| Livestock Research for Rural Development 32 (7) 2020 | LRRD Search | LRRD Misssion | Guide for preparation of papers | LRRD Newsletter | Citation of this paper |
This study aims to investigate pineapple juice as natural cock semen diluent owing to its availability and affordability in the rural farming community. Ripe pineapple fruit was processed into juice and included in dextrose saline at 0% (P0), 10% (P1), 20% (P2), 30% (P3), 40% (P4), 50% (P5) as semen diluent compared to undiluted semen as control (C). Semen pool for 30 adult roosters was allotted randomly in triplicates to the different pineapple-dextrose at 1:2 dilution rate. The diluted semen evaluated for sperm kinetics using computer-assisted sperm analyser and seminal plasma were assayed for lipid peroxidation and total antioxidant activity. The result showed that individual motility indices of semen diluted with 30-50% pineapple inclusion (P3, P4 and P5) were better (p<0.05) than undiluted semen (C). Spermatozoa liveability and kinetics properties of diluted semen compared favourably with the control. Pineapple inclusive diluents had higher antioxidant activity and reduction in lipid peroxidation up to 3 hours compared to other semen groups (C and P0 compared to P1 to P5). In conclusion, pineapple juice-dextrose is a potent cock semen diluent that sustains sperm cell mobility by reducing lipid peroxidation due to its antioxidant capacity.
Keywords: antioxidant activity, artificial insemination, lipid peroxidation, avian reproduction
Recent advances in avian reproduction have focused on the potential of reactive oxygen species (ROS) and its role as prime mediators of infertility. Physiologically, ROS are involved in many functions of spermatozoa, their excessive production may result in oxidative stress (Khan 2011). Spermatozoa membranes are vulnerable to free radical-induced damage because they are not only rich in polyunsaturated fatty acids, but also contains a low concentration of antioxidant enzymes (Zhong and Zhou 2013). During semen collection and processing, semen exposed to atmospheric oxygen, which in turn increases the susceptibility to lipid peroxidation (Bucak et al 2011). Many natural plants and their seed, leaf, or root extracts which are rich in polyphenols, flavonoids, carotenes, gallic acid, tannins, and essential oil as antioxidants are recognised to be better than synthetic antioxidants due to lower cytotoxicity and residue (Zhong and Zhou 2013). Fruits are sources of natural antioxidants, comprising many different antioxidant components (Jimoh 2020) such as pineapple. These antioxidants include carotenoids, vitamins, phenolic compounds and flavonoids and have proved to function as singlet and triplet oxygen quenchers, free radical scavengers and peroxide decomposers (Anghel et al 2010). Pineapple juice like other natural fruit juice is available and affordable in farming household and can be an economic antioxidant supplement for semen processing. This study aims to investigate the oxidative stability and spermatozoa kinetics of cock semen diluted in pineapple juice based diluent.
30 breeder cocks of 35- 40 weeks of age were obtained from a reputable breeder farm and were used for this study. All cocks were assessed for fertility, and only fertile cocks were used. Ripe pineapple fruit was purchased, washed, peeled and the fruit pulp blended. The fruit juice was clarified with a juice extractor (Mikachi model No 1706). The juice obtained was designated as pineapple juice and kept frozen (-4 °C) in disposable 5 ml sterile bottles until required for use.
The 30 cocks were trained for 2 weeks, and semen were harvested twice a week and ejaculate taken to the laboratory for in-vitro analysis. Care was taken to avoid any contamination of semen with faeces.
The experimental design was completely randomised, consisting of seven treatments and three replicate. The 7 treatments consisted of different diluents:
undiluted semen as control (C),
semen diluted in dextrose saline + 0% Pineapple juice (P0),
semen diluted in dextrose saline + 10% Pineapple juice (P1),
semen diluted in dextrose saline + 20% Pineapple juice (P2)
semen diluted in dextrose saline + 30% Pineapple juice (P3),
semen diluted in dextrose saline + 40% Pineapple juice (P4),
semen diluted in dextrose saline + 50% Pineapple juice (P5).
All diluents were of similar volume.
The pooled semen of the 30 cocks were allotted randomly to treatments in triplicates and diluted at 1:2 (semen: diluents), respectively. Diluted samples were gently mixed to allow equilibration, and semen assessment took place immediately. Semen qualitative and oxidative assay were assessed for each treatment.
Extended semen, according to treatments, were evaluated for sperm kinetics using computer-assisted sperm analyser (Sperm Analyze Win 7) and livability using convention procedures. Livability was done by placing a drop of semen on a glass slide, one drop of eosin–nigrosin stain was added and mixed gently, then smeared on a slide, air-dried and viewed under the microscope at a magnification of x400. For the seminal oxidative assay, the respective diluted semen was prepared according to their treatments. The various treatment samples constituted and stored at room temperature for three hours. Triplicate samples were taken at 0 hours and 3 hours, centrifuged at 4000 rpm for 15 min to separate seminal plasma. The seminal plasma was assayed for lipid peroxidation and total antioxidant activity as outlined in Jimoh and Ewuola (2018).
The data obtained were subjected to descriptive statistics and one-way analysis of variance at P = 0.05. Differences in mean value were separated using New Duncan's multiple range test.
