Different nutritional systems influence the tenderness and lipid oxidation of ewe lamb meat without altering gene expression

Abstract

Feeding is a determining factor in the various characteristics of sheep meat and animal performance, the objectives were to evaluate the effect of supplementation of ewe lambs finished in different nutritional planes on the gene expression of CASP3, CAPN1, CAPN2 and CAST and its possible association with meat quality. Samples of the Longissimus lumborum muscle of 24 ewe lambs were used, distributed in 3 groups (n=8): P (pasture), PS (pasture and supplement) and F (feedlot). Physicochemical analyses were performed for centesimal analysis, pH, lipid oxidation, Warner-Bratzler shear force and RT-qPCR for the analysis of relative gene expression of the following genes: CASP3, CAPN1, CAPN2 and CAST. There is an increase in daily weight gain and ethereal extract values in the meat of confined animals, due to the greater energy intake in the nutrition of these animals. Animals kept only on pasture have lower lipid oxidation in meat than other treatments because of the lower percentage of lipids. The Warner-Bratzler shear force is considerably higher in the meat of animals kept only on pasture but is still considered tender. The different nutritional systems do not interfere with the gene expression of CASP3, CAPN1, CAPN2 and CAST in ewe lambs.

Key words
μ-Calpain; calpastatin; caspase-3; Ovis aries; RT-qPCR

INTRODUCTION

Sheep farming is performed on virtually all continents; the wide use of sheep is mainly a result of animal adaptability and the lack of ethnic or religious restrictions on the consumption of its various products (Garcia et al. 2000, Viana 2008).

Thus, for successful rearing, it is necessary that the systems of breeding and feeding enhance the meat quality, and animals fed higher proportions of certain nutrients have higher fat content, which in turn increases meat succulence and tenderness (Moreno et al. 2015).

Regarding the concept of different diets, Medeiros & Ferreira (2018) affirmed that food can promote direct changes in gene transcription, since nutritional factors may be responsible for activating genes. Thus, there is great interest in exploring dietary sources that can improve specific characteristics, such as meat quality.

The different nutritional compositions of the feeding systems used in lamb’s breeding can influence the physiology and expression of various genes, this statement is since genes interact with each other in a way that reveals a more complex regulation of transcription, recovering functional information of the tissue analyzed at the molecular level (Flora et al. 2017, Hudson et al. 2012, Fuller et al. 2007).

The enzyme caspase-3 (CASP3) is responsible for the proteolytic cleavage of several proteins postmortem (Anwar et al. 2004), and the main enzymes involved in this process are calpains and their inhibitor, calpastatin. Calpains degrade myofibrillar proteins at certain internal points of the molecules according to the extracellular calcium reserve, improve the tenderness of meat, and are inhibited by calpastatin (Koohmaraie & Geesink 2006).

The objectives of this study were to analyze the relative gene expression of the target genes caspase-3, μ-calpain, m-calpain, and calpastatin and their possible relationship with meat tenderness and to evaluate lipid oxidation, pH, and centesimal composition in lambs subjected to different nutritional systems.

MATERIALS AND METHODS

Development site

The study was approved by the Ethics Committee on the Use of Animals (ECUA) of the institution of origin, under protocol number 4610, and carried out at the University of Western São Paulo - UNOESTE, Campus II, in the city of Presidente Prudente-SP, latitude 22°07’21.06” north and longitude 51°23’17.71”, tropical climate, starting in July 2018 and ending in January 2021.

Nutritional management

Twenty-four ewe lambs (7/8 Dorper), with initial live weight (ILW) of 27.5 ± 3.08 kg to pasture group (P), 27.8 ± 3.67 to pasture with supplement group (PS) and 29.12 ± 1.4 to feedlot group (F) and initial age between 6 and 7 months, and born after fixed-time artificial insemination (TAI) with semen of the same ram, were used. At the beginning of the experiment, all females were weighed, and feces were collected for examination of fecal egg count (FEC), which was repeated monthly until the end of the field experiment. An antihelminthic was applied according to the FEC value.

The Nutrient Requirements of Small Ruminants (NRC 2007) recommends 11.3% crude protein, 73% dry matter, and 58% TDN for this type of sheep, with predicted increases of 0.200 kg/day.

In this way, the females were divided into three groups at random: P, where the ewe lambs consumed between 70 and 80% of the nutritional requirement advised by the NRC (2007); PS, where the females received between 100% and 110% of the nutritional requirement, with supplementary intake as needed based on the period’s bromatological analysis and the group; and pasture with no supplement (P). F, where the females were fed separately and received a balanced meal that met 140% of their nutritional needs (NRC 2007). The animals in the feedlot were placed in separate stalls with slatted and suspended floors and had access to limitless water during the trial. Reading was used to manage the feed, resulting in feed leftovers of about 10%.

The methodology of food systems was based on the research of Carvalho et al. (2006), because the different sheep production systems in Brazil presented disuniformity in animal performance and, consequently, the age of animals at slaughter when compared animals in fattening system with grazing, grazing and supplement and feedlot (Ribeiro et al. 2009).

