1. Discussion Conclusion References Acknowledgements
EFFECTS OF EQUILIBRATION DURATION VAPOR
TEMPERATURE AND VAPOR EXPOSURE DURATION
ON MOTILITY CHARACTERISTICS OF FREEZABILITY
AFRICAN CATFISH (Clarias gariepinus) SPERM
Noor Azlina KAMARUDING1, Wan Khadijah W. EMBONG2 and *Ramli B. ABDULLAH2
1Department of Biological Sciences, Faculty of Science and Technology, Universiti Malaysia Terengganu, Kuala Terengganu. Malaysia.
2Animal Biotechnology Embryo Laboratory (ABEL), Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur. Malaysia. *
Table 1 demonstrates the highest values of total motility and progressive motility were obtained from combination factors of 120 min equilibration duration, -100oC vapor temperature and 15 min vapor exposure duration (87.44±2.07% and 28.22±2.16%, respectively). Combinations of 140 min, -100oC and 10 min showed the lowest values of total motility (59.27±8.00%), but the lowest value of progressive motility was observed at combination of 160 min, -90oC and 10 min (12.20±2.24%). Subsequent analysis of sperm motility characteristics using IVOS-analyzer at 120 min equilibration duration, -100oC vapor temperature and 15 min vapor exposure duration produced an encouraging values of rapid, medium, slow and static velocities, VAP, VSL, VCL, ALH, BCF, STR and LIN which are tabulated in Table 2.
Introduction
A pair of elongated testis
Seminal vesicles
(a)
(b)
(c)
(d)
Sperm cryopreservation is practically important technique mainly for conservation and breeding purposes, since it was first introduced by Blaxter in In aquaculture, this technique benefits primarily to aid management of controlled reproduction in fish farming, allowing storage of semen from selected species and individuals, shipment of genetic material, conservation of endangered species, self fertilisation of protandrous fish and hybridization of species with non-overlapping reproductive periods [1, 2-5]. Remarkable factors affecting sperm freezing have extensively been studied and reviewed by many researchers. The most crucial factors that are critically inter-dependent are composition of extender, cryoprotectant, equilibration duration and cooling rate. The stepwise process of temperature reduction is important to avoid cold-shock during freezing process. The present study was designed to explore into the potential of Fish-Ringer Extender (FRE) to cryopreserve the sperm of African catfish (Clarias gariepinus). Our study found FRE extender is an ideal extender that allows the sperm to a suitable environment in accordance with the seminal fluid. Previously, FRE extender with 10% DMSO gave better survivability of Mahseer (Tor khudree) [11]. Additionally, the present study was also conducted to evaluate the effects of different equilibration durations, vapor temperatures and vapor durations on sperm movement characteristics.
Discussion
The optimum combination factors were obtained at 120 min
equilibration duration, -100oC vapor temperature and 15 min vapor
exposure duration which gave the highest total motility and
progressive motility.
At 120 min equilibration duration, the trends of total motility and
progressive motility values increased as vapor temperature
reduced within 15 min vapor exposure duration.
At 140 min equilibration duration, the values of total motility and
progressive motility were at peak under -90oC vapor temperature,
but as the vapor temperature was lowered until -100oC, the total
motility and progressive motility values reduced.
There was a consistent trend in values of total motility and
progressive motility at 160 min equilibration duration. The present
study indicates equilibration duration and vapor temperature play
an important role in sperm freezing process.
Equilibration duration is required in sperm freezing process of
African catfish, for number of reasons:-
1) Membrane of fresh water fish was reported to be less resistant
than those marine fish due to the higher content of phospholipids and
lower polyunsaturated fatty acids. Cooling and warming induces
phase transitions of the lipids in the sperm cell membrane, and
influencing the special redistribution of their component [12].
2) Optimal equilibration temperature at 4oC is crucial to reduce the
occurrence of cold-shock. In addition, cryoprotectant protects the
sperm cells to form ice crystal internally by inhibiting cryo-injury
during the freezing process.
Fig. 2 (a) Finger-like testis; (b) Collected semen; (c) Equilibration of straws in the incubator; (d) Exposure of straws to liquid nitrogen vapor.
Results
Objective
Table 1
Total motility and progressive motility (mean ± S.E.M.) of post-thawed cryopreserved sperm of African (Clarias gariepinus) catfish using 10% DMSO in FRE extender for combination of equilibration duration, vapor temperature and vapor exposure duration
To determine the optimal freezing rate with special focus on equilibration
duration, vapor temperature and vapor exposure duration on sperm
motility characteristics (SMCs) of African catfish using Fish-Ringer
Extender (FRE).
