Carcass characteristics and meat quality of weaned New Zealand rabbits supplemented with Moringa oleifera leaves meal

Abstract Moringa oleifera leaves (MOL) containing phytochemicals could be used to replace synthetic growth promoters to improve products quality in sustainable rabbit production. This study explores the supplementation of MOLM on carcass characteristics and meat quality of rabbits. A total of 64, 28 days-old male weaned New Zealand white rabbits (600 ± 8.58 g live weight) were randomly allocated to four diets in completely randomised design. The rabbits were reared on iso-energetics and isoproteic diets formulated by incorporating MOLM at 0, 5, 10, and 15% and were humanely slaughtered after 6 weeks. Heart weight increased linearly with MOLM levels (R2 = 0.50; p = 0.007) whereas slaughter (SLW), hot carcass (HCW), cold carcass (CCW), legs, head and kidneys weights were increased quadratically. Also, liver decreased quadratically (R2 = 0.41; p = 0.03) with MOLM levels. Fillet lightness (L*) (R2 = 0.21; p = 0.02), redness (a*) (R2 = 0.14; p = 0.04) and yellowness (b*) (R2 = 0.14; p= <0.0001) decrease whereas peak force increased (R2 = 0.63; p = 0.02) quadratically with MOLM levels. Leg muscle a* (R2 = 0.45; p = 0.0001) and cooking loss (R2 = 0.19; p = 0.0009) decreased linearly with MOLM levels. Only aroma (R2 = 0.04; p = 0.04), juiciness (R2 = 0.05; p = 0.04) and connective tissues (R2 = 0.03; p = 0.05) increased linearly with MOLM levels. In conclusion, MOLM did not adversely affect rabbit carcass characteristics and meat quality hence it can be recommended as a nutraceutical sources for sustainably rabbit production. Highlights Moringa oleifera leaves (MOL) containing phytochemicals could be used to improve the physio-chemical quality of meat of rabbit in sustainable rabbit production. Partial inclusion of MOL in rabbit’s diets improved carcass characteristics and meat quality. Slaughter and organ weights, physio-chemical and sensory evaluation were significantly improved by MOLM inclusion.


Introduction
Rabbit farming is expanding due to its proliferous production and excellent meat.The rabbit meat is highly valued for its nutritional and dietary properties.The meat contains proteins (20-21%), unsaturated fatty acid, potassium, phosphorus, and magnesium with less fat and salts contents (Dalle Zotte and Szendro 2011).Hu and Willett (2002) reported the meat as healthy and can be consumed by people with heart related diseases.With that, China, Italy, Spain, France, Czech Republic and Germany are gaining a lot of attention (FAOSTAT 2012) on rabbit farming because rabbits can convert protein (20%) consumed into meat better than other high growth rate and short gestation period animals.However, in Sub-Saharan Africa, smallholder farmers are rearing rabbit on account of their popularity, low investment requirements, low economic risks, family nutrition, income generation and gender empowerment (Oseni and Lukefahr 2014).Moreover, a possible and most appropriate remedy for the shortage of animal protein for the increasing human population globally (Mahfuz and Piao 2019) could be solved by using fast maturing animals like rabbits.To meeting high rabbit's production, formulation of inexpensive high quality compound feeds incorporating safe, reliable and nutritious ingredients to deliver sustainable rabbit production is important.
Vast literatures reporting the use of MOL in poultry species like indigenous chickens (Sebola and Mokoboki 2019); Tutubalang et al. 2022), quails (Mulaudzi et al. 2022), pigs (Chen et al. 2021) and rabbits (Sun et al. 2018;Mankga et al. 2022) has been done.These studies concluded that MOL can be used as a good feed ingredient due to its nutritional value, antioxidant, antimicrobial properties and low antinutrients level.Thus, it is imperative to further explore the impact of MOL on carcass characteristics and meat quality of weaned rabbits.It was hypothesised that the inclusion of MOL in the diets of weaned rabbits might significantly improve their carcass characteristics and meat quality.

Study area and feed components
The research was done at Kaffirstad farm (26.34 S and 29.18 E), Mpumalanga Province, South Africa.During this time, the ambient temperatures range from 25 to 35 C. The MOLM (Table 1) was acquired from the Patience Wellness Centre (24 30 0 51.S 29 56 0 51.E), Limpopo Province, South Africa.Soybean meal, corn, molasses, lime and premix were obtained from Opti feeds, Lichtenburg, South Africa.

