Impact of Romanov breed lamb gender on carcass traits and meat quality parameters including biogenic amines and malondialdehyde changes during storage

Abstract The present study aims to evaluate the effect of Romanov breed lamb gender on carcass traits and meat quality parameters, as well as on the formation of biogenic amines (BAs) and malondialdehyde during meat storage. Obtained results revealed that lamb gender had a significant influence on sternum/breastbone, ribs, right shoulder, and bones of the back leg. Significantly higher lightness (by 3%) was found for male meat; however, higher redness of female meat was observed (by 7.7%). In all cases, a lower pH was obtained for female meat. Significantly higher cooking loss (by 38%) was found for male meat. However, gender was not a significant factor in lamb meat proximate composition, or for BAs and cholesterol content. The gender of animals had a significant influence on 10‐heptadecenoic (C17:1), linoleic (C18:2n – 6), total polyunsaturated FA, and total trans isomers content in meat. A significantly higher concentration of malondialdehyde was found in female lamb meat (by 43.4% and 56.8% in fresh and after 3 months of storage at −18°C, respectively) compared to males. Finally, the obtained results supplement the scarce database about the characteristics of Romanov breed meat of different gender and this is beneficial for lamb breeders and meat industry in order to obtain a better quality production.

productive purebred sheep breeds, originated in Russia (Dvalishvili et al., 2015). The Romanov breed is characterized by high productive and reproductive performance with a high number of juveniles, as a Romanov ewe can produce seven live and healthy lambs in a litter (Zapasnikiene & Nainiene, 2012).
In general, meat quality is influenced by animal age, gender, physiological state, post-mortem biochemistry, carcass composition, and feed contribution to flavor, protein, and fat levels, as well as the effect of genetics on tissues and metabolism, pre-and post-slaughter handling, storage, etc. (Armstrong et al., 2018).
Nowadays, there is growing interest in manipulating the carcass quality and fatty acid (FA) composition of livestock meat in order to produce meat with higher content of polyunsaturated FA (n-3) and better consumer acceptance (Vahmani et al., 2020). A number of strategies have been used to supply sheep meat according to this new consumer demand (Hopkins & Mortimer, 2014). Some animal production strategies affect the chemical and physical quality of the meat, and usually, to obtain desirable production rates and carcasses with different characteristics, animals of different gender are selected; breed also has a large effect on carcass morphology (Hopkins & Mortimer, 2014).
Q uite a range of parameters (e.g., pH, color, fat content, carcass, etc.) characterizes the quality of lamb meat and most of them are widely explored (Corazzin et al., 2019). However, other not so often mentioned compounds or processes, such as formation of biogenic amines (BAs) or malondialdehyde (MDA), could be also relevant for meat quality and consumer health. BAs have been reported in a variety of foods as the result of the microbial decarboxylation of amino acids (Papageorgiou et al., 2018). These compounds mostly participate as neurotransmitters in human body but when a high content of BAs is consumed with food, this leads to the intoxication of the organism (Ruiz-Capillas & Herrero, 2019). The most common symptoms include headache, abdominal cramps, nausea, diarrhea, breathing difficulties, low or highly increased blood pressure, and rash (Wójcik et al., 2020). Increased content of BAs could be related with the lack of hygiene and usually reflects the freshness of the food (Ruiz-Capillas & Herrero, 2019). For this reason, the control of BAs in foods is very important. Another important issue for meat quality is lipid peroxidation, which leads to undesirable changes in meat color, flavor, odor, texture, and even nutritional value during storage (Cimmino et al., 2018). Besides, lipid peroxidation, which is initiated in the subcellular membranes of the unsaturated fatty acid (UFA) fraction, is a major cause of the deterioration and reduced shelf life of meat products (Yagoubi et al., 2018). Changes that occur in the muscle during the postmortem period may create an unbalanced proportion between antioxidant and pro-oxidant capability, increasing the risk of oxidative damage (Sabow et al., 2016). MDA is one of the lipid peroxidation products and widely known as the marker of this process (Ma et al., 2020). It was reported that MDA elicits a great concern to human health (Douny et al., 2015). Therefore, the composition and content of lipid fraction is a very important indicator of meat quality (Corazzin et al., 2019).
Several reports have shown that male lambs have greater daily body gain, lower carcass fat (Bas et al., 2007), and a better feed conversion ratio and daily weight gain (Kashani & Bahari, 2017) than females. On the contrary, there is conflicting evidence about the influence of gender on meat physical parameters. On the contrary, there is conflicting evidence about the influence of gender on meat physical parameters and FA composition of muscle lipids. Tejeda et al. (2008) reported that color, pH, moisture, and intramuscular fat were not influenced by lamb gender, while total polyunsaturated fatty acid (PUFA) content was higher in females than in males. Yarali et al. (2014) found that female Kıvırcık lambs had a lower PUFA content and higher cooking loss and water loss that males; however, a significant difference in meat shear force between genders was not observed. The study of Facciolongo et al. (2015) showed that meat of the Gentile di Puglia males had higher values of final pH, brightness, and yellow index, and a lower PUFA n-6:n-3 ratio in comparison with females. In this respect, there is still a need for the more detailed studies in this field. Therefore, the present study aims to evaluate the effect of Romanov breed lamb gender on carcass traits and meat quality parameters, as well as on the formation of biogenic amines and malondialdehyde during meat storage.

