Krishna Mohan1, Devlon Brathwaite2, Nitu Kumar1, Reeza Ramgattie3, Gopalan Kathiravan2 and Uppor Krishnamoorthy2
1School of Veterinary Medicine, Department of Basic Veterinary Sciences, FMS, The University of the West Indies, St. Augustine Campus, Trinidad and Tobago (W.I.)
2Department of Food Production, Faculty of Food and Agriculture, The University of the West Indies, St. Augustine Campus, Trinidad and Tobago (W.I.)
3 Department of Biosciences and Agriculture and Food Technology, University of Trinidad and Tobago (UTT), Trinidad and Tobago (W.I.)
Krishna Mohan Kumar
Department of Basic Veterinary Sciences,
School of Veterinary Medicine, FMS, EWMSC,
The University of The West Indies, St. Augustine
Trinidad and Tobago (W.I)
E-mail: [email protected]; [email protected]
Copyright: This is an open-access article under the terms of the Creative Commons Attribution License which permits use, distribution, and reproduction in any medium, provided the original work is properly cited.
©2021 The Authors. Caribbean Medical Journal published by Trinidad & Tobago Medical Association.
This study aimed to evaluate the impact of the dietary supplement of Moringa oleifera leaves (MOL) on semen quality and characteristics in rabbits.
Eighteen (n=18) breeding bucks of New Zealand white, of similar age group, were used for the study. Three feeding regimes, (i) 100% commercial rabbit pellets (CRP)-Group I (ii) 90% CRP + 10% fresh MOL on a dry matter (DM) basis – Group II and (iii) 80% CRP + 20% fresh MOL on a DM basis – Group III, were adopted and the trial continued for 21 days. After adaptation to the diet, semen was collected from each buck and subjected to evaluation using a computer-assisted semen analyser.
In Group III, the sperm count, normal sperm morphology, and sperm motility increased (52.0%) in comparison with the control (Group I; 50.1%). The inclusion of 20% Moringa oliefera in the diet (Group III) caused a significant increase (P<0.05) in semen concentration (Control =136.2 M/mL; Group III=297.2 M/mL). There was no significant difference (P>0.05) in sperm motility and semen volume among the groups.
The results suggest that supplementing commercial rabbit pellets with 20% fresh Moringa oliefera leaves on a DM basis can improve the quality and characteristics of semen in breeding bucks.
Key Words: Moringa oliefera, Morphology, Motility, Rabbit Bucks, Semen,
Improvement of reproductive efficiency in the rabbit bucks is dependent on many factors such as feeding, genetic strain, health status, management condition, season, and age. These reasons are contributing to the variation in semen qualities.1, 2, 3 Moringa oleifera, generally called horse radish tree and used as a potential cheap protein source for livestock feeding.4 It is rich in carotene, ascorbic acid, vitamin B, calcium, potassium, selenium, zinc and iron.
The most used part of plants is the leaves, which are rich in vitamins, carotenoids, polyphenols, phenolic acids, flavonoids, alkaloids, glucosinolates, isothiocyanates, tannins and saponins. The plant survives best under the insular tropical climate. It can grow well in the humid tropics or hot, dry lands and can survive in less fertile soils, and it is also little affected by drought.5 Due to this fact, Moringa oleifera can be grown for inclusion into an animal’s diet. This plant has reportedly been extensively used in ethnoveterinary and traditional medicine. The leaves are well known for its antimicrobial effects.