Experimental designs, treatments diets and proximate analyses
A total of 64 male rabbits (600 ± 8.58 g live weight) were randomly allocated to four treatment diets (Table 1) according to NRC (National Research Council) 1977.A completely randomised design with four treatment diets replicated times and amounting to a total of 16 floor cages was used.The experimental unit was a floor cage (0.07 m 2 per weaned rabbit) holding 4 rabbits.The four diets were constituted by diluting a commercial rabbit diet with grounded air-dried MOLM.The diets were as follows: MOLM0 ¼ rabbit diet without MOLM as substitute (control), MOLM5 ¼ rabbit diet with 5% MOLM as substitute; MOLM10 ¼ rabbit diet with 10% MOLM as substitute and MOLM15 ¼ rabbit diet with 15% MOLM as substitute as described by Mankga et al. (2022).
The formulated diets (MOLM0, MOLM5, MOLM10 and MOLM15) were milled (Polymix PX-MFC 90 D) to pass through a 1 mm sieve for chemical analyses.For laboratory dry matter (Association of Official Analytical Chemists (AOAC) 2005; method no.930.15) determination, approximately 1 g of each sample was placed into pre-weighed crucibles and placed in an oven set at 105 C for 12 h.The loss in weight was measured as moisture content and dry matter (DM) was calculated as the difference between the initial sample and moisture weights.Organic matter (OM) content (Association of Official Analytical Chemists (AOAC) 2005; method no.924.05) was determined by ashing the dried samples in a muffle furnace set at 600 C for 12 h.The loss in weight was measured as OM content and the residue as ash.Total nitrogen content was determined by the standard macro-Kjeldahl method (Association of Official Analytical Chemists (AOAC) 2005; method no.984.13) and was converted to crude protein by multiplying the percentage N content by a factor of 6.25.Crude fibre was determined using the ANKOM 2000 Fibre analyser (ANKOM Technology, New York) with 0.255 N crude fibre acid solution and then with 0.313 N crude fibre base solution.

Preparation of the house, experimental rabbits and management
The rabbit's house and cages were cleaned thoroughly with water and detergent and thereafter was disinfected with F10.All drinking nipples and feeders were cleaned before they could be utilised.The house was then given adaptation period for 2 weeks post-cleaning.Feed and water were offered ad-libitum under a constant lighting.

Carcass characteristics and internal organs
At day 35 of experiment, all rabbits were starved for 12h and humanely slaughtered Carltonville rabbit abbatoire (Carltonville, Johannesburg, South Africa).The carcasses of different dietary treatments were tagged and put in plastic bags for proper identification.The weight of the hot carcasses (HCW) was taken instantly post slaughter at the abattoir using electronic scale (Explorer EX224, OHAUS Corp).After 24 hrs of chilling, the carcasses were re-weighed for cold carcass (CCW).The front, hind legs, heads and fillets were measured and recorded.The livers, hearts, kidneys, lungs and the intestines were then removed and weighed.The carcass samples were placed into polythene storage bags and put in the cold room (± 4 0 C) awaiting the meat physico-chemical qualities.They were then cut apart in line with the standards of the World Rabbit Science Association (Blasco and Ouhayoun 1996).Fillet samples and thighs were collected 24 hrs post slaughter for evaluation of the meat quality traits.

Meat colour and pH measurement
Meat colour was determined within an hour post slaughter and at 24 h (PH u ) after slaughter.The colour (L Ã ¼ lightness, a Ã ¼ Redness and b Ã ¼ Yellowness) was measured from fillet muscle and the thigh muscle using a spectrophometer (CM 2500c model, Konica Minolta, Inc. Japan).The three distinct points of the fillet muscle were measured by rotating the spectrophometer between each measurement to get the mean value of the colour.
A digital pH metre (CRISON pH25, CRISON Instruments SA, Spain) with a penetrating conductor was utilised to get the pH of the fillet and the thigh muscle of each individual rabbit at 45 min for initial pH (pH i ) and 24 hrs post mortem to obtain the ultimate pH (pH u ) as described by Manyeula et al. (2020).