| Animals used in the study
The animals were cared for in accordance with the Lithuanian State B1-866). Lambs of Romanov breed were reared at the commercial sheep farm in Kaunas district (Lithuania). Lambs were born by natural way during March and April and reared on the same farm where they were born, until slaughter. All animals were kept with their mothers on natural pasture under the same rearing conditions, and they were fed ad libitum with ewe's milk and grasses. Water was given freely to all animals. At 6 months of age, the lambs were divided into two groups according to their gender (group I -male lambs, and group II -female lambs) and transported to a commercial slaughterhouse. A total of 34 lambs (17 males and 17 females) were randomly selected for this study.

| Evaluation of lamb meat physicochemical parameters
The Gluteus medius muscle was used for meat chemical analysis (protein, fat, ash, and dry matter), determination of cholesterol, biogenic amines, malondialdehyde, and fatty acids profile, as well as for the assessment of meat quality traits (pH, color, water holding capacity, cooking loss, and shear force). Until analysis of meat quality, samples were vacuum packed and kept at 4°C in the refrigerator. The The analysis of meat physicochemical parameters was carried out as described by Rozanski et al. (2017) and AOAC (2019).  Klupsaite et al. (2020). WHC was determined using filter paper press method. Briefly, sample (2 g) was placed on a filter paper (Whatman filter paper 41/ashless), compressed between two plexiglass sheets, and received a pressure exerted by a weight of 1 kg for 10 min. DL was measured as the weight loss during suspension of a standardized (40-50 g and approximately 30 × 60 × 25 mm) muscle sample (in an airtight container over 24 h at 4°C). The CL corresponded to the weight difference between the samples (in a plastic container) before and after cooking in a water bath (internal temperature of 70°C for 30 min). For the evaluation of SF, three cylindrical samples with a diameter of 1.27 cm were removed from each sample that was used to determine CL. SF was measured using TA-XT Plus texturometer (TA.XT plus Texture Analyzer, Stable Microsystems Ltd., Surrey, England) coupled to a Warner-Bratzler device. The dry matter content of muscles was determined after oven drying the samples at 105°C for 24 h. Ash was determined by burning meat samples in a muffle furnace at 550°C for 4 h. Intramuscular fat content was determined using a Soxhlet SE 416 Columbus macro automatic system for fat extraction (Gerhardt, Germany). The protein content in meat samples was analyzed by the Kjeldahl method (ISO 937:1974).

