, antioxidant defense, and immune system, especially in stressful situations, which occurs at the end of the ewes' gestation. The action of Cr on carbohydrate and lipid metabolism may explain the improvement in performing ruminants in pregnancy, lactation, growth, and fattening [13,14]. Cr has been used in mineral supplementation to enhance stressed ruminants' nutritional and health status, but unstressed animals have reported inconsistent results.
The study hypothesized that alternative energy sources in the diet of pregnant ewes are positive in the generation of progeny. Therefore, we aimed to evaluate the effect of the inclusion of chromium propionate or palm oil Ca salts on the nutrition of pregnant and lactating ewes on the progeny.

Animal
The experiment was carried out at the Faculty of Animal Sciences and Food Engineering of the University of Sao Paulo, Brazil. From an initial flock of 60 sheep, we selected forty-three pregnant ewe, single gestating, Santa Ines and Dorper crossbreed, 3 ± 1 year old, weighing 57 ± 10 kg. The ewes were distributed in a completely randomized block in three treatments according to the feed, CTL (n = 15) with starch from corn; CR (n = 15) diet CTL plus 0.5 mg of Cr propionate/kg dry matter; PF (n = 13) diet CTL plus Ca salts of palm oil (Table 1). Feed supply was the same between treatments, provided at 800 h and 1600 h, and consumption was calculated daily. Ewes received the experimental diet during the last 50 days of gestation and 70 days of lactation. At the end of this period, weaning was complete.
The samples of the diet offered and orts were chemically analyzed according to AOAC (2000) for DM: dry matter, ID 930.15; OM: organic matter, ID 942.05; CP: crude protein, as 6.25 × N, ID 954.01; ADF, acid detergent fiber, ID 973.18; and NDF, neutral detergent fiber by Mertens et al. (2002). In addition, chromium analysis [17,18] was carried out in a mineral solution using dry hydrochloric acid 1:1, and the reading was in plasma spectrometry (ICPE-9000 from Shimadzu) at wavelength 267.716 using SpecSol brand standard. Finally, for calculation, the metabolizable energy (ME, Mcal) was using the Small Ruminant Nutrition System program [19].
The ewes were weighed on an electronic weighing scale at 135 ± 3 days of gestation. In the sequence, the body condition score (BCS) was measured using a scale from 1 to 5, with one being extremely thin and five being extremely fat [20].
Blood samples were collected at 135 days of gestation to analyze non-esterified fatty acids (NEFA), betahydroxybutyrate (BHB), insulin, and glucose. The procedure for blood collection was by venipuncture of the jugular vein in specific sterile tubes suitable for each test. The serum insulin and glucose levels were determined with Kit Insulin (Monobind, Accubind EIA Kit, Code. 2425-300) and glucose by the kit with Ref. 133-1; by the ELISA technique and reading through the Lasystems Multiskan MS equipment. NEFA and BHB were quantified by colorimetric enzymatic methodology in an Automatic Biochemical Analyzer brand RX Daytona®-Randox Laboratories, UK, using a commercial kit branded Randox®, reference FA115 and RB 1007, respectively.

Animals and Local
The lambs remained with their dams for 70 days and, in the sequence, were confined. We used 23 lambs, seven for CTL, nine for CR, and seven for PF. The males were not castrated and stayed in individual stalls (1.70 × 2.30 cm) with slatted flooring, access to water ad libitum, and shade for 56 days. Diet and management were the same for all lambs (Table 2), so only the dam's diet was assessed on the animal performance. They were offered daily, at 800 h and 1500 h, and daily feed consumption control. Every 14 days, lambs were weighed, and the average daily weight gain was calculated.
To calculate feed efficiency, we used the equation of weight gain by dry matter consumption.

