The effect of extenders and removal of seminal plasma on semen of South African

34 with Bioxcell^® (37.9±28.6) extender than the semen extended with Triladyl^® (59.1±33.1) extender.

Table 4:4 Effect of extenders (Bioxcell^® & Triladyl^®) and seminal plasma (non-washed &

washed) on equilibrated spermatozoa acrosome, chromatin and mitochondrial membrane potential (mean ± S.E.)

Treatment Normal acrosome (%)

Reacted acrosome (%)

Normal chromatin (%)

Reacted chromatin (%)

Δψm^high (%)

Δψm^low (%)

Raw NW 62.8±13.7^a 37.2±13.7^a 82.2±16.5^a 17.8±16.5^d 83.4±14.2^a 16.6±14.2^c Biox NW 2h 71.1±13.8^a 28.9±13.8^a 79.8±21.9^ab 20.2±21.9^cd 39.5±23.2^c 60.5±23.2^a Tril NW 2h 60.1±15.9^a 40.3±16.0^a 67.5±24.4^abc 32.5±24.4^bcd 51.2±31.4^bc 48.8±31.4^ab Raw W 68.4±19.0^a 31.6±19.0^a 65.1±17.5^bc 34.9±17.5^bc 65.0±20.8^ab 35.0±20.8^bc Biox W 2h 71.3±22.7^a 28.7±22.7^a 56.8±13.6^cd 43.2±13.6^ab 37.9±28.6^c 62.1±28.6^a Tril W 2h 70.1±13.4^a 29.9±13.4^a 46.3±9.4^d 53.7±9.4^a 59.1±33.1^bc 40.9±33.1^ab Different superscripts (a, b, c and d) within the same column indicates significant differences among means (P < 0.05)

Raw NW = raw non-washed, Biox NW 2 h = Bioxcell^® non-washed 2 hours, Tril NW 2 h = Triladyl^® non- washed 2 hours, Raw W = raw washed, Biox W 2 h = Bioxcell^® washed 2 hours, Tril W 2 h = Triladyl^® washed 2 hours, Δψm^high = High mitochondrial membrane potential, Δψm^low = Low mitochondrial membrane potential.

4.2 The effect of extenders and removal of seminal plasma on semen of South

35 Table 4.5: Effect of extenders (Bioxcell^® & Triladyl^®) and seminal plasma (non-washed &

washed) on frozen-thawed spermatozoa motility parameters (mean ± S.E.)

Treatment TM (%) PM (%) NPM (%) RAP (%) MED (%) SLW (%) STC (%) Raw NW 98.9±1.2^a 92.9±4.7^a 6.0±3.7^d 91.6±5.6^a 6.3±4.0^c 1.0±0.7^c 1.1±1.1^d Biox NW FT 85.0±3.4^b 58.5±10.0^b 26.6±8.5^ab 49.9±11.5^b 27.5±7.2^a 7.6±3.2^b 15.0±3.4^c Tril NW FT 73.9±13.8^c 45.4±11.2^c 28.5±5.7^a 40.1±11.0^bc 23.9±6.1^a 9.9±1.4^a 26.1±13.8^b Raw w 98.3±2.7^a 91.4±7.9^a 6.8±5.3^d 89.7±9.0^a 7.1±4.6^c 1.4±1.8^c 1.8±2.7^d Biox W FT 68.2±13.5^cd 47.0±15.7^c 21.1±5.8^c 42.1±16.0^bc 17.8±5.3^b 8.2±2.7^ab 31.9±13.5^ab Tril W FT 63.1±15.1^d 39.9±13.1^c 23.2±3.2^bc 36.1±12.4^c 18.1±4.3^b 8.9±1.4^ab 36.9±15.1^a Different superscripts (a, b, c and d) within the same column indicate significant differences among means (P

< 0.05)

Raw NW = raw non-washed, Biox NW FT = Bioxcell^® non-washed frozen-thawed, Tril NW FT = Triladyl^® non- washed frozen-thawed, Raw W = raw washed, Biox W FT = Bioxcell^® washed frozen-thawed, Tril W FT = Triladyl^® washed frozen-thawed, TM = total motility, PM = progressive motility, NPM = non-progressive motility, RAP= rapid, MED = medium, SLW = slow, STC = static.