| Evaluation of biogenic amine content in lamb meat
The analysis of BAs content was performed at 48 h post-mortem.
After that, these samples were vacuum packed, stored at −18°C

| Analysis of fatty acid content in lamb meat
Extraction of lipids for FA analysis was performed with chloroform/ methanol (2:1 v/v) as described by Pérez-Palacios et al. (2012).
Fatty acid methyl esters (FAME) were prepared by esterification with 2 mol/L of KOH in methanol and shaking using laboratory shaker for 1 hr, upper layer was filtered using 0.22 µm membrane syringe filter and used for the analysis. Fatty acid composition was determined using gas chromatograph GC-2010 Plus (Shimadzu

corp.) equipped with Mass Spectrometer GCMS-QP2010
(Shimadzu corp.). Separation was carried out on a Rxi-5ms column (30 m length, 0.25 mmID, and 0.25μm df (Restek). Mass spectrometer was operated at full scan mode. Analyte was injected in split mode at 1:60 split ratio. Oven temperature programming started at 40°C, it was raised 8°C/min to 220°C, held for 1 min at 220°C, increased again at 20°C/min to 270°C, and held for the last 10 min. Injector temperature was 240°C, interface −270°C, and ion source 220°C. The carrier gas was helium at a flow rate of 0.91 ml/min. Fatty acid methyl esters (FAME) concentration was determined using calibration curve and results were expressed as percentage of total FAME concentration in sample. The calibration curve was prepared using standard Supelco 37 Component FAME Mix (Merck & Co., Inc.). The extraction of lipids was performed once and all analytical determinations were performed at least in triplicate.

| Evaluation of cholesterol and malondialdehyde content in lamb meat
The analysis of cholesterol was performed according to the method of Polak et al. (2008) with some modifications. Lamb meat sample (2.00 ± 0.01 g) and 10 ml of saturated KOH were mixed and heated for 60 min at 60°C. The sample tube was cooled and 10 ml of hexane was added. The distilled water (3 ml) was poured into top layer of liquid and then the tube was shaken for 10 times. The sample was then centrifuged at 1000 g for 2 min (Sigma 2-5, Germany). An aliquot of the hexane extract (1 ml) was transferred to vial, dried with a nitrogen flush, and dissolved in 2 ml of mobile phase. The HPLC instrumentation was used the same as mentioned for BAs analysis.
The mobile phase comprised a mix of 2-propanol and acetonitrile The analysis of MDA was performed at 48 hr post-mortem. After that, these samples were vacuum packed, stored at −18°C in freezer for 3 months, and used for further determination of MDA. MDA was analyzed according to method described by Mendes et al. (2009).

| Statistical analysis
All measurements were carried out in triplicate. Statistical analysis was performed using the descriptive statistics and univariate analysis of variance (ANOVA) (statistical program R 3.2.1; R Core Team, 2015) to determine the significance effect of lamb gender on the carcass traits and meat quality parameters. The mentioned parameters were set as dependent variables and gender as an independent variable. The assumption of normality and homogeneity of variance of data were assessed by the Shapiro-Wilk and Levene's tests, respectively. Results were considered statistically significantly different at p ≤ .05.

| Influence of lamb gender on carcass traits
The effect of lamb gender on carcass traits is shown in Table 1.
According to the results obtained, lamb gender has a significant influence on the sternum/breastbone (male sternum/breast was 13.9% heavier), ribs (male ribs were 12.6% heavier), right shoulder (male right shoulder was 15.2% heavier), and bones (left and right) of the rear foot (male bones were 10.3% and 20.5% heavier, respectively). Carcass weight, yield, and fat measurements are parameters of great importance to producers; weight and fat measurement can also determine the price paid for lamb carcasses (Gardner et al., 2015). Many studies have been conducted in relation to the evaluation of lamb carcass characteristics, and it is important to estimate the impacts on the quantity and quality of muscles in a carcass (De Brito et al., 2016). Gender can influence carcass composition because female lambs have a greater proportion of fatty tissue than males. However, the effect on meat quality traits is less clear (Santos et al., 2015). Ram lambs are favored in lamb production systems for their faster growth rates compared to ewe, castrated, and cryptorchid lambs (Kenyon & Schreurs, 2017). The meat from ram lambs has been associated with poorer tenderness and color characteristics, in comparison with meat from ewe lambs (Stempa et al., 2018). An influence of animal gender is evident in the regulation of adiposity and muscularity, and these differences have been attributed to gender steroid hormones because testosterone promotes an increase in body mass (de Araújo et al., 2017). From an economic point of view, the carcass is the most valuable part of the animal, and at certain weights it can be largely breed dependent (Mateo et al., 2018).