With regards to reproduction, nutrition is one of the most important factors in sperm viability, and poor nutrition may result in semen with questionable quality. Improper environmental condition leads to decreases in semen quality and fertility of farm animals.6 Accumulation of free radicals has been associated with significant decreases in sperm motility and sperm plasma membrane integrity and significant increases in sperm abnormalities and DNA damage, leading to infertility.7
For practical, safe and economical applications, different natural feed resources have been used to counter negative impacts on semen quality of male rabbits.8, 9 Previous studies showed the ability of Moringa Oleifera ethanolic extract (MOEE) to enhance libido and sperm quality and to improve sexual activity and testosterone level in immobilisation in stressed male rats.10 As it relates to semen characteristics and its quality, it is very important that semen and its compositions remain of the best quality at all time, for good reproductive performance. Determination of the concentration in a semen sample is important because this parameter is used to determine the number of spermatozoa that will be used if the quality of the ejaculate is good enough for insemination.11
Sperm motility is definitely one of the most important features associated with semen fertilising ability. Sperm motility was the first and continues to be the most widely used indicator of sperm function.12 Sperm viability is also a key determinant of sperm quality and a prerequisite for successful fertilization. Besides visual evaluation, sperm concentration, motility morphology and volume can be evaluated using photographic analysis, or computer-assisted semen analysis (CASA) can be used.13, 14 The shortage of grain and the high cost of imported feed ingredients were also the major force to exploit other alternatives.
Therefore, this study aimed to investigate the level of inclusion of fresh Moringa oleifera leaves supplementation with commercial rabbit pellets and its impact on semen characteristics of rabbit buck under the tropical climate in Trinidad.
MATERIALS AND METHODS
The experiment was carried out using eighteen (n=18) sexually matured New Zealand white, California white, and Flemish Giant rabbit’s bucks weighing 3200 g- 3900 g. The animals were acquired from the rabbitry unit of the field station of the University of the West Indies, Trinidad and Tobago; and were placed in hutches with a size of 30″× 25″. Throughout the period of the experiment, bucks were exposed to subtropical conditions, where the average minimum temperature ranged from 21.0 to 23.0°C, and the average maximum temperature was 32.0 to 35.0°C. Good ventilation and fresh air were provided to minimize ammonia levels in the house, as well as hygienic elimination of waste.
Plant Collection and Preparation of Moringa oleifera leaves
Fresh leaves of Moringa oleifera were collected in the morning from the University field station farm, Mt. Hope, UWI. The plants were identified and fresh leaves were manually removed from the stem, cleaned and made free of sand and other impurities. The fresh leaves were harvested green a day before to feeding and air-dried under shade to reduce the moisture content.
Supplement of M. oleifera leaves in rabbit’s diet
All the rabbits were fed on commercial food pellets (Master Mix® Ltd.) and provided ad libitum drinking water during whole experimental periods. The diet composition on a dry matter basis contains 17.0% crude protein, 15% crude fibre, 2.5 % crude fat along with trace elements such as zinc, copper sulphate, niacin, trace of cobalt and many other elements.
The rabbits were divided into three groups of 6 rabbits each. The first group received 100% commercial food pellets (concentrate) and served as control (Group 1). The second group received a dosage of 90% concentrate and 10% (40g) fresh Moringa oleifera leaves (Group 2). The third group received 80% concentrate and 20% (80g) fresh Moringa oleifera leaves (Group 3). The bucks were fed with the respective diets for three weeks, following which semen was collected artificially after eight weeks of feeding.
Rabbit’s semen was collected from six sexually mature male rabbits in each group by using a specially constructed artificial vagina, which was filled with a warm liquid. A matured doe was placed in the mating pen with the buck to stimulate libido and to increase sperm concentration. After a couple of strokes done by the buck on the does, the penis was then guided into the artificial vagina for collection of semen. Immediately after collection, the ejaculates were transferred to the laboratory, and care was taken to avoid exposure of the semen to any unfavourable conditions during or after collection. A different artificial vagina was used for each collection and was collected under hygienic conditions to prevent bacterial and environmental contamination. The animals were ejaculated twice a week in the morning hours. Semen volume was read off the collection tube and recorded in millilitres.
The semen collection was carried out at Artificial Breeding Centre (ABC), Aripo, which is a government farm specially designed for the collection of animal semen for distribution via artificial insemination to the farming public.
