Effect of Moringa oleifera seed extract on antioxidant activity and sperm characteristics in cryopreserved ram semen

Figures & data

Figure 1. Scheme summarizing the methodology used to assess the effects of Moringa oleifera seed extract supplementation on the antioxidant activity and spermatic characteristics of cryopreserved ram semen. M. oleifera: Moringa oleifera, mg/mL: milligrams per millilitre, spz/mL: spermatozoa per millilitre, mL: millilitre, °C: Celsius degrees, s: seconds, CASA: computer assisted semen analysis, PI: propidium iodide, FITC-PNA: fluorescein isothiocyanate-coupled peanut agglutinin, FRAP: ferric reducing antioxidant power.

Table 1. Settings of the CASA system* used to evaluate ram spermatozoa.

Variable	Setting
Resolution horizontal/vertical	1024 pixels × 1024 pixels
Pixel size horizontal/ vertical	5.5 μm × 5.5 μm
Frame rate	Up to 101 frames per second
Area	10–100
Tail detection	5 μm
Camera depth	20 μm
Pixel relation	0.54 μm
Reference volume
Width	555.12 μm
High	555.12 μm
In-motile	ALH < 4.00 μm and BCF < 4.00 μm
Motility
Local motility	VCL < 20.00 μm s^−1 and VSL < 10.00 μm s^−1
Progressive motility
Circle motility	Radius > 10.00 μm s^−1 to < 80.00 μm s^−1 and rotation > 0.70 μm s^−1
Slow motility	VCL < 80.00 μm s^−1
Fast motility	VCL > 80.00 μm s^−1

*AndroVision, Minitube, Germany.

ALH, Amplitude of lateral head displacement; BCF, Beat cross frequency; VCL, Curvilinear velocity; VSL, Straight-line velocity.

Table 2. Total polyphenolics and flavonoids in Moringa oleifera seed extract.

	Polyphenolics (mg GAE/g)	Flavonoids (mg CE/g)
	3.11 ± 0.11	2.43 ± 0.11

mg GAE/g: milligrams of gallic acid equivalents per gram, mg CE/g: milligrams of catechin equivalents per gram. Data are presented as mean ± SEM.

Figure 2. Antioxidant activity in ram semen following cryopreservation. FRAP: ferric reducing antioxidant power, mmol TE: millimoles of Trolox equivalents, Control: control group, M0.5: 0.5 mg/mL of Moringa oleifera seed extract, M5: 5 mg/mL of M. oleifera seed extract, M10: 10 mg/mL of M. oleifera seed extract. Data are presented as mean ± SEM. ^a,b Different literals across bars indicate significant differences (P < 0.05).

Figure 3. Sperm membrane damage, acrosome damage, and mitochondrial activity in ram semen following cryopreservation. Control: control group, M0.5: 0.5 mg/mL of Moringa oleifera seed extract, M5: 5 mg/mL of M. oleifera seed extract, M10: 10 mg/mL of M. oleifera seed extract. Data are presented as mean ± SEM. ^a,b Different literals across bars of the same colour indicate significant differences (P < 0.05).

Table 3. Quality of cryopreserved ram semen supplemented with Moringa oleifera extract at 0.5, 5.0, and 10.0 mg per mL.

	Viability (%)	Progressive motility (%)	Fast motility (%)	Slow motility (%)
Control	41.44 ± 2.17^b	35.84 ± 2.15^b	7.75 ± 0.92^a	28.06 ± 1.40^a
M0.5	51.33 ± 3.31^a	45.61 ± 3.47^a	11.69 ± 1.40^a	33.89 ± 2.16^a
M5	48.76 ± 3.77^ab	43.31 ± 3.82^ab	11.16 ± 1.79^a	32.08 ± 2.16^a
M10	44.71 ± 2.96^ab	38.71 ± 2.88^ab	8.81 ± 0.83^a	29.90 ± 2.10^a

M0.5: 0.5 mg/mL of Moringa oleifera seed extract, M5: 5.0 mg/mL of M. oleifera seed extract, M10: 10.0 mg/mL of M. oleifera seed extract. Data are presented as mean ± SEM. ^a,b Different literals within columns indicate significant differences (P < 0.05).

Figure 4. Improvement of cryopreserved ram semen quality (in vitro analysis) upon supplementation of Moringa oleifera seed extract prior to freezing.