| Differences in meat quality parameters and chemical composition between lambs of different gender
Male and female lamb meat technological parameters are shown in Table 2. In all cases, lamb gender was a significant factor influencing lamb meat color coordinates (L* p = .045; a* p = .023; b* p = .006).
Higher lightness (L* coordinates higher by 3.0%) of male meat was found. Opposite to the lightness, higher redness and yellowness coordinates of female meat were obtained (7.7% and 32.0% higher, respectively). The color coordinates of lamb meat can be related to animals' diet, production system, slaughter weight, breed, gender, and muscle type (Kuchtík et al., 2012). In general, consumers rate Lamb meat pH was significantly influenced by the gender factor, and, in all cases, lower pH was obtained for female meat (pH of male meat after 24 and 48 h was higher by 2.4% and 2.7%, respectively, compared to female lamb meat). An increase in the pH of meat can increase the activity of cytochrome oxidase by reducing the uptake of oxygen by myoglobin, which results in a purplish red color (Calnan et al., 2016). pH is the main meat quality parameter; it is very important commercially and has effects on meat texture, color, and water holding capacity (WHC), as well as sensory properties and overall acceptability of meat (Popp et al., 2015). High pH values of meat influence storage quality and can adversely affect flavor and aroma.

TA B L E 1 Effect of Romanov lamb gender on carcass traits
Meat shelf life is reduced when pH exceeds 5.8, and as pH increases, meat becomes darker and affects consumer purchase decisions (Hopkins & Mortimer, 2014). A faster decrease in pH in females than in males can be explained by a higher concentration of glycogen in females due to lower sexual activity (de Lima et al., 2016).
Gender was not a significant factor in lamb meat WHC and tenderness; however, a significant influence of gender on meat cooking loss (CL) was found (38.0% higher CL for male lamb meat). In literature, the reports are inconsistent about the effect of gender on mentioned parameters. Some studies have shown that meat from male lambs was harder than that from the female lambs, whereas other reported no significant differences in meat quality traits between ewes and rams (Hopkins & Mortimer, 2014). Velasco et al. (2000) observed the impact of gender on WHC of Talaverana lamb meat. However, no gender effect on CL and tenderness of lamb meat was reported in that study.
The differences in the CL and shear force (SF) between female and male lamb meat could be partly explained by the higher intramuscular fat content in female meat (Facciolongo et al., 2018).
The proximate chemical composition of the lamb meat is given in  Jaworska et al., 2016). In another study, female lambs showed a higher percentage of carcass fat than males (Armero & Falagán, 2015). It was published that Texel variety male and female lambs have a significantly (p < .01) different muscle fat content adjusted to the same carcass weight (de Lima et al., 2016).
No significant differences in intramuscular fat content between the goat breeds Moxotó and Canindé were found (Ablikim et al., 2016).
Variations in meat fat content occur mainly due to changes in the balance between dietary energy and nutrient requirements (de Araújo et al., 2017).