Semen was evaluated for determination of semen volume, sperm motility, sperm morphology, sperm concentration, and live sperm count using Computer Assisted Semen Analyser (CASA), a computerised program called HT CASA II, Hamilton Thorone, USA.
This equipment gives a detailed evaluation of a semen sample immediately after collection. The first measure of evaluation done is the volume of the semen ejaculated. Semen was then diluted 1:5 in an extender using a commercial buffer solution. Before the buffer was used, it was placed in a water bath at a temperature of 30°c as to prevent shock. Semen was then aspirated from collecting tube using a micro pipette and then gently placed in a four- chambered slide for evaluation.
All data produced from the experiment were analysed using the linear models Statistical Package for Social Sciences (SPSS) software. The SPSS (version 22, IBM Corp; USA) was used for the statistical analysis in this study. Semen characteristics were analysed using one- way analysis of variance (ANOVA) tests for comparisons among means between the three treatments. Duncan is multiple range tests were used to locate points of significant differences between groups.
Results of semen quality assessment, total volume (mL), sperm concentration, motility (%), abnormality (%), and total sperm output (106/ ejaculate) are presented in Table 1. Significant differences (P<0.05) were observed on the effects due to inclusion levels of Moringa oleifera leaves (MOL) in rabbit diet on the sperm concentration and total sperm output. There were no significant differences (P>0.05) observed on the effects due to the inclusion levels of Moringa oleifera leaves on the other parameters like reaction time, semen volume, forward motility, live sperm and semen abnormalities and semen morphology. The results illustrated that there is no influence of treatment recorded in semen appearance and semen viscosity.
This research demonstrates that feeding of Moringa oleifera leaves at a level of 10% and 20% in commercial rabbit pellets in male rabbit (bucks) caused a significant increase in sperm qualities in the rabbit.
Semen concentration and total sperm output (TSO) were significantly (P<0.05) influenced by treatment. The highest sperm concentration and TSO (297.2×106/mL) were observed in Group 3, followed by Group 2 and control Group 1 (136.2×106/mL).
The effect of Moringa oleifera leaves on semen morphology is shown in Figure 1 and Table 1. The effect of MOL on semen morphology manifested in treatment groups 2 and 3 compared to group 1 (control). The result illustrated that MOL inclusion in the rabbit diet increased the normal morphology of rabbit sperm (Figure 1). It also shows that MOL actively reduced sperm abnormality like bent tail, coiled tail, DMR (distal mid-piece reflex), distal droplet, and proximal droplet, as observed in the difference between group 1 (control) and rabbit fed with MOL (group 2 and 3). Overall, the effect of MOL on sperm abnormalities (tail abnormalities, head abnormalities) manifested in treatment Groups 2 and 3, compared to Group 1 (control). Overall, the results illustrated that M. oleifera reduced the abnormality as observed in the difference between treatment Group 1 (control) and Group 2 and 3.
The results of sperm motility were also influence by dietary treatment. The forward motility was higher (89.9%) in rabbit fed with the highest level (20%; Group 3) of MOL followed by 89.4% (10%; Group 2) and 81.6% in control (Group 1). Also, the results show that there was an increase in live spermatozoa percent between rabbit fed varying levels of M. oleifera in treatment Group 2 (88.3%) and Group 3 (91.2%) when compared to those in the control group (76.0%).
Table 1: Effect of different levels of Moringa oleifera leaves on physical parameters of semen of rabbit bucks
|Parameters||Group 1||Group 2||Group 3|
|(Control)||(10% inclusion of MOL)||(20% inclusion of MOL)|
|Reaction time (s)||2.6||2.4||2.1|
|Sperm concentration (106/mL)||136.2a||221.0b||297.2b|
|Forward motility (%)||81.6||89.4||89.9|
|Live sperm (%)||76||88.3||91.2|
|Abnormal sperm (%)||6.4||5.2||5|
|Tail abnormality (%)||5.3||4.8||4.4|
|Head abnormality (%)||1.1||0.4||0.7|
|*Total sperm output (TSO) = semen ejaculate volume (mL) × semen concentration (106/mL)
a,b Means within a row with different superscript letters are significantly different (P<0.05).
