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Abstract

Chicken semen cryopreservation is a tool for programs of genetic diversity management and endangered breeds conservation. Due to physiological features, the fertility rates of cryopreserved poultry sperm are lower than mammal species. Thus, improvement of the semen cryopreservation methods is required. A first study was performed by a 2 × 2 factorial design consisting of two methods of adding the cryoprotectant [Direct or Diluted (mixed with extender medium)] and two cryoprotectants (glycerol and dimethylacetamide). Then sperm quality indicators were evaluated after freezing. A second study with a 2 × 2 design was conducted to evaluate the effectiveness of bovine serum albumin (BSA) on the optimization of two different extenders (Lake and ASG). Viability and motility variables were evaluated before and after freezing. There was no significant difference in sperm viability and motility variables between Direct or Diluted methods. Supplementation of extenders with BSA improved most of the sperm motility variables in both extenders before and after freezing. Progressive sperm, non-progressive sperm before freezing, and all post-thaw sperm motility parameters, except ALH and BCF, were increased in BSA-supplemented extenders (P<0.05), and BSA improved sperm viability in ASG extender after thawing (P<0.05). After thawing, the interaction between extender and BSA (P<0.05), eliminated the differences between the two BSA-supplemented media in VCL, VSL, VAP, and ALH which were higher in non-supplemented ASG extender than non-supplemented Lake medium. In conclusion, the direct or diluted methods of adding glycerol or dimethylacetamide, did not significantly affect the post-thaw sperm characteristics. BSA positively affected most of the post-thaw sperm motility indicators regardless of the type of extender and resulted in significantly higher post-thaw sperm viability in ASG medium.
Journal Pre-proof
Research Note: Evaluation of two methods for adding cryoprotectant
to semen and effects of bovine serum albumin on quality
characteristics of cryopreserved rooster spermatozoa
Alireza Behnamifar , Berenice Bernal , Olga Torres ,
H´
ector Luis-Chincoya , Mar´
ıa G. Gil , Pedro Garc´
ıa-Casado ,
Shaban Rahimi , Henri Woelders , Juli´
an Santiago-Moreno
PII: S0032-5791(21)00127-9
DOI: https://doi.org/10.1016/j.psj.2021.101093
Reference: PSJ 101093
To appear in: Poultry Science
Received date: 8 October 2020
Accepted date: 4 March 2021
Please cite this article as: Alireza Behnamifar , Berenice Bernal , Olga Torres ,
H´
ector Luis-Chincoya , Mar´
ıa G. Gil , Pedro Garc´
ıa-Casado , Shaban Rahimi , Henri Woelders ,
Juli´
an Santiago-Moreno , Research Note: Evaluation of two methods for adding cryoprotectant to
semen and effects of bovine serum albumin on quality characteristics of cryopreserved rooster
spermatozoa, Poultry Science (2021), doi: https://doi.org/10.1016/j.psj.2021.101093
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1
RESEARCH NOTE
Research Note: Evaluation of two methods for adding cryoprotectant to semen and effects
of bovine serum albumin on quality characteristics of cryopreserved rooster spermatozoa
Alireza Behnamifar, Berenice Bernal*1, Olga Torres*, ctor Luis-Chincoya§, María G. Gil*,
Pedro García-Casado#, Shaban Rahimi, Henri Woelders‖‖, and Julián Santiago-Moreno
Department of Poultry Science, Faculty of Agriculture, Tarbiat Modares University,
1411713116 Tehran, Iran
*Departamento de Mejora Genética Animal, INIA, 28040 Madrid, Spain
§Recursos Genéticos y Productividad-Ganadería. Colegio de Postgraduados, 56230, Mexico
#Zoitechlab (Arquimea Group), R&D Department, 28400, Madrid, Spain
‖‖Wageningen University & Research, Animal Breeding and Genomics, P.O. Box 338, 6700 AH,
Wageningen, the Netherlands
Departamento de Reproducción Animal, INIA, 28040 Madrid, Spain
1Corresponding author: berenice.bernal@inia.es; berenice.bernal.juarez@gmail.com
Avda. Puerta de Hierro km 7.5,28040, Madrid, Spain
Telephone: +34 913474020
Scientific Section: Physiology and Reproduction
2
Abstract
1
Chicken semen cryopreservation is a tool for programs of genetic diversity management and
2
endangered breeds conservation. Due to physiological features, the fertility rates of