Equilibration duration (min)
Vapor temperature
(oC)
Vapor exposure duration
(min) N*
Total
motility
(%)
Progressive
120
-80 5 10 15 13
74.00 ±4.69abc
62.73 ± 6.95ab
59.92 ± 9.11a
20.00 ± 2.59abc
12.80 ± 2.49a
15.77 ± 3.63a
-90
71.38 ±9.76abc
80.80 ±4.54abc
72.60 ±7.45abc
15.62 ± 2.54a
19.93 ± 2.73abc
19.93 ± 2.71abc
-100 20 18
84.00 ± 2.86bc
80.95 ±3.71abc
87.44 ± 2.07c
25.95 ± 2.36bc
25.95 ± 2.74bc
28.22 ± 2.16c
140 7 6
62.86 ± 7.74ab
59.67 ± 13.56a
78.29 ±7.51abc
14.00 ± 3.51a
16.00 ± 6.54a
19.57 ± 3.72abc
73.73 ±5.17abc
68.00 ±5.89abc
76.33 ±4.59abc
18.73 ± 2.83ab
16.13 ± 2.35a
20.00 ± 2.18abc 11
61.27 ± 8.10a
59.27 ± 8.00a
60.80 ± 9.43a
13.27 ± 3.21a
13.53 ± 2.87a
13.80 ± 3.21a
160 14 12
76.07 ±5.57abc
78.75 ±6.04abc
73.75 ±4.76abc
15.64 ± 2.67a
20.08 ± 2.74abc
16.08 ± 2.82a 19
74.15 ±6.40abc
60.20 ± 7.12a
73.21 ±4.50abc
16.08 ± 1.95a
12.20 ± 2.24a
14.05 ± 2.23a
71.45 ±6.69abc
80.00 ±3.65abc
73.75 ±4.86abc
18.60 ± 2.66ab
20.16 ± 2.04abc
17.80 ± 2.00ab
Materials and Methods
Injection of gonadotrophin (Ovaprim, Aqualife, Syndel Int. Inc., Canada) into the dorsal muscle of catfish 24 hours before freezing experiment
Male catfish was anaesthetized by salt immersion (euthanasia)
Collection of milt by incising and squeezing of testes from sacrificed catfish
Dilution of semen with FRE extender +10% DMSO in a ratio of 1:10
Slow freezing (vaporization) at three vapor temperatures (-80, -90 or -100oC) with three vapor exposure duration (5, 10 or 15 min)
Rapid freezing (plunging directly into liquid nitrogen at
-196oC)
Thawing at 30oC for 30 sec
Analysis of sperm motility characteristics using Semen Analyzer (IVOS)
Diluted semen was loaded into 0.5 ml or 0.25 ml French straws
Equilibration in incubator at 4oC with three duration interval (120, 140 or 160 min)
Conclusion
Under the present conditions, the optimal frozen-thawed sperm motility characteristics in African catfish using FRE extender was obtained with combination of 120 min equilibration duration, -100oC vapor temperature and 5 to 15 min exposure vapor duration. It is recommended that FRE extender using DMSO as cryoprotectant apparently is suitable to cryopreserve the sperm of African catfish (Clarias gariepinus).
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N* = Total number of observations (straws).
abcMeans with different superscripts within a column were significantly
different (P<0.05).
Table 2
Sperm motility characteristics of post-thawed cryopreserved sperm of African catfish (Clarias gariepinus) using 10% DMSO in FRE extender for combination of 120 min equilibration duration, -100oC vapor temperature and 15 min vapor exposure duration
Motility characteristics
Value1
Rapid (%)
38.56 ±3.10
Medium (%)
10.22 ±0.53
Slow (%)
38.56 ±1.75
Static (%)
12.56 ±2.07
VAP (μm/s)
64.50 ±2.79
VSL (μm/s)
55.72 ±2.25
VCL (μm/s)
89.50 ±3.45
ALH (μm)
5.08 ±0.20
BCF (Hz)
13.68 ±0.70
STR (%)
85.78 ±0.70
LIN (%)
62.72 ±0.78
Acknowledgements
The authors wish to thank ABEL Members and the staff of Institute of
Biological Sciences Mini (Livesotck) Farm, who helped in this project.
This project is financially supported by PPP Research Grant
(P0060/2008), University of Malaya.
Fig. 1. Flowchart of semen collection and sperm freezing
technique.
1Values (mean±S.E.M) of respective SMCs were obtained from total number of observations (straws), N = 18.