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Table 1. Cock semen kinetics in pineapple-dextrose saline diluent |
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| Parameters | C | P0 | P1 | P2 | P3 | P4 | P5 | SEM | p value |
|---|---|---|---|---|---|---|---|---|---|
|
Percentage motility (%) |
91.6a |
88.9a |
83.0b |
91.3a |
89.8a |
91.1a |
90.9a |
1.10 |
0.40 |
|
Progressive motility (%) |
77.0b |
84.1a |
77.0b |
78.1b |
82.0a |
84.1a |
83.2a |
1.23 |
0.26 |
|
Non- progressive motility (%) |
14.6a |
4.8c |
6.0b |
13.2a |
7.8b |
7.0b |
7.7b |
1.14 |
0.32 |
|
Curvilinear velocity (VCL) (μm/s) |
19.5a |
17.0b |
5.1c |
18.7ab |
18.4ab |
18.1ab |
18.1ab |
1.06 |
0.00 |
|
Average path velocity (VAP) (μm/s) |
11.2a |
9.1a |
4.8b |
10.9a |
10.0a |
9.8a |
9.9a |
0.49 |
0.00 |
|
Straight-line velocity (VSL) (μm/s) |
4.95a |
3.73a |
3.42b |
4.68a |
4.16a |
4.17a |
4.18a |
0.16 |
0.17 |
|
Linearity (%) |
25.2b |
21.9c |
67.0a |
25.1b |
22.6c |
23.1c |
23.1c |
3.42 |
0.00 |
|
Straightness (%) |
44.1b |
41.1b |
73.2a |
42.9b |
41.8b |
42.5b |
42.1b |
2.44 |
0.00 |
|
Amplitude of lateral head (ALH) (μm) |
0.51ab |
0.48b |
0.21c |
0.54a |
0.49ab |
0.48b |
0.50ab |
0.02 |
0.00 |
|
Beat cross frequency (BCF) (Hz) |
4.44ab |
4.31b |
0.37c |
4.27b |
4.66a |
4.55ab |
4.55ab |
0.32 |
0.00 |
|
Wobble (%) |
56.9bc |
53.4c |
91.8a |
58.5b |
54.0bc |
54.3bc |
54.9bc |
2.91 |
0.00 |
|
Liveability (%) |
92.1 |
94.6 |
90.5 |
87.7 |
85.7 |
91.2 |
92.3 |
1.10 |
0.53 |
|
abc: means in the same row with different superscripts are different ar p<0.05 |
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The semen kinetics of cock semen diluted in pineapple-dextrose saline diluent is shown in Table 1. The sperm motility was significantly (p>0.05) similar across the various diluent except for P1. The individual motility indices (progressive and non-progressive motility) of semen diluted with 30-50% pineapple inclusion were (p<0.05) better than the control (C). Other semen kinetics properties compared favourably with the control. The liveability of sperm cells was not affected by the diluents as similar statistical values across the treatments.
|
|
| Figure 1. Lipid peroxidation of cock semen in Pineapple-dextrose based diluent |
The rate of lipid peroxidation in cock semen diluted with pineapple juice is shown in Figure 1. The pineapple inclusive semen diluents had marked reduction in lipid peroxidation between 0 and 3 hours in comparison with C and P0. The rate of lipid peroxidation in undiluted semen and 0% pineapple juice-dextrose diluted semen increased from 0 hours to 3 hours.
 |
| Figure 2. Total antioxidant activity of cock semen in Pineapple-dextrose based diluent |
The total antioxidant activity of cock semen in pineapple dextrose based diluent is shown in Figure 2. At 0 hour, undiluted semen had comparably high total antioxidant activity and declined rapidly at 3 hours. However, pineapple inclusive diluents had higher antioxidant activity up to 3 hours compared to the positive (C) and negative controls (P0).
The individual motility indices (progressive and non-progressive motility) of semen diluted with 30-50% pineapple inclusion were better than the control (P1). This result indicates the potential of pineapple-dextrose saline to maintain viability and kinetics of spermatozoa. The inclusion of pineapple juice at 30-50% produced better spermatozoa kinetics than the control. This result could be due to the antioxidants present in pineapple that reduce free radical and reactive oxygen species accumulation, thereby inhibiting lipid peroxidation. The reduction in spermatozoa friction occasioned by the dilution, which increases surface area for sperm cell motility could also account for the results obtained.
Fruits contain phytoconstituents with an array of antioxidant potency which confers fruit juices as good sources of antioxidants for semen preservation. The reduction in lipid peroxidation between zero and 3 hours in pineapple inclusive semen diluents in comparison with the increased rate of lipid peroxidation in undiluted semen and 0% pineapple juice-dextrose diluted semen shows the potential of pineapple inclusion. This result shows the beneficial ability of pineapple to reduce lipid peroxidation in cock semen. It also emphasises the need to control lipid peroxide accumulation in cock semen. At 0 hour, undiluted semen had comparably high total antioxidant activity, which indicates the antioxidant contribution of the accessory gland section in the seminal fluid, but it rapidly declined at 3 hours. The rate of lipid peroxidation and antioxidant activity of undiluted semen, suggests its inadequacy to meet the demand for free radical and reactive oxygen species scavenging. The highest antioxidant activity in pineapple juice-dextrose diluents was obtained at 30 (P3) and 50% (P5) inclusion. This, coupled with other results, shows that pineapple juice due to its antioxidant enrichment enhanced antioxidant activity and semen kinetics of cock semen by reducing the rate of lipid peroxidation. The result of this study agrees with the suggestion of Adekunle et al (2018) that flavonoids, ferulic acid and vitamins present in fruit juices acted synergistically to protect sperm cells from lipid peroxidation and accounts for improvements in semen parameters.
The research outcome presented in this article was funded by the Tetfund 2018 institution-based research intervention of the Nigeria government.
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Received 1 May 2020; Accepted 20 May 2020; Published 1 July 2020