MOL = Moringa oleifera leaves
Figure 1. Effect of Moringa oleifera supplement on abnormality of spermatozoa in rabbit’s semen
DMR = distal midpiece reflex
The present finding suggests that the supplement of Moringa oliefera leaves could be integrated at a 10 to 20% level without any a negative effect on reproductive efficiency and performance of rabbit bucks. EL Deeb Mariam et al has suggested that Moringa Oleifera leaves (MOL) could be integrated at 4% without negative effect for reproductive efficiency and performance of male rabbits intended for breeding purposes.3 Also, Ewoula et al stated that inclusion of MOL in rabbit’s diet up to 7.5% improved semen quality but also recommended that the inclusion level of 2.5% MOL can enhance reproductive efficiency in male rabbits.15 Meanwhile, Abu Ahemen et al also reported that using MOL at a 15 % level doesn’t cause any adverse effect on semen quality; these supplement ranges are in agreement with the current findings.16
In the present study, all semen quality parameters as; concentration, motility, live sperm count, and total sperm output (TSO) tend to follow an upward trend in bucks received 20% MOL compared to control animals. It was stated that the number of sperms and their motility are the most important parameters about fertility.17,18 The high nutritive value of MOL incorporated into buck’s diets possesses the potential to improve the reproductive efficiency of rabbit bucks and subsequently enhance fertility performance. In the present study, not many differences were recorded in the volume, appearance and viscosity due to either treatments.
In the current finding, the sperm concentration of rabbit bucks significantly (P <0.05) influenced by the experimental diet containing MOL at 20% level. The values of sperm concentration observed in this research ranged from 136.2 to 297.2 x106/ mL and were more or less similar to the recorded values of 136.00 to 184.00 x106/mL.19 An increased concentration of spermatozoa is a signal to a possible high fertility rate by the reason of the number of spermatozoa available during service or insemination.20 Oyeyemi et al also declared that quality nutrition with high percentage of protein will improve motility and concentration of spermatozoa and Moringa leaves is known to have high crude protein content.20
The percentage normal sperm cells in this research ranged from 93.6 to 95.0% which showed increased pattern at 20% inclusion level but were not significantly affected by experimental inclusions of MOL. The percentage normal sperm cells value was significantly (P<0.05) higher in T4 (90.50%) compared with T1 (84.67%), T2 (85.50%) and T3 (88.00%) of Moringa Oleifera leaves (MOLE) treatment, which supports our findings.21 The percentage of live sperm cells in this study range from 76.0 to 91.2% and were not significantly influenced by dietary supplement of Moringa oleifera leaves. Arthur et al discovered that high quality semen samples show an average of less than 25% dead sperm and the average value of percentage normal sperm cells reported in present study was within the range of high quality samples.22 The percentage live sperm cells are those present for use during fertilization.23 The percentage of abnormal sperm in present study ranged from 5.0 to 6.4 %. The value obtained were more or less within the range of 9.50 to 15.33% and 6.00 to 16.00%.19, 21 Whereas the percentage of abnormal sperm cells in the present finding were lower than the upper limit of 20% abnormality20, which suggests that the semen could be recommended for good reproductive potential and fertility in either normal mating or in artificial insemination. On the other side, this improvement in buck reproductive performance may be due to the fact that Moringa oleifera leaves are valuable in minerals such as Fe, vitamins A, B complex, C, K and E that are of importance in enhancing semen quality and reaction time.24, 25, 26 In another study, D’cruz and Mathur proved that the sperm cytoplasm contained very low concentrations of scavenging enzymes therefore causing an increase in the antioxidant enzyme system levels by MOL treatment can favour the reproductive process and also enhances spermatogenesis.27