| Biogenic amine content in lamb meat
The BA content in fresh lamb meat and after 3 months of storage is given in Figure 1. Gender was not a significant factor in BA concentration in lamb meat, except for spermine and putrescine after 3 months of storage (p ≤ .05). Histamine and tyramine were not detected in all analyzed samples. Putrescine was not found in fresh male samples, while its content in fresh female meat was 2.84 mg/kg. After storage, spermine and putrescine contents were significantly higher in female lamb meat compared to male meat (by 33.6% and 8.9-fold, respectively). In lamb meat, BA concentration was found to be in the order tryptamine < putrescine < spermine <spermidine < cadaverine < phenylethylamine. BAs are generally formed by amination or transamination of aldehydes and ketones or decarboxylation of free amino acids (Jain et al., 2015). The content of BAs is highly affected by the presence of decarboxylase-positive bacteria, meat composition (the content of free amino acids, pH, and additives), endogenous or microbial proteases, and handling and storage conditions (time/temperature, packaging, temperature abuse, etc.) (Triki et al., 2018). The most significant BAs in meat and meat products are tyramine, cadaverine, putrescine, and histamine (Jairath et al., 2015). In fresh meat, spermine and spermidine can be found naturally in significant amounts (20-60 mg/kg and less than 10 mg/kg, respectively) (Stadnik & Dolatowski, 2010). Similar levels of spermine were also observed in our study; however, the levels of spermidine were higher than 10 mg/kg. Usually, tyramine, cadaverine, and putrescine are formed during meat storage (Jairath et al., 2015). Coliform microflora and lactic acid bacteria produce tyramine, whereas Enterobacteriaceae and Pseudomonas are known to produce putrescine, cadaverine, and histamine (Hutařová et al., 2013). According to our results, only cadaverine was found in higher levels, while histamine and tyramine were not detected in all samples.
Meanwhile, phenylethylamine was the predominant BAs in all lamb meat. The determined level of this Bas is slightly higher than the set limit (30 mg/kg) (Triki et al., 2018). The higher content of phenylethylamine could be related with the presence of microflora with phenylalanine decarboxylation capacity or increased availability of free phenylalanine in lamb meat (Triki et al., 2018). However, a lower storage temperature minimizes BAs accumulation through inhibition of microbial growth and the reduction in enzyme activity.
This tendency was also observed in most of the analyzed lamb meat samples of this study. It has been reported that in over 4 months' storage at 1°C, only tyramine was detected consistently in beef carcasses among all the other BAs (histamine, phenylethylamine, tryptamine, and tyramine) (Jairath et al., 2015). The biogenic amines index (BAI), which is the sum of histamine, cadaverine, putrescine, and tyramine, is calculated to assess the quality of the meat (Wójcik et al., 2020

| Influence of lamb gender on lamb meat fatty acid composition
The FA composition of meat from lambs of different gender is given in Table 3. The significant effect of lamb gender was found on the levels of 10-heptadecenoic (C17:1), linoleic (C18:2n -6), and total polyunsaturated acids, as well as total trans isomers in analyzed lamb meat (p = .003; p = .018; p = .005, p = .005, respectively). The significantly higher levels of 10-heptadecenoic (C17:1) and linoleic (C18:2n -6) acids were found in the female lamb meat (by 72.5% and 16.0%, respectively) compared to male meat. Moreover, the level of polyunsaturated acids was higher by 10.7% in the female meat compared to male. However, the meat from female lambs contained the higher amount of total trans isomers (by 26%).
The FA composition has a significant influence on the sensory attributes of meat and its nutritional value (Wood & Enser, 2017).
The data about FA composition in lamb meat are varied. Camacho et al. (2017) reported that Canarian Hair female lambs had a higher content of monounsaturated FA (mainly oleic acid) than males. Erasmus et al. (2017) found that females had more α-linolenic acid (C18:3n -3) and polyunsaturated FA in their subcutaneous fat than male lambs. In our study, a higher content of polyunsaturated FA was also found in female meat, while the content of C18:3n -3 was similar for both genders. Moreover, the small amount of trans FA was found in female and male meat. Trans FA naturally occur in ruminant meat due to anaerobic bacteria fermentation in rumen (Lichtenstein, 2016).

| CON CLUS IONS
The Romanov lamb breed is characterized by a high productive performance and is frequently crossed with other lamb breeds.
However, there is little or no information about how the gender could influence the carcass traits and quality parameters of Romanov lamb meat. The outcome of this study showed that Romanov lamb gender had a significant influence on several carcass parameters (sternum/breastbone, ribs, right shoulder, and bones (left and right) of the rear foot). Male meat had a significantly higher lightness, lower redness, and higher values of pH and cooking loss. The content of 10-heptadecenoic (C17:1), linoleic (C18:2n -6), total polyunsaturated FA, and total trans isomers were higher by 72.5%, 16.0%, 10.7%, and 26%, respectively, results, further preparation of lamb meat can be planned in order to prepare the highest quality and acceptability of meat products for consumers.

ACK N OWLED G M ENTS
There are no acknowledgments.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L A PPROVA L
The animals were cared for in accordance with the Lithuanian State