Carcass Parameters
Before slaughter, the lambs were fasted from solids for 16 h and weighed. Next, they were stunned with a pressure gun and bled by cutting the jugular veins and arteries. Then, we removed the paws, head, leather, and viscera, washed the carcass, and toilet, weighed the hot carcass, and sent them to the cooling chamber, where they remained for 24 h at 2 °C. After this period, we weighed the carcass and calculated hot and cold yield carcass and chilling loss. The muscle conformation score was from 5 to 1, were 5, exceptional; 4, very good; 3, good; 2, fair; 1, poor; and the fatness score was a 5-point scale of fat cover (1, low; 2, slight; 3, average; 4, high; 5, very high). At the time of slaughter, the non-carcass components were weighed with an analytical balance and measured in the feet, head, leather, lung, heart, respiratory tract, liver, intestines, and rumen. To calculate the proportion of these components, we used body weight at slaughter.
We sectioned the chilled carcass in half, with the left side cut between the 12th and 13th ribs, exposed to the Longissimus Dorsi muscle, and measured the fat thickness with a digital caliper (Digital Caliper, Western®). To calculate the loin eye area (Eq. 1), we demarcate the shape of the muscle in this same region, and measurements of A (medial-lateral direction) and B (dorsoventral direction).
The loin and leg, from the half-carcass, were sampled. The loin corresponded to the last eight thoracic vertebrae. The tenderloin is the cut of the muscle masses attached to the ventral surface of the last three thoracics, six lumbar, iliac, and femur (third trochanter) vertebrae. The leg cut is between the femoral head and the acetabulum. All parts were weighed with a semi-analytical scale, accurate to 10 g. Subsequently, we dissected the loin and leg into muscle, bone, and fat. We used the weight of muscle, bone, and fat in relation to cut weight to calculate the ratio.

Bone Density
After boning the leg, we weighted the femur with a precision scale, with a capacity of 20 g to 3 kg and precision of 0.0001 g (BL3200H, Shimadzu), and measured its length with a millimeter tape measure. Then, we calculate the Seedor index, which serves as an indication of bone density and corresponds to the weight of the bone (mg), divided by its length (mm) [21].
Subsequently, the femur was frozen in a freezer at − 20 °C for further analysis. Radiographs of the left femur were performed in the dorsopalmar and lateromedial direction. The bone was positioned on top of the digital cassette, and the X-ray machine was at a distance of 100 cm. An aluminum scale was placed on the projection for color system calibration and bone density determination. The scale used has 25 steps, with a difference of 1 mm in height between them, the 1st step being 1 mm high and the 25th step being 25 mm. The standardized technique was a voltage of 72 kV and an exposure time of 3.2 mAs. A digital X-ray machine from Sound Eklin®, Carlsbad, USA, was used and the emitter was the JOB X-Ray®, 380 HF, Job do Brasil® São Paulo, BR. Then, the images were analyzed using the ImageJ® program, and the bone density was determined on an aluminum scale (mmAl) in the epiphysis region.

Statistical Analysis
The statistical design was in randomized complete block design and treatments were considered fixed effects, according to the model: where Yij is the dependent variable, (i, treatment and j, repetition), μ is the overall mean, Ti is the fixed effect of the treatment (i = 1-3), B is the block (age of the sheep) and eijk is the residual error.
The statistical program used was SAS [22]. Data were checked for normality (Shapiro-Wilk and Levene's tests) and then analyzed using PROC GLM and the Tukey test at 5% significance.

Ewes
The CTL, CR, and PF treatments did not differ (P > 0.05, Table 2) for body weight (BW) with a mean value of 76.50 kg, BCS (mean 3.83), glucose (mean 66.65 mg/DL) at 135 days of gestation. However, the insulin concentration (P = 0.0272) was higher for PF than for CTL. The concentration of NEFA (P = 0.0446) was highest in CR and lowest in PF. BHB (P = 0.0101) was higher in CR and PF.
Lambs from ewes fed with Cr propionate or Ca salts of palm oil had higher body weight at the feedlot (P = 0.0150) and slaughter (P = 0.0040) than lambs from the CTL treatment. The values for BW at the initial feedlot for Cr were 26.92 kg and PF 28.20 kg, and for slaughter, 46.81 kg and 47.62 kg, respectively.

Carcass
There was a difference in hot and cold carcass yield between treatments (P < 0.05, Table 4), with a higher value for lambs from CR treatment (54.58% and 52.41%, respectively), followed by CTL (52.13% and 50.13%, respectively) and lower for PF (50.74% and 48.39%, respectively). However, the parameter refrigerated losses (3.13% mean), loin eye area (19.59 cm 2 mean), and fat thickness (1.92 mm mean) were similar among lambs regardless of maternal diet (P > 0.05).
There was a tendency (P = 0.0704, Table 7) for lambs from the CR and PF treatments to have better muscle conformation (average of 3) compared to the CTL (2.8). The maternal diet did not change the fat conformation, the internal and external length of the carcass, the thorax measurements, and the leg length (P > 0.05). However, the diet altered the leg dimensions: the perimeter was 22 cm for CR and PF groups and 20 cm for CTL, and the width was approximately 70 cm for CR and PF and 65 cm for CTL (P = 0.0379).