The pasture underwent three bromatological analyses at the beginning, middle, and end of the trial. During the beginning, middle, and last thirds of the experiment, dry matter (DM), ether extract (EE), ash content, crude protein (CP), total digestible nutrients (TDN), neutral detergent fiber (NDF), and acid detergent fiber (ADF), were assessed (Table I). According to Capelle et al. (2001), the analyses were carried out using the methods recommended by Silva & Queiroz (2002), total digestible nutrient (TDN) values (2001). The fiber in acid detergent (ADF) contents according to the methodology obtained by the method of van Soest (1963). Commercial feed® (FORT OVINOS 16, FORTSAL®) was utilized as a supplement and a complete diet for the PS and F groups, respectively, the nutritional experiment lasted 180 days and the bromatological analyses were performed in three periods: the beginning between day 0 (D 0) and day 60 (D 60), in the middle of the experiment between days 61 (D 61) and days 120 (D 120) and at the end of the experiment between days 121 (D 121) and days 180 (D 180) (Table I).

Table I
Chemical composition of pasture and commercial feed used as feed for the ewe lambs during the experiment.

	PASTURE (%)			Comercial feed® (%)
	Beginning (D 0 – D 60)	Middle (D 61 – D 120)	End (D 121 – D 180)
DM	26,22	45,12	25,11	88,0
EE	1,32	1,29	1,49	3,3
Ashes	11,38	9,38	11,05	5,0
CP	6,31	7,55	9,39	16,0
TDN	53,24	48,58	49,51	72,0
NDF	75,81	79,33	80,45	10,0
ADF	45,78	51,76	50,56	7,0

• DM: dry matter/ EE: ether extract / ashes/ CP: crude protein/ TDN: total digestible nutrients/ NDF: neutral detergent fiber / ADF: acid detergent fiber.

Thus, the different dietary systems of the experimental groups were initially established according to the worst grazing of Brazilian pasture. Currently, many Brazilian pastures are insufficient, and the animals remain in a nutritional deficit. The animals in group P (n=8) were maintained exclusively on pasture (Panicum maximum cv. Tanzânia), with access to water and mineral salt ad libitum. The PS group (n=8) was also maintained on pasture (Panicum maximum cv. Tanzânia), but animals also received 1.5% of their live weight in commercial feed® (FORT OVINOS 16, FORTSAL®) containing 16% crude protein and 72% TDN.

The P and PS ewe lambs were kept on pasture in a rotation module, previously sealed for 150 days, for helminth control and quality and grass availability, totaling an area of 0.8 hectares. This was subdivided into 4 padocks of approximately 200 m², where the animals remained for approximately 9 days in each padock, with a 27-day grass rest period (Hegarty et al. 2007).

Feedlot ewe lambs (n=8) were confined throughout the experimental period and were administered a diet with a forage/concentrate ratio of 20:80, composed of Tifton hay and commercial feed. The average intake was 4.5% of the live weight, according to trough reading (Parente et al. 2009, Rogério et al. 2007). Throughout the experiment, the ewe lambs in groups PS and F were fed twice a day at eight o’clock in the morning and four o’clock in the afternoon and were weighed every 15 days to adjust the diet according to live weight.

Slaughter schedules as a function of live weight

Upon reaching an average weight of 37.26 ± 3.25 kg/BW, the ewe lambs were slaughtered after fasting for 18h, due to the kg/BW requirement, the animals in the F group reached slaughter weight before the animals in the PS and P groups due to the different nutritional intakes offered. Table II shows the slaughter order of the ewe lambs according to the sequence in which they reached the pre-stipulated weight.

Table II
Order, date, quantity, group belonging and age per slaughter (days after initial experiment) to the slaughtered ewe lambs and average weight per slaughter of the groups.

Order of slaughter	Slaughter days	P (grass)	PS (grass + supplement)	F (feedlot)
1st	27/09/2018	01	01	04
2nd	11/10/2018	-	02	04
3rd	29/10/2018	02	03	-
4th	06/12/2018	03	01	-
5th	10/12/2018	02	01	-
Age per slaughter (days after initial experiment)	-	145.66±16.6	116.28±27.16	86.28±8.87
Average weight per slaughter experimental group	-	34.37±1.13	35.84±2.64	40.9±1.32

The slaughter took place in a commercial slaughterhouse, following the rules of ORDINANCE No. 62, OF MAY 10, 2018, published by the Ministry of Agriculture, Livestock and Supply (MAPA).

Physicochemical analyses of meat

Centesimal analyses of the Longissimus lumborum muscle (% moisture, % crude protein, % ethereal extract, and % mineral matter) were performed according to AOAC (2005) methodology.

The pH of the meat was determined using a portable potentiometer (peagameter) with a penetration electrode (Hanna Instruments, Brazil), 1 cm deep in the slice muscle (Gomide et al. 2013). The lipid oxidation index was determined using the T’bars test, with a spectrophotometer reading at 538 nm (Pikul et al. 1989).

The carcasses of each animal were kept in cold storage for the proper measurement of rigor mortis and pH drop, and the samples were prepared for the evaluation of the shear force (WBSF) the following day. They were then placed into an electric oven that was preheated to 180 °C. The steaks were roasted until the internal temperature reached 71 °C (Pikul et al. 1989). The samples were collected using a cylindrical sampler.

Six readings per animal were obtained from each of the three samples that were used, each of which had two subsamples that were each roughly 1.25 cm thick (Koohmaraie et al. 2002). A 3mm thick Warner-Bratzler blade and a Brookfield® CT3 Texture Analyzer (Brookfield engineering, Middleboro, MA) were used to evaluate the shear force objectively (Ramos & Gomide 2007).