Motility is the movement of spermatozoa in the semen medium during reproduction and it is a good indicator of viability. Motility basically estimates the proportion of active spermatozoa in the semen; fifty percent (50%) or more of the sperm should be moving. In the current study the sperm motility was influenced by the dietary treatment with MOL. However, motility was found to be higher in rabbit fed with the highest level (20%) of Moringa oleifera. George et al also observed a similar finding and reported that significant improvement in sperm motility (90.7%) was noticed in rabbit fed with the highest level (30 g) of Moringa oleifera.28 This is in agreement with our results. However, Abu et al has observed that Moringa oleifera leaf meal did not significantly influence sperm active motility, which is not in agreement with our finding.16
Our results shows that appearance of the rabbit semen and viscosity was not influenced by the treatments, as semen usually exhibit milky and homogeneous stickiness. Herbert also reported that the appearance of semen is a part of important characteristic of quality. Good quality semen should have a uniformly milky appearance which gives the indication of high sperm concentration.29 Semen viscosity is the liquid flow of semen. After ejaculation, it should take 30 minutes to an hour for semen to liquefy from its gel-like state. High semen viscosity causes low sperm mobility, if the sperm does not liquefy or remains very thick, fertilization maybe difficult due to the sperm not being able to reach an egg. Our results also shows viscosity of the rabbit semen was not influenced by either of the treatments.
The present results confirm that Moringa oleifera leaf meal produces positive effects on semen characteristics of rabbits. It also lends support to the claims for traditional usage of Moringa oleifera as a sexual function enhancing medicine. This may also point to the fact that Moringa oleifera leaf meal are relatively safe and also serve as a supportive treatment in the nutritional management to improve semen production of rabbit bucks, and consequently increasing reproductive performance of rabbit does mated by this semen. Thus, Moringa oleifera may prove to be an effective and safe alternative locally available feed to improve the reproductive efficiency. Therefore, Moringa oleifera leaf meal can be included in rabbits’ diets up to 20% without any detrimental effect on the performance.
Ethical Approval statement: Ethics approval was granted by the committee of the Faculty of Food and Agriculture, University of the West Indies, St. Augustine.
Conflict of interest: The authors declare no conflict of interest.
Informed Consent statement: Not Applicable
Funding statement: Campus Research and Publication Fund Committee, The University of The West Indies, St. Augustine.
Author contributions: All the authors have contributed in research work, data interpretation and drafting of manuscript. Krishna Mohan, Role: Writing- original draft, editing, supervision and contributed to research work. Devlon Brathwaite, Role: Sample collection, conducting animal trial, and writing and contributed equally to this work. Nitu Kumar, Role: Editing, graphical presentation and data analysis, and contributed equally to this work. Reeza Ramgattie, Role: Sample collection and laboratory analysis of samples and contributed equally to this work. Gopalan Kathiravan, Role: Data analysis, graphical presentation and contributed equally to this work. Uppor Krishnamoorthy, Role: Co- supervisor, writing, editing and contributed equally to this work.
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- Castellini C. Semen Production and Management of Rabbit bucks. In Proc. 9th World Rabbit Congress, June 2008, Verona, Italy, 265-277.
- EL Deeb Mariam A, Afifi OS, Mahmoud HA, Refay MS. Effect of Nutritional and Functional Properties of Moringa Oleifera Leaves (MOL) on: I. Semen Quality and Offspring Performance of New Zealand White (NZW) Bucks. Assiut J. Agric. Sci. 2015; 46 (2): 120-134.
- Sarwatt SV, Kapange SS, Kakengi AMV. Substituting sunflower seed-cake with Moringa oleiferaleaves as a supplemental goat feed in Tanzania. Agroforestry Systems. 2002; 56 (3): 241-247.
- Anwar F, Sajid L, Muhammad A, Anwarul HG. Moringa oleifera: A Food plant with Multiple Medicinal Uses. Res. 2007; 21: 17- 25.