Bone Density
The thickness of the femoral epiphysis was altered by the diet (P = 0.0307,

Ewes
During the late gestation and early lactation, the nutritional requirement of the ewe increases [23]. Therefore, nutritional strategies are used to meet these demands and decrease negative energy balance. The BCS is an easy and accurate method to estimate the nutritional condition of the sheep. The BCS suitable for late pregnancy is between 3.5 and 4, and at the end of lactation, between 2.5 and 3  [20]. The results obtained for BCS and BW in CTL, CR, and PF diets were the same and are within the recommended range.
The use of grain in the diet of ruminants at late gestation and lactation aims to provide ingredients with a higher concentration of non-structural carbohydrates and thus meet the nutritional requirements. Corn is a commonly used energy ingredient in ruminant production due to the high amount of starch in its composition. The digestive processes of starch by the rumen microbiota increase propionate production, which is essential in maintaining plasma glucose levels. Shortly after eating, serum glucose levels rise, and the pancreatic beta cells react by releasing insulin into the circulation. By binding to insulin receptors on the cell wall, the hormone activates the glucose transport pathways, enabling its circulation inside the cells.
In sheep, the placenta-fetal unit uses 30 to 40% of the maternal glucose [24]. During inadequate dietary energy consumption periods, the concentration of hormones that stimulate lipolysis, such as glucagon, beta-adrenergic catecholamines, and cortisol, increases while insulin concentration decreases. The high mobilization of triglycerides substantially increases the amount of NEFA [25]. Usually, a small amount of acetyl-CoA turns into acetone, acetoacetate, and beta-hydroxybutyrate in the liver. The production of ketone bodies is abnormally high when the degradation of carbohydrates does not accompany the degradation of triglycerides.
Calcium salts of palm oil increased the concentration of insulin and BHB in the blood, indicating energy generation by lipid metabolism. Supplementation with chromium propionate increased the concentration of NEFA and BHB, which are common findings in a highly metabolizable energy diet [26]. In addition, chromium propionate days before parturition increases lipolysis [27].

Performance
The sheep's diet had the same concentration of metabolizable energy but with different feeds, such as Cr propionate and calcium salts of palm oil. During pregnancy and lactation, ewes fed with these two feeds produced heavier lambs at the beginning and the end of the feedlot, with approximately a difference of 4 kg of BW more than the lamb in the CTL treatment. Therefore, there was a beneficial effect of fetal programming of weight in the progeny growth phase.
Daily weight gain and feed efficiency in the confinement of lambs were similar between treatments (P > 0.05). However, the lambs entered the feedlot with different body weights, and there was no compensatory gain, so the difference in BW remained until slaughter. CR and PF increase the energy availability of ewes during the gestation phase, which promotes more significant fetal growth at the end of pregnancy. The better availability of feed energy for the ewe during pregnancy can affect milk production and consequently the weaning weight of the lamb. The effect of fetal programming can extend to the lactation period. Therefore, the best energy use of the diet by the ewe fed either with CR or with PF may have increased the milk production of the ewes, which allowed better nutrition of the lambs and thus influenced the weight at weaning and slaughter. The beneficial effect of Cr propionate or calcium salts of palm oil has already been proven with dairy cows [26].