For the oxidative analysis of the meat, the Thiobarbituric Acid Assay (TBA) was performed as described by Pikul et al. (1989). Samples were collected during slaughter, frozen and stored in a freezer at -80 °C until processing, and evaluated one week after the last slaughter, in which 10g of lamb meat with 50 mL of trichloroacetic acid 7.5% was homogenized for 1 minute in turrax. Then the mixture was filtered, and 5 mL of the extract was transferred to a tube containing 5 ml of 2-thiobarbituric acid 0.02 M. The tubes were heated in a boiling water bath for 40 minutes and cooled in running water for 10 minutes, for the measurement of reactive substances to 2-thiobarbituric acid (TBARS) malonaldehyde quantification was estimated by comparing absorbance values of samples and standards at 538 nm. The values were expressed in mg of malonaldehyde (MDA)/kg.

Gene expression

During slaughter, Longissimus lumborum muscle samples were collected for RNA extraction for gene expression analysis. Muscle samples were collected immediately after slaughter, deposited in liquid nitrogen (-196 °C), and stored in a freezer at -80 °C until processing for RT-qPCR. These fragments (~40 mg) were crushed in a tissue homogenizer and subjected to the TRIzol ® (Thermo Fisher Scientific®) extraction protocol for total extraction.

The total RNA concentration recovered and the 260/280 ratio were measured using a NanoDrop® (Thermo Fisher Scientific, Brazil). All total RNA samples were treated with DNAse before being submitted to RT-qPCR, according to the instructions of the DNAse I - Amplification Grade (Invitrogen) protocol®.

Reverse transcription was performed using the High-Capacity protocol (Applied Biosystems^TM, Thermo Fisher Scientific, Brazil) following the manufacturer’s protocol. qPCR was performed for quantitative analysis of relative gene expression. The primers and probes of target genes correlated with meat quality were for CASP3 (Oa0481763_m1), CAPN1 (Oa04658113_g1), CAPN2 (Oa04659692_m1), and CAST (Oa0456608_m1). Three endogenous genes were used as internal controls for real-time qPCR reactions in real time, and 3 endogenous genes were used: B2M (Oa04818291_m1), HMBS (Oa04838105_g1), and TBP (Oa04666642_m1) (Table III).

Table III
Primers, genes, TaqMan® assays, reference sequence and products used in qPCR.

Primers	Gene	TaqMan® Assays	Seq. of reference	Product
CASP3	Alvo	Oa0481763_m1	XM_015104559.2	77 pb
CAPN1	Alvo	Oa04658113_g1	NM_001127267.1	53 pb
CAPN2	Alvo	Oa04659692_m1	NM_001112817.1	63 pb
CAST	Alvo	Oa0465608_m1	NM_001009788.1	56 pb
B2M	Endógeno	Oa04900279_mH	XM_027971438.1	84 pb
HMBS	Endógeno	Oa04704013_g1	XM_004016091.4	62 pb
TBP	Endógeno	Oa04818075_m1	XM_015097549.2	66 pb

For data normalization, a combination of HMBS and B2M was used, which was shown to be more stable by the NormFinder software® program (MOMA, Denmark), to normalize the results obtained for the target gene. Primers for endogenous and target genes and their respective probes were obtained from standardized TaqMan® assays (Applied Biosystems®, Foster, USA).

Statistical analyses

The initial experiment consisted of three completely randomized treatments, with eight replications. For initial weight, average daily gain, slaughter weight, centesimal analysis: moisture, crude protein (CP), ashes, and ethereal extract (EE), pH, WBSF, and lipid oxidation, homogeneity of variance was tested using the Bartlett test (p > 0.05), and residue normality was verified using the Shapiro-Wilk test. The data were submitted to variance analysis and when significant, were submitted to the Tukey test at a level of 5%. All statistical evaluations were performed using the RStudio Software.

The qPCRs were conducted in duplicate for each sample, and expression was determined by quantification in relation to the endogenous gene. The Pfaffl (2001) method was used for relative quantification of amplifications. Gene expression data were analyzed for Shapiro-Wilk normality and then subjected to variance analysis (ANOVA). Differences were considered statistically significant at p < 0.05.

RESULTS

The average daily gain (ADG) was higher in the confined animals (p < 0.05) and lower in the animals of the PS and P groups. The lowest weight gain in the PS and P groups occurred because of the food system to which the animals were subjected.

Consequently, as expected for ADG, there was a difference in the total weight gain (p < 0.05) with an increase in concentrate, according to the nutritional plan (Table III). The values found in the centesimal analysis of lamb meat showed no statistical difference between treatments for most variables, except ether extract (EE), which showed a linear increase as a function of dietary system (Table IV).

Table IV
Initial live weight, daily average gain, mean values (%) for moisture, CP, ash, EE and pH of ewe lamb meat, in three groups with different feeding systems.

Variables	TREATAMENTS			Average	Standard error	p value*
	P	PS	F
ILW	22.24	22.56	20.14	21.64	1.264	0.4611
ADG	0.0539c	0.178b	0.222a	0.151	0.0083	2.1231
Moisture	71.76	71.57	68.93	70.91	0.71	0.2328
CP	23.74	23.20	23.52	23.50	0.58	0.9345
Ash	1.25	1.03	1.18	1.16	0.04	0.1309
EE	1.77a	3.56b	5.10c	3.29	0.39	<0.001
pH	5.62	5.47	5.54	5.54	0.03	0.05

• P = pasture / PS = pasture and supplement / F = feedlot / ILW = initial live weight / ADG = average daily gain / CP = crude protein / Ash = ash content / EE = ether extract.
  * (p < 0.05).