3
cryopreserved poultry sperm are lower than mammal species. Thus, improvement of the semen
4
cryopreservation methods is required. A first study was performed by a 2 × 2 factorial design
5
consisting of two methods of adding the cryoprotectant [Direct or Diluted (mixed with extender
6
medium)] and two cryoprotectants (glycerol and dimethylacetamide). Then sperm quality
7
indicators were evaluated after freezing. A second study with a 2 × 2 design was conducted to
8
evaluate the effectiveness of bovine serum albumin (BSA) on the optimization of two different
9
extenders (Lake and ASG). Viability and motility variables were evaluated before and after
10
freezing. There was no significant difference in sperm viability and motility variables between
11
Direct or Diluted methods. Supplementation of extenders with BSA improved most of the sperm
12
motility variables in both extenders before and after freezing. Progressive sperm, non-
13
progressive sperm before freezing, and all post-thaw sperm motility parameters, except ALH and
14
BCF, were increased in BSA-supplemented extenders (P<0.05), and BSA improved sperm
15
viability in ASG extender after thawing (P<0.05). After thawing, the interaction between
16
extender and BSA (P<0.05), eliminated the differences between the two BSA-supplemented
17
media in VCL, VSL, VAP, and ALH which were higher in non-supplemented ASG extender
18
than non-supplemented Lake medium. In conclusion, the direct or diluted methods of adding
19
glycerol or dimethylacetamide, did not significantly affect the post-thaw sperm characteristics.
20
BSA positively affected most of the post-thaw sperm motility indicators regardless of the type of
21
extender and resulted in significantly higher post-thaw sperm viability in ASG medium.
22
Key words: sperm motility, sperm viability, dimethylacetamide, glycerol, BSA
23
3
Introduction
24
The degree of cryodamage can be affected by the composition of the extender, the type
25
and concentration of cryoprotectant, and the cooling rate (Woelders et al., 2006). Cryoprotectants
26
in the freezing medium mitigate the adverse effects of freezing. Notably, they prevent
27
excessively high intra- and extracellular concentrations of electrolytes and excessive shrinking of
28
the cells during freezing (Mazur and Rigopoulos, 1983). The addition of cryoprotectants straight
29
from the bottle could lead to brief exposure of cells to very high local cryoprotectant
30
concentrations, which could aggravate osmotic events and lead to chemical toxicity effects. The
31
latter seems especially relevant for dimethylacetamide (DMA), which is not tolerated very well
32
by poultry sperm at very high concentrations. Thus optimizing the cryoprotectant addition
33
method may reduce the potential stress of cryoprotectant addition and may overall reduce sperm
34
injury during the cryopreservation protocols.
35
Serum albumin, the most or one of the most abundant proteins of the chicken seminal
36
plasma, stimulates chicken sperm motility at the normal concentrations found in seminal plasma
37
(Santiago-Moreno and Blesbois, 2020). It has been reported that its addition after thawing of
38
poultry semen is also shown to give higher motility values (Woelders, 2021).
39
The objectives of this study were to 1) compare two different methods of adding the
40
cryoprotectant (direct vs. diluted) to extended rooster semen on in vitro post-thaw sperm quality
41
and 2) to test bovine serum albumin (BSA) as a supplement to poultry extenders.
42
Material and methods
43
Chemicals, Extenders
44
4
All Chemicals used in this study were obtained from Merck (Darmstadt, Germany) and
45
Sigma Co. (St. Louis, MO, USA). Extenders used in this study were Lake and ASG. Lake
46
extender contains 1.92g sodium L-glutamate monohydrate, 0.5 g potassium acetate, 0.08 g
47
magnesium acetate tetrahydrate, 0.8 g glucose, 0.3 g polyvinylpyrrolidone (Mr 10000), and 100
48
mL of water (343 mOsm/kg, pH 7.08). ASG medium contains 1.211 g sodium L-glutamate
49
monohydrate, 0.102 g tripotassium citrate monohydrate, 0.064 g magnesium acetate tetrahydrate,
50
0.526 g glucose, 2.43 g BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), 0.185 g
51
sodium hydroxide, and water to make a total volume of 100 mL (325 mOsm/kg, pH 7.1).