Drip loss
Approximately 30 g meat strips were sampled from the fillet muscle parallel to the fibre direction then weighed (W i ) by digital weighing scale (Explorer EX224, OHAUS Corp) sensitive to 0.01 g.The samples were suspended in a cold room (4 C) for 72 h and reweighed again (W F ) following method by Honikel and Hamm (1994) using the following equation:

Cooking loss
Raw meat cubes were cut from the fillet muscles, initial weighed (W i ) and then placed in an oven heated at 75 C for 45 min.The samples were then cooled down at a room temperature for 15 min, dried with the soft tissue and weighed (W F ) (Sanka and Mbanga 2014).Cooking loss was then determined as a lost weight percentage through cooking comparative to the uncooked muscle mass (Gopinger et al. 2014) according to the following equation:

Sensory evaluation
Approximately 5 cm portions of meat were cut and baked for 30 min in an oven set at 105 C. The meat was then assessed for aroma, first bite, juiciness, sustained juiciness, overall tenderness, connective tissue, overall flavour and typical flavour preferences using 35 panellists to rank each part on 8-point position scale (1 ¼ extremely bland; 2 ¼ very bland; 3 ¼ fairly bland; 4 ¼ slightly bland; 5 ¼ slightly intense; 6 ¼ fairly intense; 7 ¼ very intense and 8¼ extremely intense).

Statistical analysis
Dietary effects on carcass characteristics and meat quality data were analysed using the general linear model procedure of SAS ( 2010) for a completely randomised experimental design with pen as the experimental unit.Data from all measured parameters were evaluated for linear and quadratic effects using polynomial contrasts.Response surface regression analysis (RSREG) (Statistical Analysis Software Institute (SAS) 2002-2012) was applied to describe the responses of white weaned rabbit to inclusion levels of MOLM using the following quadratic model:

Carcass characteristics and internal organs
Diets significantly affected carcass characteristics and internal organs except lungs and fillets (Table 2).
Rabbits fed diet MOLM10 had longer small intestine than those fed on diet MOLM5.However, those fed on MOLM5 and MOLM10 had similar small intestine length to those fed on diets MOLM0 and MOLM15.Longest large intestine length was observed on rabbits fed MOLM0 whereas the shortest were observed on rabbits fed other diets.Rabbits fed diet MOLM15 had the heaviest heart compared to those fed MOLM0 and MOLM5.Neither quadratic nor linear effects (p > 0.05) were observed for small intestines, large intestine, lungs and fillet with MOLM inclusion levels except for the heart weight.Heart increases linearly (y ¼ 0.35 (±0.04)À0.008(±0.01) x; R 2 ¼ 0.50; p ¼ 0.007) with MOLM levels.
All carcass characteristics and internal organs parameters quadratically increased with the inclusion levels of MOLM (Table 3).
Dietary treatments had significantly effects on all carcass characteristics and internal qualities except SLW (Table 4).HCW and CCW of meat from rabbits on diets MOLM5 and MOLM15 were higher (p < 0.05) than those on diets MOLM0 and MOLM10 which were similar (p > 0.05).Rabbits on diet MOLM15 had heavier fillet leg than diets MOLM5 and MOLM10, which were similar (p > 0.05).Likewise, diet MOLM15 promoted the lighter (p < 0.05) hind leg weights compared to diet MOLM10 and MOLM5.However, head weight was higher (p < 0.05) in rabbits on diet MOLM15 than those on diets MOLM0, MOLM5 and MOLM10, which did not differ (p > 0.05).Diet MOLM10 promoted  ,b,c In a row, means with common superscripts do not differ (p < 0.05); d Diets: MOLM0: rabbit grower diet with 0% M. oleifera as substitute; MOLM5: rabbit grower diet with 5% M. oleifera as substitute; MOLM10: rabbit grower diet with 10% M. oleifera as substitute; MOLM15: rabbit grower diet with 15% of M. oleifera as substitute; e SEM ¼ standard error of the mean.MM: millimetre.higher liver weight than diets MOLM0.More so that, diets MOLM5 and MOLM15 were similar to diets MOLM0 and MOLM15.Nonetheless, the MOLM0 and MOLM15 promoted heavier kidney weights than other diets.