The pH remained within the expected pattern for ewe lamb meat, and there was no significant difference between treatments, with an average value of 5.54.

In relation to lipid oxidation, there was a statistical difference between treatment groups, and the group where the animals remained on pasture presented the lowest values, with an average of 1.1998 mg MDA/kg of meat (Figure 1).

Figure 1
Lipid oxidation in the meat of ewe lambs fed with different nutritional systems: P (pasture, in which lambs received between 70 and 80% of the nutritional requirement [NRC 2007], n=8); PS (pasture and supplementation, in which females received 100 to 110% of the nutritional requirement (NRC 2007), n=8); and F (feedlot, in which the lambs were fed in an individual trough and provided 140% of the nutritional requirement [NRC 2007], n=8).

For WBSF, there was a significant difference between the three treatment groups, and group F presented a value of 27.06 N, followed by the PS group (31.28 N), which showed a lower WBSF than that in treatment group P (50.40 N) (Figure 2).

Figure 2
WBSF of the meat of ewe lambs fed with different nutritional systems: P (pasture, in which ewe lambs received between 70 and 80% of the nutritional requirement [NRC 2007], n=8); PS (pasture and supplementation, in which females received 100 to 110% of the nutritional requirement (NRC 2007), n=8); and F (feedlot, in which the lambs were fed in an individual trough and provided 140% of the nutritional requirement [NRC 2007], n=8).

With the results duly corrected for qPCR efficiency, there was no statistical difference in any of the genes evaluated in the relative gene expression analysis (p > 0.05) (Figure 3a-d).

Figure 3
Relative gene expression of CASP3 (a), CAPN1 (b), CAPN2 (c), and CAST (d) genes evaluated in the Longissimus lumborum ewe lamb muscle, using the mean endogenous HMBS and B2M levels as normalizers, in three groups with different feeding systems: P (pasture, in which the lambs received between 70 and 80% of the nutritional requirement [NRC 2007]; PS (pasture and supplementation, in which females received 100 to 110% of the nutritional requirement [NRC 2007]; and C (confinement, in which the ewe lambs were fed in an individual trough and provided 140% of the nutritional requirement [NRC 2007]).

DISCUSSION

When breeding animals exclusively using grazing feeding systems, the nutritional quality often does not meet the requirements of production or reproduction, especially in genetically modified animals, and there is a need to seek alternatives to address this problem, such as supplementation (Borton et al. 2005).

In relation to the performance of ewe lambs in the feedlot fattening system, as expected, they reached slaughter weight in a shorter time because of the nutritional contribution provided by this system, and the animals also likely reached their optimum health status (Pelegrini et al. 2008). According to Gomes et al. (2018), feedlots show better results because, although the costs of feeding are higher than those in grazing alone, the higher production of body weight and reduction in the slaughter age dilute these costs, and the decision to use the confinement system offers the possibility of achieving more cycles in the year, since the conversion into animal weight is optimized (Berwanger & Amorim 2022).

According to Guimarães et al. (2020, 2021), the average centesimal composition of the meat of lambs consuming pastures supplemented with different energy sources is 73% to 76% moisture, 20% crude protein, 1.1% to 4.9% ethereal extract, and 1% to 1.6% mineral matter, which may be influenced by feeding and type of termination.

Ewe sheep fed more energy-dense diets had increased values of ethereal extract in their meat. According to Gois et al. (2017) and Oliveira et al. (2020), the mean value for this variable was 3% to 3.75% in the meat of confined lambs.

Araújo et al. (2014) compared animals on pasture with and without supplementation and observed higher lipid values in the meat of supplemented animals, with values of 3.67% and 2.02%, respectively. Furthermore, Santos (2019) found lipid values of 2.2% in the meat of animals fed pasture and supplement and 3.44% for confined animals, corroborating the results of the present study.

According to Souza et al. (2009), diets with a higher lipid content lead to an improvement in the metabolic efficiency of anabolic reactions in adipose tissue, with this fatty acids readily available for deposition causing a reduction in the energy cost of fat synthesis from short-chain fatty acids.

For sheep meat, the final pH is expected to be between 5.5 and 5.8, which is important for the quality of the product, especially tenderness, because it is related to the transformation of muscle into meat by the enzymatic degradation that occurs in the myofibrillar structure of the muscle (Silva Sobrinho et al. 2005). The observed pH values corroborate recent data for the meat of sheep and lambs and demonstrate that slaughter was carried out within the animal welfare standards, allowing correct rigor-mortis (Valadez-Garcia et al. 2021).

The average lipid oxidation value observed in the meat of the animals in treatment group P (1.19 mg of malonaldehyde kg^-1) was within the desired range, because results below 1.59 mg of malonaldehyde kg^-1 are considered acceptable and safe for the consumer (Torres & Okani 1997). On the other hand, the animals in groups PS and F presented higher than desired averages.

This may be related to the diet of the animals, correlating with Yamamoto’s (2006) findings that showed increasing ethereal extract values as a function of concentrate levels in the diets of confined ewe lambs (3.41% and 3.96%). In other words, the lower concentration of malonaldehyde detected in animals on pasture may also be related to the lower proportion of fat found in these animals, since the TBARS methodology analyzes lipid oxidation, and the lower percentage of fat in these (Girgih et al. 2015). Concomitantly, the average lipid oxidation also increased (0.9 to 1.71 mg MDA/kg).