The Spermatozoa VCL was not significantly different (P > 0.05) in non-washed semen extended with Bioxcell^® (50.2±11.9) from the semen extended with Triladyl^® (53.0±8.6) extender (Table 4.6). The Spermatozoa VSL was not significantly different (P

> 0.05) in washed semen extended with Bioxcell^® (33.7±12.7) from the semen extended with Triladyl^® (25.8±5.0) extender. The Spermatozoa VAP was not significantly different (P > 0.05) in non-washed semen extended with Bioxcell^® (33.3±12.7) from the semen extended with Triladyl^® (32.8±7.2) extender. The Spermatozoa LIN was not significantly different (P > 0.05) in washed semen extended with Bioxcell^® (57.7±16.5) from the semen extended with Triladyl^® (48.1±8.6) extender.

Table 4.6: Effect of extenders (Bioxcell^® & Triladyl^®) and seminal plasma (non-washed &

washed) on frozen-thawed spermatozoa kinematic parameters (mean ± S.E.) Treatment VCL

(µm/sec)

VSL (µm/sec)

VAP (µm/sec)

LIN (%)

STR (%)

WOB (%)

ALH (µm)

BCF (Hz) Raw NW 96.2±7.8^a 47.2±6.5^a 69.7±5.3^a 49.5±8.0^abc 67.6±5.8^b 72.7±5.8^ab 2.9±0.4^a 7.3±0.4^ab Biox NW FT 50.2±11.9^b 24.1±12.1^c 33.3±12.7^c 46.1±13.8^bc 70.0±9.4^b 64.6±10.9^c 2.1±0.3^d 6.1±1.3^bc Tril NW FT 53.0±8.6^b 23.3±6.7^c 32.8±7.2^c 41.2±6.2^c 66.9±6.1^b 61.3±3.9^c 2.5±0.1^bc 7.1±2.2^ab Raw w 95.6±3.7^a 53.3±10.8^a 73.8±7.8^a 55.9±11.5^ab 71.6±7.9^ab 77.3±8.0^a 2.7±0.4^ab 7.4±0.2^ab Biox W FT 57.7±11.9^b 33.7±12.7^b 43.8±12.2^b 57.7±16.5^a 75.3±11.3^a 75.1±12.2^ab 2.1±0.3^d 5.9±0.9^c Tril W FT 53.8±4.8^b 25.8±5.0^bc 36.4±4.6^bc 48.1±8.6^abc 70.5±7.1^ab 67.6±6.6^bc 2.3±0.2^cd 7.6±2.3^a

Different superscripts (a, b, c and d) within the same column indicate significant differences among means (P

< 0.05)

36

Raw NW = raw non-washed, Biox NW FT = Bioxcell^® non-washed frozen-thawed, Tril NW FT = Triladyl^® non- washed frozen-thawed, Raw W = raw washed, Biox W FT = Bioxcell^® washed frozen-thawed, Tril W FT = Triladyl^® washed frozen-thawed, VCL = velocity on the curve line, VSL = velocity on the straight line, VAP = average path velocity, LIN = linearity, STR = straightness, WOB = wobble, ALH = amplitude of lateral head displacement, BCF = beat cross frequency.

There was a higher (P < 0.05) live and normal spermatozoa percentage in non- washed semen extended with Bioxcell^® (45.7±21.2) extender than the semen extended with Triladyl^® (24.5±22.2) extender (Table 4.7). There were a higher (P < 0.05) percentage of spermatozoa with head abnormalities in non-washed semen extended with Triladyl^® (20.4±10.2) extender than the semen extended with Bioxcell^® (18.3±12.4) extender. There were a higher (P < 0.05) percentage of spermatozoa with head abnormalities in washed semen samples extended with Triladyl^® (34.0±16.0) extender than the semen extended with Bioxcell^® (10.1±7.0) extender. The percentage of spermatozoa with coiled tail abnormalities were reduced significantly (P < 0.05) in washed semen extended with Triladyl^® (35.9±21.6) extender compared to the semen extended with Bioxcell^® (65.4±25.0) extender.

