1. CRYOSURVIVAL AND ENZYME ACTIVITIES OF
ROOSTER SEMEN DILUTED WITH TRIS EGG-YOLK
EXTENDER FORTIFIED WITH JUICES AND MIXED
CRYOPROTECTANTS
Balogun A.S.1,3, Narang. R1., Cheema R. S2, Shakti kant Dash1, and
kashyap Neeraj1
1Department of Animal genetics and Breeding, College of Veterinary Science, Guru
Angad Dev Veterinary and Animal Science University, Punjab, India.
2School of Animal Biotechnology, Guru Angad Dev Veterinary and Animal Science
University, Punjab, India.
3Directorate of livestock farms, Guru Angad Dev Veterinary and Animal Science
University, Punjab, India
2. Sperm Banking of Poultry Genetic Resources
Semen Freezing procedures significantly reduce
sperm quality
Low progressive motility;
increase acrosome damage
high morphological damage
spermatozoa
very important role in the fertilization
•Today, semen cryopreservation seems to be the only effective method of storing
reproductive cells for the ex-situ management of genetic diversity in birds (Kowalczyk
and Łukaszewicz, 2015 Blesbois, 2011;)
•Research efforts and success are still fluctuating and yet to be properly standardized
in poultry
•However, we cannot rule out a combination of many complex factors, including
phospholipid lysis, endogenous metabolism, insufficient level of antioxidant enzyme
and lipid peroxidation in the semen (Kotłowska et al., 2007; Douard et al., 2004),
exogenous metabolism (Balogun, 2019)
•Which are as a result of reactive oxygen species (ROS) competition with sperm cells
for available nutrient in the semen (Balogun, 2019)
3. JUSTIFICATION
•Frozen semen has numerous practical and field advantages for
poultry production
•However, despite the good progress made in the cryopreservation of
semen in cattle, this preservation method has not been as successful in
avian species
•Moreover adequate knowledge about mechanism of action of cell
protecting agents, ditto antioxidant activities
•Knowledge of requirement for poultry semen exogenously both
during and after semen processing for cryopresvation, resulting into
low semen quality and consequently the fertility levels achievable
with frozen/thawed spermatozoa are limited
4. AIM AND OBJECTIVES
This experiment therefore aimed at evaluating the effects of tomato
and orange juice supplemented in Tris egg-yolk extender
cryoprotected with two mixed slow permeating cryoprotectants
5. Tris Egg-yolk diluent supplemented with juices preparation
Diluents +
(DMF+MA)
Semen collection
Pooled semen; Mass activity
&Motility test 80% above
TEYE
T1, T2, T3, T4, T5 & T6
Dilution
Post thawed semen evaluation after 48hrs of
preservation;
Motility, viability, HOST & Acrosome integrity
Equilibration of diluted semen
samples; 2hrs-2hrs
Vapour freezing -80° C ;
12mins
Deep In liquid
nitrogen;
-196°C
Thawed
semen at
4°C
TEYE, TEYE+ 15%T & TEYE + 15%O
TEYE, TEYE+ 15%T & TEYE + 15%O
Mix 2
Mix 1
Antioxidant enzymes evalution ; GR, SOD & catalase
LPO evaluation
18%
13%
METHODOLOGY; EXPERIMENTAL PROCEDURES
6. POULTRY SEMEN FREEZING PROCEDURE
S E
Stage 1
Refrigerator
D E
D E
Stage 5; 2h
Stage 2; 2h Stage 3 Stage 4
Refrigerator
Liquid N2
Stage 7 Stage 6; 12mins
4°C
4°C
-80°C
4Cm
-196°C
Experimental Procedure: Stage 1: Semen dilution at 37 °C with TEYO® (A-fraction without
cryoprotectant). Stage 2: Equilibration in refrigerator for 2h. Stage 3: Semen dilution @ 4°C
with TEYO® (B-fraction with cryoprotectant). Stage 4: filling and sealing of semen in straws.
Stage 5: Final equilibration in refrigerator for 2h. Stage 6: Vapour freezing on a platform
above liquid nitrogen @ (-80°C). Stage 7: storage in liquid nitrogen @ ( – 196 °C).
7. RESULTS
Egg-yolk Egg-yolk + 15% Tomato
juice
Egg-yolk + 15% orange
juice
Parameters 13%(DMF
+MA)
18%(DMF
+MA)
13%(DMF
+MA)
18%(DMF
+MA)
13%(DMF
+MA)
18%(DMF
+MA)
SEM
Motility 23.33bc 15.00c 31.67ab 13.33c 31.00ab 40.00a 2.78
Viability 52.67 48.80 56.67 37.83 46.87 67.43 0.42
Membrane
Integrity
48.33ab 39.83ab 61.93a 26.21b 43.57ab 62.90a 0.44
Acrosome
Integrity
76.13 73.50 81.2 66.36 71.5 73.7 0.32
Superscripts indicate significant difference at 5 % level with in the columns (a - c)
Table 1: Microscopic semen quality of post thawed rooster semen cryopreserved with Tris- egg yolk tomato and orange
juice extenders
8. Table2: Lipid peroxidation and antioxidant enzymes activity of post thawed rooster
semen cryopreserved with Tris- egg yolk tomato and orange juice extenders
Egg-yolk Egg-yolk + 15% Tomato
juice
Egg-yolk + 15% orange
juice
Parameters 13%(DMF
+MA)
18%(DMF
+MA)
13%(DMF
+MA)
18%(DMF
+MA)
13%(DMF
+MA)
18%(DMF
+MA)
SEM
SOD(umols/1
06sper)
0.83 0.90 1.47 0.88 2.37 2.37 0.25
GR (KU/106) 0.73 0.47 0.17 0.47 0.30 0.37 0.00
Catalase
(IU/106)
0.54 0.77 0.41 1.32 0.47 1.08 0.14
LPO(umols/
109sperm)
84.33 79.00 67.67 84.00 69.33 93.00 4.19
9. CONCLUSION
• It appears that antioxidant enzymes in tomato and orange juice were
not effectively utilized by frozen sperm, as most parameters observed
revealed no significant different across the treatments
• Probably due to short duration of exposure of sperm to metabolic
activites
• However, most treatment with juice performed better, moreover
orange juice treatment had higher values compare to tomato juice
treatment irrespective of cryoprotectant concentrations
• Conclusively, antioxidant capacity of orange and tomato juice could
modulate sperm function irrespective of different cryoprotectants
concentrations and significant influence may be more evident only
when the sperm undergoes metabolic activities for longer duration.
10. REFERENCES
• Balogun A S. 2019. Evaluation of antioxidant potentials of tomato &
orange juices on extended poultry semen preserved with egg-yolk
& coconut water diluents to improve selection intensity Ph.D
Dissertation
• Blesbois E. 2011. Freezing avian semen. Avian. Biol. Res., 4: 52
• Douard V., Hermier D., Magistrini M., Labbé C., Blesbois E. 2004.
Impact of changes in composition of storage medium on lipid
content and quality of turkey spermatozoa. Theriogenology, 61: 1–
13
• Kotłowska M., Dietrich G., Wojtczak M., Karol H., Ciereszko A. 2007.
Effects of liquid storage on amidase activity, DNA fragmentation and
motility of turkey spermatozoa. Theriogenology, 67: 276–286.
• Kowalczyk A., Łukaszewicz E. 2015. Simple and effective methods of
freezing capercaillie (Tetrao urogallus L.) semen. PLoS ONE, 10: 1–
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