In ruminant meat reared in the pasture system, pasture with supplementation, and feedlot, other authors also found lower values of lipid oxidation for animals fed only with feed from pasture (pasture: 0.09 mg MDA/kg; confined: 0.28 mg MDA/kg), which present, consequently, lower fat content in meat (Daley et al. 2010, Descalzo et al. 2005, Santé-Lhoutellier et al. 2008).

Although lipid oxidation is dependent on polyunsaturated fatty acids, which tends to be higher per g of tissue in animals in fattening system with grazing, Luciano et al. (2012) emphasized that lamb meat in a fattening system with grazing presented a higher amount of unsaturated fatty acids, lower lipid oxidation and greater color stability when compared to the meat of confined lambs. Also in the same study, it can be concluded that the diet affected the fat content of the meat, because it expressed the classes of fatty acids as percentages of total fat and this fact may not be particularly relevant in the discussion of the susceptibility of the muscle to lipid oxidation, because the animals that received concentrate had a higher content of ether extract and, therefore, a higher absolute content of easily oxidisable fatty acids.

In the same way that Insani et al. (2008) found that lipid oxidation was higher for confined animals, in addition, the results showed that the high level and synergistic action of α-tocooferol and β-carotene found in animals in a fattening system with grazing contributed to oxidative stability and meat color.

The significant difference in SF may be explained physiologically by the fact that grazing animals move more in search of food, consequently exercising more, producing more collagen, which brings greater resistance to the meat causing increased hardness (Field 1996). Another factor is that confined lambs were butchered with a higher percentage of fat, since fat is responsible for the sensation of tenderness of the meat favoring juiciness, in addition to the fact that confined animals have less intramuscular collagen (Hocquette et al. 2010, Listrat et al. 2016, Lorenzo 2019).

According to the SF, Boleman et al. (1997) categorized the meat’s tenderness as follows: highly tender (22.55 to 35.30 N), moderately tender (40.20 to 52.95 N), and not very tender (57.85 to 70.6 N). The meat from treatment P may be categorized as tender using the values indicated previously, whereas the meat from treatments PS and F can be categorized as very tender. For lambs and ewe lambs, values between 34.9 N and 57.8 N were close to those reported in the literature (Valadez-Garcia et al. 2021, Zhao et al. 2017).

Regarding factors that affect tenderness, gene expression has been the target of many studies (Lonergan et al. 2010, Chung & Davis 2012, Picard et al. 2015). Souza (2012) used Nelore bulls in his experiment to track genes related to meat quality, and all animals were reared in confinement until the time of slaughter. Hence, there was no influence of different feeds on the abundance levels of genes of interest, showing that different nutritional plans can alter gene expression levels.

The CASP3 gene was investigated because it is involved in the process of muscle proteolysis; that is, when there is an increase in the level of its expression, it is possible to verify an increase in the abundance of CAPN1 and CAPN2 RNA, which are responsible for the breakdown of muscle myofibrils, leading to a higher percentage of tenderness (Ouali et al. 2006). However, in this study, no alterations were observed in the expression of this gene in the different groups.

In the evaluation of the tenderness of the meat of discarded cows, there was no association of shear force with the activity of caspase 3. Despite being related to phenotype, this fact demonstrates that even with its proper action on proteolysis, caspase 3 does not influence the tenderness of the meat (Underwood et al. 2008).

According to Guillemin et al. (2011), deprivation of nutrients and oxygen in cells and tissues after slaughter presumably involves muscle cells in the process of cell death via apoptosis rather than necrosis. The response to this stimulus has a 12% interaction with caspase 3; that is, this mechanism of action stimulus is defined as a change in the state or activity of an organism (in terms of secretion, enzyme production, or gene expression).

For the CAPN1 and CAPN2 genes, the lack of difference in expression in the different systems may be because the animals were from the same genetic and contemporary group, corroborating Ferraz (2009), and the fact that they remained on different diets for a short period of time. In beef cattle, when examining the relative gene expression of these calpains between distinct breeds (Nelore and Angus) fed with two nutritional sources, a statistical difference was observed.

Bagatoli et al. (2013) evaluated sheep and found greater expression of the CAST gene in the muscle samples of Santa Inês lambs than that in the meat of crossbred White Dorper × Santa Inês and Dorper × Santa Inês. The authors suggested that the expression of this gene in the Santa Inês breed leads to a higher production of the calpastatin enzyme and consequent inhibition of calpain, resulting in lower meat tenderness.

In another study using goats with the same racial pattern and different ages, there was no difference (p > 0.05) in the expression of the CAPN1 gene in the meat of post-pubescent goats when compared to that in the meat of younger goats (Saccà et al. 2019). The low age difference between the animals at slaughter in the present study was not a determining factor, so the results of gene expression could be manifested.

CONCLUSIONS

The confined animals had higher average daily weight gain and shorter slaughter time than animals raised exclusively on pasture and pasture with supplementation. There was an increase in the ethereal extract in the meat of confined animals because greater energy intake increases this variable. Animals kept only on pasture had lower lipid oxidation in meat compared with that in other treatment groups because of the lower ethereal extract content. The WBSF was considerably higher in the meat of animals kept only on pasture, but the meat was still considered tender. The nutrition of ewe lambs did not interfere with the expression of CASP3, CAPN1, CAPN2, and CAST. More research should be performed in different sheep breeds using longer experimental periods.