Table 4.7: Effect of extenders (Bioxcell^® & Triladyl^®) and seminal plasma (non-washed &

washed) on frozen-thawed spermatozoa viability and morphology parameters (mean ± S.E.)

Treatment Viability Head abnormal

(%)

Bent mid-piece (%)

Coiled tail (%)

Live & normal (%)

Dead (%)

Raw NW 71.7±12.3^a 28.3±12.3^d 14.1±16.4^bc 23.6±11.9^c 62.3±23.2^a Biox NW FT 45.7±21.2^b 54.3±27.2^c 18.3±12.4^bc 30.9±15.5^abc 50.9±15.5^ab Tril NW FT 24.5±22.2^c 74.5±22.2^b 20.4±10.2^b 41.1±18.0^ab 38.5±16.1^b Raw w 60.7±22.9^ab 39.3±22.9^cd 8.2±10.7^c 43.8±12.1^a 48.0±12.5^ab Biox W FT 5.2±4.9^d 94.8±4.9^a 10.1±7.0^bc 25.5±20.4^bc 65.4±25.0^a Tril W FT 6.9±8.6^d 93.1±8.6^a 34.0±16.0^a 28.0±25.1^abc 35.9±21.6^b Different superscripts (a, b, c and d) within the same column indicate significant differences among means (P

< 0.05)

Raw NW = raw non-washed, Biox NW FT = Bioxcell^® non-washed frozen-thawed, Tril NW FT = Triladyl^® non- washed frozen-thawed, Raw W = raw washed, Biox W FT = Bioxcell^® washed frozen-thawed, Tril W FT = Triladyl^® washed frozen-thawed.

The spermatozoa acrosome integrity was not significantly different (P > 0.05) in non-washed semen extended with Bioxcell^® (50.4±23.5) extender and semen extended with Triladyl^® (55.6±22.1) extender (Table 4.8). The spermatozoa chromatin integrity was

37 not significantly different (P > 0.05) in washed semen extended with Bioxcell^® (48.7±21.3) extender and semen extended with Triladyl^® (37.9±24.3) extender. There was a lower percentage (P < 0.05) of spermatozoa with high mitochondrial membrane potential in non-washed semen extended with Bioxcell^® (49.8±20.1) extender than the semen extended with Triladyl^® (68.7±26.8) extender.

Table 4.8: Effect of extenders (Bioxcell^® & Triladyl^®) and seminal plasma (non-washed &

washed) on frozen-thawed spermatozoa acrosome, chromatin, and mitochondrial membrane integrity (mean ± S.E.)

Treatment Normal acrosome (%)

Reacted acrosome (%)

Normal chromatin (%)

Reacted chromatin (%)

Δψm^high (%) Δψm^low (%)

Raw NW 62.8±13.7^ab 37.2±13.7^ab 79.9±15.6^a 20.1±15.6^c 83.4±14.2^a 16.6±14.2^c Biox NW FT 50.4±23.5^b 51.7±23.5^a 63.7±9.1^ab 36.3±9.1^bc 49.8±20.1^c 50.2±20.1^a Tril NW FT 55.6±22.1^ab 44.4±22.1^ab 51.2±18.7^bc 48.8±18.7^ab 68.7±26.8^ab 31.3±26.8^bc Raw w 68.4±19.0^a 31.6±19.0^b 65.1±17.5^ab 34.9±17.5^cb 65.0±20.8^cb 35.0±20.8^ab Biox W FT 65.4±11.4^ab 34.6±11.4^b 48.7±21.3^bc 51.3±21.3^ab 65.3±14.9^cb 34.7±14.9^ab Tril W FT 65.5±15.4^ab 34.5±15.4^b 37.9±24.3^c 62.1±24.3^a 78.4±16.8^ab 21.6±16.8^bc Different superscripts (a, b and c) within the same column indicate significant differences among means (P <

0.05)

Raw NW = raw non-washed, Biox NW FT = Bioxcell^® non-washed frozen-thawed, Tril NW FT = Triladyl^® non- washed frozen-thawed, Raw W = raw washed, Biox W FT = Bioxcell^® washed frozen-thawed, Tril W FT = Triladyl^® washed frozen-thawed, Δψm^high = High mitochondrial membrane potential, Δψm^low = Low mitochondrial membrane potential.

38