Meat quality
Physico-chemical quality   ,b,c In a row, means with common superscripts do not differ (p < 0.05).d Experimental diets: MOLM0: rabbit grower diet with 0% M. oleifera as substitute; MOLM5: rabbit grower diet with 5% M. oleifera as substitute; MOLM10; rabbit grower diet with 10% M. oleifera as substitute; MOLM15: rabbit grower diet with 15% of M. oleifera as substitute.e Parameters: SLW: slaughter weight; HCW: hot carcass weight; CCW: cold carcass weight; f SEM: standard error of the mean.  b,c In a row, means with common superscripts do not differ (p < 0.05); d Diets: MOLM0: rabbit grower diet with 0% M. oleifera as substitute; MOLM5: rabbit grower diet with 5% M. oleifera as substitute); MOLM10; rabbit grower diet with 10% M. oleifera as substitute; MOLM15: rabbit grower diet with 15% of M. oleifera as substitute); e SEM: standard error of the mean.

Carcass characteristics and internal organs
Carcass characteristics and meat quality are influenced by feed consumed by the animal.However, including MOLM in rabbit diets at higher inclusion rate may be necessary to improve the quantity and quality of meat.It is known that MOL contains bioactive compounds such as quercetin which are responsible for fat reduction, muscle growth and anti-oxidation (Wang et al. 2022;Zhu et al. 2022).Despite its high crude fibre (19.3%) and condensed tannins (12 g/kg dry matter basis) content (Moyo et al. 2011), current results shows that the highest inclusion level of MOLM promoted HCW and CCW signifying the potential of MOLM to improve edible meat and this could be due to the presents of quercetin.Indeed, quercetin as flavone was reported by literatures (Wang et al. 2022;Zhu et al. 2022) to promote muscle growth and reduced fats.Rabbits fed the MOLM-containing diets had higher fillet, hind, head, liver and kidneys weights when compared to those in the control group, which could have been influenced by the high SLW (Mankga et al. 2022).This suggests that the inclusion of MOLM as a functional ingredient in rabbit's diets did not affect the functionality of these organs.Obviously, increasing graded level of MOLM in the diets lead to increased nutrients density (protein, fatty acids, minerals, vitamins, and several bioactive) that promotes growth (Mahfuz and Piao 2019) hence increased SLW.But, literatures on MOLM inclusion at 25% (Abubakar et al. 2015;Helal et al. 2017) and 200 g/kg (Khalil et al. 2019) in the Rabbit weaner's diet reported lack of significant effect on carcass weight, intestinal length, liver and kidneys.Contrarily to this findings, Selim et al. (2021) reported increased in intestinal length with 25% of MOLM inclusion levels.MOL contains fibre (19.3%) which is known to increase the size of internal organs when added at a higher inclusion levels (Manyeula et al. 2020).This is an adoptive mechanism that enhances nutrients absorption and utilisation in monogastric animals.In this study, it was expected that higher levels of MOLM inclusion would increase the size and length of internal organs leading to rabbits with larger organs than those fed on the MOLM0 diet.However, in this study rabbits fed standards control promoted longer intestines suggesting that gastro intestinal tract of a rabbits has the capacity to utilise fibre in MOLM containing diets, which is not our expectations.The significant linear increases in heart weight as MOLM levels increased could indicate the increases in body weight as shown by slaughter weight.Miya et al. (2020) reported an increase in body weight leading to increases in heart weight due to the fact that heavy body demand more oxygen and blood resultant in heavy heart.Lack of significant different in lungs and fillet agreed with the findings of Abubakar et al. 2015;Chia et al. 2018) who reported lack of significant difference in the intestine, lungs and fillets of rabbits when MOLM was included in the diets.5.0 4.3 5.5 5.5 0.4 0.12 0.46 a Diets: MOLM0: rabbit grower diet with 0% M. oleifera as substitute; MOLM5: rabbit grower diet with 5% M. oleifera as substitute; MOLM10: rabbit grower diet with 10% M. oleifera as substitute; MOLM15: rabbit grower diet with 15% of M. oleifera as substitute; b SEM: standard error of the mean.