ACKNOWLEDGMENTS

The authors thank the Universidade do Oeste Paulista, which funded this research, where the entire project was developed, and qPCR analysis was performed. The authors would also like to thank Laís Belan for their collaboration on meat quality analyses.

REFERENCES

• ANWAR S, AMBROS RA, JENNINGS TA, ROSS JS, BEZA A, MIAN B & NAZEER T. 2004. Expression of cysteine protease protein 32 in prostatic adenocarcinoma correlates with tumor grade. Arch Pathol Lab Med 128(6): 649-652.
• AOAC - ASSOCIAÇÃO DOS QUÍMICOS ANALÍTICOS OFICIAIS. 2005. Métodos Oficiais de Análise. 18ª ed. Arlington, VA, EUA.
• ARAÚJO FE, SILVA FILHO AS, MOUSQUER CJ, OLIVEIRA MA, MEXIA AA & GERON LJV. 2014. Características qualitativas de carcaças de cordeiros mestiços santa inês x pantaneiro terminados em pastagem recebendo suplementação. Rev Bras Hig San An 8(2): 263-278.
• BAGATOLI A, GASPARINO E, SOARES MAM, AMARAL RM, MACEDO FAF, VOLTOLINI DM & DEL VESCO AP. 2013. Expression of calpastatin and myostatin genes associated with lamb meat quality. Gen Mol Res 12(4): 6168-6175.
• BERWANGER D & AMORIM DIM. 2022. Economic and financial viability of cutting sheep farming activity in the Baixada Cuiabana region. Res Soc Dev 11(4): 309.
• BOLEMAN SJ ET AL. 1997. Consumer evaluation of beef of known categories of tenderness. J An Sci 75(6): 1521.
• BORTON RJ, LOERCH SC, MCCLURE KE & WULF DM. 2005. Comparison of characteristics of lambs fed concentrate or grazed on ryegrass to traditional or heavy slaughter weights. I. Production, carcass, and organoleptic characteristics1. J An Sci 83(3): 679-685.
• CAPELLE ER, VALADARES FILHO SC, DA SILVA JFC & CECON PR. 2001. Estimativas do Valor Energético a partir de Características Químicas e Bromatológicas dos Alimentos. R Bras Zootec 30(6): 1837-1856.
• CARVALHO S, BROCHIER M, CAPPELATTI L & PIVATO J. 2006. Avaliação econômica de três sistemas alimentares utilizados na terminação de cordeiros. Arch Latinoam Prod Anim 14(3): 86-87.
• CHUNG H & DAVIS M. 2012. PCR-RFLP of the ovine calpastatin gene and its association with growth. Asian J Anim Vet Adv 7(8): 641-652.
• DALEY CA, ABBOTT A, DOYLE PS, NADER GA & LARSON S. 2010. A review of fatty acid profiles and antioxidant content in grass-fed and grain-fed beef. Nut J 9(10): 1-12.
• DESCALZO AM, INSANI EM, BIOLATTO A, SANCHO AM, GARCÍA PT, PENSEL NA & JOSIFOVICH JA. 2005. Influence of pasture or grain-based diets supplemented with vitamin E on antioxidant/oxidative balance of Argentine beef. Meat Sci 70(1): 35-44.
• FERRAZ ALJ. 2009. Análise da expressão gênica no músculo esquelético de bovinos das raças Nelore e Aberdeen Angus e sua relação com o desenvolvimento muscular e a maciez da carne. Thesis (Doctorate), Universidade Estadual Paulista. Available at: http://hdl.handle.net/11449/104894.
• FIELD R, MCCORMICK R, BALASUBRAMANIAN V, SANSON D, WISE J, HIXON D, RILEY M & RUSSELL W. 1996. Growth, carcass, and tenderness characteristics of virgin, spayed, and single-calf heifers. J Anim Sci 74(9): 2178.
• FLORA RPD, DIONELLO NJL, BENITEZ L, GERMANO JM, GOTUZZO AG & FREITAS S. 2017. Expressão gênica de IGF-I e GHR no fígado e no músculo do peito de codornas de corte suplementadas com diferentes níveis de metionina em duas gerações sucessivas. Arq Bras Med Vet Zootec 69(1): 205-213.
• FULLER TF, GHAZALPOUR A, ATEN JE, DRAKE TA, LUSIS AJ & HORVATH S. 2007. Weighted gene coexpression network analysis strategies applied to mouse weight. Mamm Gen 18(6-7): 463-472.
• GARCIA IFF, BONAGURIO S & PEREZ JRO. 2000. Comercialização da carne ovina. In: Encontro Mineiro de Ovinocultura. Lavras: Universidade Federal de Lavras, 177 p.
• GIRGIH AT, HE R, HASAN FM, UDENIGWE CC, GILL TA & ALUKO RE. 2015. Evaluation of the in vitro antioxidant properties of a cod (Gadus morhua) protein hydrolysate and peptide fractions. Food Chem 173: 652-659.
• GOIS GC, SANTOS EM, SOUSA WH, RAMOS JPF, AZEVEDO PS, OLIVEIRA JS, PEREIRA GA & PERAZZO AF. 2017. Qualidade da carne de ovinos terminados em confinamento com dietas com silagens de diferentes cultivares de sorgo. Arq Bras Med Vet Zootec 69(6): 1653-1659.