Carcass Parameters
The CR treatment had a higher carcass yield (hot and cold) than PF. The Cr ingested by the ewe may have enabled better energy partition between the ewe and the fetus, with higher energy support for the fetus. In the adult phase, this great muscular development results in lambs with higher carcass yield.
The maternal nutrition did not influence the fat thickness and loin eye area. Animals fed directly with Cr may have a reduction in the amount of fat in the Carcass, despite this being controversial information. Lambs fed different doses of Cr did not have a different fat thickness but had a change in mesenteric fat [28]. Rumen-protected fat in lamb nutrition did not increase carcass fat thickness [29].
Maternal diet did not change the proportions of loin and tenderloin cuts by about half carcass weight (P > 0.05). However, they changed the leg proportion, higher for CR, intermediate for CTL, and lower for PF (P = 0.0255). Within the leg cut, there was a difference in the proportion of fat, with a higher value for PF, intermediate for CTL, and lower for CR (P = 0.0359). Another study observed a higher proportion of the hind limb in the Carcass of lambs fed with Cr, explaining greater insulin activity and glucose utilization for muscle development [30].
Regarding the non-carcass components, the maternal diet influenced the spleen and the respiratory tract (P < 0.05). The animals in the control group had a larger spleen, intermediate for CR, and smaller for PF. Among the main functions of the spleen is the crucial formation of cells of the immune system and filtering of the body's red cells. Dietary Cr supplementation can stimulate genes in the spleen that stimulate the production of defense cells in animals under some stress conditions. The chromium requirement for sheep is not yet established [23], demonstrating the need for further studies. Different types and concentrations of rumenprotected fat in the diet of lambs altered spleen weight [31], as in this experiment.
The deposition of Cr in body tissues of lambs was measured and concluded that there is a more significant accumulation in the heart, testes, and lungs [32]. In our study, CR treatment generated progeny with a smaller respiratory tract, so the effect on the fetus remained until adulthood.
The hind perimeter and width were more significant for the CR and PF than for the CTL (P > 0.05, Table 7). The CR treatment had better leg development, which influenced the evaluation of carcass conformation (P = 0.0704). The CR and PF treatments had greater rump width and perimeter (Table 5), which agrees with the higher body weight at slaughter (Table 3). Maternal nutrition in early pregnancy affects the number of muscle fibers; at the end of pregnancy, it interferes with muscle hypertrophy, that is, fiber size. The diets that allowed the best energy use for the ewe were CR and PF, which resulted in heavier lambs at weaning, and this advantage extended until slaughter, hence the more significant measures of the shank. The allometric growth of the leg can be affected by genetics, but in this study, the racial pattern was similar; therefore, the positive effect of the larger size of this cut is related to maternal nutrition.

Density Bone
Bone tissue presents an intense vascularization that allows the exchange of nutrients and minerals, therefore, helping to regulate mainly the amount of calcium and plasma phosphorus by the action of hormones, such as parathyroid hormone (PTH), calcitonin, and vitamin D. Bone cells, osteoclast, act on bone resorption and present a large number of mitochondria, supporting the premise that energy utilization is an essential element in bone remodeling [33], reaffirming the hypothesis that diets with more energy are capable of producing the most offspring and bone heavy. The smaller density bone in the epiphysis region found in treatments with CR and PF may be related to increased production or activation of insulin and better energy use of the sheep with these diets benefiting the progeny.
The primary event leading to insulin secretion increases ionized calcium in the beta-cell cytosol. At the beginning of pregnancy, the treatment with PF contained a higher concentration of Ca since fatty acids were protected by adding calcium salts, which may have caused this group to release more insulin.
In theory, Cr supplementation had better glucose activation. Insulin is released rapidly into the bloodstream when blood sugar levels rise and bind to an outer α subunit of the transmembrane protein insulin receptor, causing a change in receptor conformation. The receptor auto phosphorylates tyrosine residues in the inner portion of its β subunit, transforming the receptor into an active kinase. Chromodulin is stored in its apo form in the cytosol and the nucleus of insulin-sensitive cells. Chromium enhances insulin receptor activation so that more glucose enters the cell. A maximum of four Cr molecules can bind to an insulin receptor, leading to an eight-fold difference in insulin receptor activation [34].
Insulin acts on bone cells by inhibiting the expression of Opg (osteoprotegerin), which in turn acts by inhibiting the activation and maturation of osteoclasts. Therefore, insulin, by inhibiting Opg, stimulates the action of osteoclasts and, consequently, bone resorption. Diets with Cr and protected fat in the rumen may have shown an imbalance in bone remodeling due to greater insulin activation and, therefore, greater bone resorption, resulting in lower bone density on X-ray. Although bone resorption is not greater than bone formation, causing osteoporosis, it is necessary to evaluate the interaction of chromium with calcium and vitamin D [33]. Therefore, these minerals and vitamins' interaction with bone tissue should be further studied.
However, regulating bone formation in the fetal phase and maternal undernutrition during pregnancy can result in a lighter, smaller femur with less bone density [35]. Therefore, even with the restriction during pregnancy, the lamb's femur can present compensatory growth, increase in weight, and become equal to lambs without fetal restriction, especially after weaning [6]. In this study, however, the sheep had no difference in the level of nutrition; only the source of energy used was different. This is likely the reason there was no effect on the weight and length of the femur.
We concluded that despite maternal diets having the same amount of metabolizable energy, supplementation with chromium propionate or calcium salts of palm oil altered the progeny, producing heavier lambs at slaughter, with better carcass yield and a better proportion of prime cut. Chromium increased the blood NEFA content of sheep. In lamb, the chromium diet reduced the amount of fat in the leg. It is essential to understand better the effect of these feeds on the spleen and their effects on the immune system and the density and length of the femur.