- Rasooli A, Taha Jalali M, Nouri M, Mohammadian B, Barati F. Effects of chronic heat stress on testicular structures, serum testosterone and cortisol concentrations in developing lambs. Animal Reproduction Science 2010; 117(1-2): 55-59.
- Potts RJ, Notarianni LJ, Jefferies TM. Seminal plasma reduces exogenous oxidative damage to human sperm, determined by the measurement of DNA strand breaks and lipid peroxidation. Mutat Res. 2000; 447:249– 256.
- Elnagar Royal jelly counteracts bucks’ ‘summer infertility’. Animal Reproduction Science. 2010; 121: 74–80.
- HashemNM, AbdEl-Hady A, Hassan Effect of vitamin E or propolis supplementation on semen quality, oxidative status and hemato-biochemical changes of rabbit bucks during hot season. Livestock Science. 2013; 157: 520–526.
- PrabsattrooT, Wattanathorn J, Iamsaard S, Somsap P, Sritragool O, Thukhummee W, Muchimapura Moringa oleifera extract enhances sexual performance in stressed rats. Journal of Zheijang University Science B. 2015; 16: 179–190.
- Di-Iorio M, Marchisi A, Rocco M, Chrenek P, Iaffaldano Comparison of different extenders on the preservability of rabbit semen stored at 5 °C for 72 hours. Ital J Anim Sci. 2014; 13:710–714.
- Partyka A, Niżański W, Ochota M. Methods of assessment of cryopreserved semen. In: Katkov II (ed) Current Frontiers in Cryobiology 2012; InTech Open Access Publisher, Rijeka, Croatia, pp 547-574.
- Verstegen J, Iguer-ouada M, Onclin K. Computer assisted semen analyzers in andrology research and veterinary practice. Theriogenology 2002; 57:149–179.
- Rijsselaere T, van Soom A, Maes D, de Kruif A. Effect of technical settings on canine semen motility parameters measured by the Hamilton-Thorne analyzer, Theriogenology 2003; 60:1553–1568.
- Ewuola E, Adeyemi A, Tewe O. Reproductive response of rabbit bucks to supplemental Moringa oleifera leaf meal. In “Bridging the gap between increasing knowledge and decreasing resources” Tropentag 2014; Prague, Czech Republic. Abs.
- Abu AH, Ahemen T, Ikpechukwu P. Testicular Morphometry and Sperm Quality of Rabbit Bucks Fed Graded Levels of Moringa Oleifera Leaf Meal (MOLM). African Journal Online 2013; 13 (1): 49-56.
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- Ojo OA, Abdurahman KO. Effect of Moringa Oleifera Leaf Extract (Mole) on some Reproductive Parameters of Rabbits Reared in a Semi-Humid Environment. Global J Sci Front Res: D Agriculture and Veterinary 2017; 17: (4) 7-12.
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- Ajala OO, Oyeyemi MO, Akusu MO, Eimunjeze HE. The effects of scrotal insulation on the testicles and spermatozoa characteristics of WAD goats. Sokoto J Vet Sci 2001; 3 (1): 44-50.
- Odeyinka SM, Oyedele OJ, Adeleke TO. Odedire JA. Productive performance of rabbits fed Moringa oleifera as a replacement for centrosema pubescens. 9th World Rabbit Congress – 2008, June 10-13– Verona – Italy, pp: 411-415
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- D’cruz SC, Mathur PP. Effect of piperine on the epididymis of adult male rats. Asian J. Androl. 2005; 7(4): 363-368.
- George OS, Ologbose FI. Akintola OAI. Sperm Characteristics of Rabbit Bucks Fed Graded Levels of Moringa (Moringa Oleifera) Leaf Meal. Agri. 2017; 20 (3): 67-70.
- Herbert U. Growth and reproductive characteristics of rabbits fed on Leucaena (Leucaena leucocephala) and (Gliricidia sepium) foliage. 1992. PhD. Thesis. Genous oxidative damage to human sperm, determined by the measurement of DNA strand breaks and lipid peroxidation. Mutation Research 44, 249–256.