• GOMES FHT, CÂNDIDO MJD, CARNEIRO MSS, FURTADO RN, PEREIRA ES, FONSECA DM, POMPEU RCFF & SOMBRA WA. 2018. Viabilidade econômico-financeira do confinamento de ovinos alimentados com rações contendo torta de mamona. Rev Cienc Agrovet 17(3): 383-395.
• GOMIDE LAM, EM RAMOS & PR FONTES. 2013. Ciência e qualidade da carne: fundamentos. Viçosa: Editora UFV.
• GUILLEMIN N, BONNET M, JURIE C & PICARD B. 2011. Functional analysis of beef tenderness. J Proteomics 75(2): 352-365.
• GUIMARÃES LJ, SANTIAGO LF, NICOLAU JP, REGO FCA, CASTILHO C, CUNHA FILHO LF, GIOTTO FM & ZUNDT M. 2020. Effect of yeast (Saccharomyces cerevisiae) associatedor not with microminerals on chemical composition, tissue composition, lipid oxidation and meat quality of confined lambs. Res Soc Dev 9(11): e1539119563.
• GUIMARÃES LJ, SILVA IG, AMBIEL C, REGO FCA, CASTILHO C, CUNHA FILHO LFC, SENA GC, GIOTTO FM & ZUNDT M. 2021. Effects of different energy source diets, as corn substitutes, on carcass characteristics and meat quality of feedlot lambs. Rev Fac Cienc Agr 53(2): 243-251.
• HEGARTY RS, GOOPY JP, HERD RM & MCCORKELL B. 2007. Cattle selected for lower residual feed intake have reduced daily methane production. J An Sci 85(6): 1479-1486.
• HOCQUETTE JF, GONDRET F, BAÉZA E, MÉDALE F, JURIE C & PETHICK DW. 2010. Intramuscular fat content in meat-producing animals: development, genetic and nutritional control, and identification of putative markers. Animal 4(2): 303-319.
• HUDSON NJ, DALRYMPLE BP & REVERTER A. 2012. Beyond differential xpression: the quest for causal mutations and effector molecules. BMC Gen 13: 356.
• INSANI EM, EYHERABIDE A, GRIGIONI G, SANCHO AM, PENSEL NA & DESCALZO AM. 2008. Oxidative stability and its relationship with natural antioxidants during refrigerated retail display of beef produced in Argentina. Meat Sci 79: 444-452.
• KOOHMARAIE M & GEESINK GH. 2006. Contribution of postmortem muscle biochemistry to the delivery of consistent meat quality with particular focus on the calpain system. Meat Sci 74: 34-43.
• KOOHMARAIE M, KENT MP, SHACKELFORD SD, VEISETH E & WHEELER TL. 2002. Meat tenderness and muscle growth: is there any relationship? Meat Sci 62(3): 345-352.
• LISTRAT A, LEBRET B, LOUVEAU I, ASTRUC T, BONNET M, LEFAUCHEUR L, PICARD B & BUGEON J. 2016. How Muscle Structure and Composition Influence Meat and Flesh Quality. Sci World J 2016: 3182746.
• LONERGAN EH, ZHANG W & LONERGAN SM. 2010. Biochemistry of postmortem muscle—Lessons on mechanisms of meat tenderization. Meat Sci 86(1): 184-195.
• LORENZO JM, MUNEKATA PES, BARBA FJ & TOLDRÁ F. 2019. More than Beef, Pork and Chicken – The Production, Processing, and Quality Traits of Other Sources of Meat for Human Diet. Cham: Springer International Publishing.
• LUCIANO G, BIONDI L, PAGANO RI, SCERRA M, VASTA V, LÓPEZ-ANDRÉS P, VALENTI B, LANZA M, PRIOLO A & AVONDO M. 2012. The restriction of grazing duration does not compromise lamb meat colour and oxidative stability. Meat Sci 92: 30-35.
• MEDEIROS IG & FERREIRA IAA. 2018. Epigenética Nutricional: Alimentação que nos molda. Rev Trab Acad: Univ Recife 5(1).
• MORENO GMB, BORBA H, ARAÚJO GGL, SAÑUDO C, SILVA SOBRINHO AG, BUZANSKAS ME, LIMA JÚNIOR DM, DE ALMEIDA VVS & BOAVENTURA NETO O. 2015. Meat Quality of Lambs Fed Different Saltbush Hay (Atriplex nummularia) Levels. Ital J Anim Sci 14(2): 3302.
• NRC - NUTRIENT REQUIREMENTS OF SMALL RUMINANTS. 2007. Washington, D.C.: National Academies Press.
• OLIVEIRA FG, SOUSA WH, CARTAXO FQ, BATISTA ASM, RAMOS JP & CAVALCANTE ITR. 2020. Quality of meat from Santa Ines sheep with different biotypes and slaughtering weights. Rev Bras Saúde Prod Anim 21: e210732020.
• OUALI A, HERRERA-MENDEZ CH, COULIS G, BECILA S, BOUDJELLAL A, LAURENT AUBRY L & SENTANDREU MA. 2006. Revisiting the conversion of muscle into meat and the underlying mechanisms. Meat Sci 74(1): 44-58.
• PARENTE HN, MACHADO TMM, CARVALHO FC, GARCIA R, ROGÉRIO MCP, BARROS NNN & ZANINE AM. 2009. Desempenho produtivo de ovinos em confinamento alimentados com diferentes dietas. Arq Bras Med Vet Zootec 61(2): 460-466.
• PELEGRINI LFV, PIRES CC, GALVANI DB, BOLZAN AMS & SILVA GCF. 2008. Características de carcaça de ovelhas de descarte das raças Ideal e Texel terminadas em dois sistemas de alimentação. R Bras Zootec 37(11): 2024-2030.