Physico-chemical
Moringa oleifera leaves is high in antioxidant compounds, tocopherol, ascorbic acid, carotenoids, polysaccharide, flavonoids, saponins, phenolics, tannins, and proanthocyanidins and are important in the action of antioxidant enzymes (Makkar and Becker 1996).Meat colour at the point of purchase influence consumer's preference (Font-I-Furnols and Guerrero 2014) and it is influenced by diet, consequence, modifies the metabolism of glycogen, storage, pH, and antioxidant accumulation (Mancini et al. 2018).It is known that low pH reduces myoglobin to absorb green light, resultant in meat appeared less red and more yellow (Sun et al. 2018).Our results, on fillet a Ã and b Ã showed quadratic decreased which is not our expectation but inline with study in rabbits fed 30% MOLM (Sun et al. 2018) but contradicts similar research in rabbits fed 15 g/kg MOLM (Selim et al. 2021).However, it is important to note the difference in inclusion levels used in these studies might alter the physicochemical parameters of the diets.The rate at which glycogen levels in muscle prior to slaughter is converted to lactic acid after slaughter determines muscle pH u (Dyubele et al. 2010).In the current study, lack of significant effects on pH u imply that MOLM inclusion did not affect glycogen levels.This is in accordance with what is previously reported by Castrica et al. (2020) when Lycium barbarum were included in the rabbit's diets.Anti-oxidants or bioactive compounds in the diets from MOL is known to change meat colour, and it has been reported that the MOL has flavonoids and antioxidant activity (Leone et al. 2015).Moreover, in this study inclusion of MOLM in rabbits diets linearly decreased the leg muscle a Ã implying that the highest inclusion levels may have interfered with flavonoids thereby reducing the myoglobin of the meat which could be a results of low pH.It is known that low pH reduces the importance of myoglobin by selectively absorbing green light, resulting in meat that appears less red and more yellow (Dougnon et al. 2012;Han et al. 2012).On the contrary to our results, Selim et al. (2021) recorded linear increase on leg muscle a Ã when 15 g/kg of MOLM is supplemented rabbits which is inline with our expectations.An exciting results shows that diets containing MOLM significantly decreases cooking loss linearly and improved the peak force (tender) of the meat confirming that including MOLM in rabbit diets positively affected meat texture.Likewise, this is regarded as the most preferred trait for cooked meat by the consumer (Glitsh 2000).Furthermore, diets had no influence on fillets a Ã , leg muscle L Ã and b Ã , pH u , and drip loss, findings that are consistent with earlier reports on the effects of MOLM containing diets on meat quality traits on Rabbits (Selim et al. 2021).This indicates that supplementing MOLM in rabbit's diets has no negatives effects on the muscle quality.It is also worth noting that greater drip loss of the muscle reduce water holding capacity, which is associated with lipids peroxides of the meat hence reduced shelf life (Schaefer et al. 1995).

Sensory evaluation
Little is known about the effects of MOLM on sensory evaluation of meat from rabbits.It was expected that higher levels of MOLM inclusion would improve meat quality leading to improved sensory quality from rabbits fed high inclusion level.Inclusion of MOLM quadratically increased aroma, juiciness and connective tissue, resulting in higher scale number compared to those of the control diets.These could be attributed by the presence of antioxidant in MOLM impacted on meat quality by enhancing water holding capacity.Similar findings in chickens fed MOLM were reported by Ologhobo et al. (2014).Also, Castrica et al. (2020) reported increased juiciness, tenderness and overall acceptance with Lycium barbarum inclusion in rabbit diets.Evenly through other parameters were affected by MOLM inclusion, nonetheless, first bite, sustained juiciness, overall tenderness and favour were not affected by the diet indicating that supplementation of MOLM has no major negative effect on the aforementioned parameters.

Conclusions
We concluded that MOLM can be used as a supplement in the rabbit diets without compromising carcass characteristics and meat quality.Based on the results, inclusion levels of 15% MOLM could be recommended to rabbit farmers.Nonetheless, further research is required on MOLM inclusion at higher levels than 15% as feed additives in sustainably rabbit production.

Table 1 .
Ingredients and proximate analysis of diets and Moringa oliefera meal in % as fed basis.

Table 2 .
Carcass characteristics and relative internal organs weight (% of hot carcass weight, unless otherwise stated) of New Zealand white weaned rabbits fed diet containing Moringa oleifera leave meal.

Table 3 .
Regression equations for Carcass characteristics and internal organs of New Zealand white weaned rabbits fed diet containing Moringa oleifera leave meal.

Table 4 .
Effect of Moringa oliefera leaves meal-containing diets on carcass charactestics and internal organs of New Zealand white weaned rabbits (%, unless stated otherwise).

Table 5 .
Meat quality of New Zealand white weaned rabbits fed diet containing Moringa oleifera leave meal.

Table 6 .
Sensory evaluation of meat of New Zealand white weaned rabbits fed diet containing Moringa oleifera leave meal.