• PFAFFL MW. 2001. A new mathematical model for relative quantification in realtime RT-PCR. Nucleic Acids Res 29(9): 445.
• PICARD B, LEBRET B, CASSAR-MALEK I, LIAUBET L, BERRI C, LE BIHAN-DUVAL E, HOCQUETTE JF & RENAND G. 2015. Recent advances in omic technologies for meat quality management. Meat Sci 109: 18-26.
• PIKUL J, LESZCZYNSKI DE & KUMMEROW FA. 1989. Evaluation of three modified TBA methods for measuring lipid oxidation in chicken meat. J Agric Food Chem 37(5): 1309-1313.
• RAMOS IN & GOMIDE LAM. 2007. Evaluation of meat quality: fundamentals and methodologies. Viçosa: Universidade Federal de Viçosa.
• RIBEIRO TMD, MONTEIRO ALG, PRADO OR, NATEL AS, SALGADO JA, PIAZZETTA HL & FERNANDES SR. 2009. Desempenho animal e características das carcaças de cordeiros em quatro sistemas de produção. Rev Bras de Saúde e Prod Anim 10(2): 366-378.
• ROGÉRIO MCP ET AL. 2007. Valor nutritivo do resíduo da indústria processadora de abacaxi (Ananas comosus L.) em dietas para ovinos. 1. Consumo, digestibilidade aparente e balanços energético e nitrogenado Zootecnia E Tecnologia E Inspeção De Produtos De Origem Animal. Arq Bras Med Vet Zootec 59(3): 773-781.
• SACCÀ E, CORAZZIN M, BOVOLENTA S & PIASENTIER E. 2019. Meat quality traits and the expression of tenderness-related genes in the loins of young goats at different ages. Animal 13: 2419-2428.
• SANTÉ-LHOUTELLIER V, ENGEL E, AUBRY L & GATELLIER P. 2008. Effect of animal diet (lamb) and meat storage on the oxidation of myofibrillar proteins and in vitro digestibility. Sci Flesh 79(4): 777-783.
• SANTOS IJ. 2019. Estratégias nutricionais para terminação de cordeiros em pastagem ou em confinamento. 2019. Available at: https://pesquisa.bvsalud.org/portal/resource/pt/vtt-212374>.
  » https://pesquisa.bvsalud.org/portal/resource/pt/vtt-212374
• SILVA DJ & QUEIROZ AC. 2002. Food Analysis (chemical and biological methods). 2ª Ed. Viçosa: Universidade Federal de Viçosa.
• SILVA SOBRINHO AG, PURCHAS RW, KADIM IT & YAMAMOTO SM. 2005. Meat quality characteristics of sheep meat of different genotypes and ages at slaughter. Rev Bras Zootec 34(3): 1070-1078.
• SOUZA ARDL, MEDEIROS SR, MORAIS MG, OSHIRO MM & TORRES JÚNIOR RAA. 2009. Dieta com alto teor de gordura e desempenho de tourinhos de grupos genéticos diferentes em confinamento. Pesqui Agropecu Bras 44: 746-753.
• SOUZA MM. 2012. Avaliação dos efeitos de polimorfismos e da origem parental do alelo na expressão de genes candidatos à característica maciez da carne em bovinos da raça Nelore. Tese de Mestrado. Universidade Federal de São Carlos. https://repositorio.ufscar.br/handle/ufscar/5500.
• TORRES EAFS & OKANI ET. 1997. Teste de TBA: Ranço em alimentos. R Nac Carne 243: 68-76.
• UNDERWOOD KR, MEANS WJ & DU M. 2008. Caspase 3 is not likely involved in the postmortem tenderization of beef muscle. J Anim Sci 86(4): 960-966.
• VALADEZ-GARCÍA KM, AVENDAÑO-REYES L, DÍAZ-MOLINA R, MELLADO M, MEZA-HERRERA CA, CORREA-CALDERÓN A & MACÍAS-CRUZ U. 2021. Free ferulic acid supplementation of heatstressed hair ewe lambs: Oxidative status, feedlot performance, carcass traits and meat quality. Meat Sci 173: 108395.
• VAN SOEST PJ. 1963. Use of Detergents in the Analysis of Fibrous Feeds. II. A Rapid Method for the Determination of Fiber and Lignin. J AOAC Int 46(5): 829-835.
• VIANA JGA. 2008. Governança da cadeia produtiva da ovinocultura no Rio Grande do Sul: estudo de caso à luz dos custos de transação e produção. Santa Maria: Universidade Federal de Santa Maria. Available at: <https://repositorio.ufsm.br/handle/1/8819 >.
  » https://repositorio.ufsm.br/handle/1/8819
• YAMAMOTO MS. 2006. Performance and carcass and meat characteristics of lambs finished in feedlot with diets containing fish residue silgens. Available at: <https://repositorio.unesp.br/handle/11449/106613>.
  » https://repositorio.unesp.br/handle/11449/106613
• ZHAO J, LI K, SU R, LIU W, REN Y, ZHANG C, DU M & ZHANG J. 2017. Effect of dietary Tartary buckwheat extract supplementation on growth performance, meat quality and antioxidant activity in ewe lambs. Meat Sci 134: 79-85.

Publication Dates

• Publication in this collection
  30 Oct 2023
• Date of issue
  2023

History

• Received
  01 July 2022
• Accepted
  07 Mar 2023