52
Experimental Birds
53
Animals were handled according to procedures approved by the INIA Ethics Committee
54
(Reference number PROEX 046/16) and were performed in accordance with the Spanish law of
55
animal protection RD53/2013 which is in accordance with the European Union Directive
56
2010/63/UE.
57
Sperm Collection, Processing, Freezing, and Thawing
58
We used 10 males from two Spanish breeds of chickens (five Birchen Leonesa roosters
59
and five Red Villafranquina roosters). Semen was collected using the abdominal massage
60
method two to three times a week from the 10 roosters. The semen was pooled directly during
61
semen collection for 10 times (ten replicates). In order to eliminate the effect of the breed, the
62
semen of both breeds were mixed in the pooled samples. Samples were transferred immediately
63
to the laboratory and kept warm in the hand to prevent cold shock. In the laboratory, the pooled
64
semen was diluted 1:1 (v/v) at field temperature using the respective extender as described below
65
for experiments 1 and 2. The extended semen samples were transferred to a refrigerator at 5 ºC.
66
5
Cooling down to 5 ºC lasted about 1 h. During this period the evaluation of the fresh sperm
67
variables were performed. Afterwards, extender and cryoprotectant were added, and were
68
allowed to equilibrate for 10 min at 5°C. After equilibration, the samples were loaded into 0.25-
69
mL straws (Minib, Germany) and then frozen using a Computer Freezer-Icetube 1810 freezer
70
unit (Minitüb, Tiefenbach, Germany) with a medium freezing rate (from 5°C to −35°C at
71
7°C/min and then from −35°C to −140°C at 60°C/min). Then the frozen straws were submerged
72
into and maintained in liquid nitrogen (at -196°C). For thawing, the straws containing glycerol
73
and DMA were warmed for 3 min and 30 s, respectively, in a water bath at 5°C with continuous
74
stirring. Then, post-thaw motility and viability were evaluated.
75
Experiment 1 (Methods of adding cryoprotectant). In this experiment two methods of
76
addition of the cryoprotectant were compared, with glycerol or DMA as cryoprotectant. Each
77
pool was extended 1:1 (v:v) with Lake extender and cooled to 5ºC. To evaluate whether the
78
samples were of sufficient quality to be frozen, evaluation of the fresh sperm variables was
79
performed before division of the pool. Four equal aliquots of the semen were placed in tubes.
80
The first aliquot was mixed 1:1 (v/v) with Lake medium containing 16% of glycerol (Diluted-
81
glycerol). The second aliquot was first mixed 1:0.84 (v/v) with Lake medium, and then glycerol
82
(0.16v) was added directly (Direct-glycerol). As a result, both aliquots eventually contained 8%
83
glycerol that was added by two methods: diluted (Diluted-Method) and directly (Direct-Method).
84
The third aliquot was mixed 1:1 (v/v) with Lake medium containing 8% of DMA (Diluted-
85
DMA); and the fourth aliquot was first extended 1:0.92 (v/v) with Lake medium, and then DMA
86
(0.08v) was added directly (Direct-DMA). Therefore, both aliquots contained 4% DMA that was
87
added by two methods: diluted (Diluted-Method) or directly (Direct-Method). Thus, the four
88
treatments were: Diluted-glycerol, Direct-glycerol, Diluted-DMA, and Direct-DMA. After
89
6
equilibration time, aliquots were frozen in the morning and were thawed in the afternoon on the
90
same day.
91
Experiment 2 (Supplementation with BSA). In this experiment, semen was frozen with
92
8% (v/v) glycerol as cryoprotectant. After measuring sperm concentration, the pooled semen was
93
split into four aliquots. Each aliquot was extended 1:1 (v/v) with either Lake or ASG extender
94
with or without BSA (10 mg/mL), i.e. Lake, Lake-BSA, ASG, ASG-BSA and cooled to 5 ºC for
95
1h, while the sperm parameters of each aliquot were determined (Table 2, first section).
96
Afterward, each aliquot was extended 1:1 (v/v) with their specific extender (with which it had
97
been previously extended) containing 16% glycerol. Finally, all aliquots contained 8% glycerol.
98
After equilibration time, the semen samples were frozen in the morning and were thawed in the
99
afternoon on the same day for assessment of post-thaw sperm motility and viability. For motility
100
evaluations, frozen/thawed semen was diluted with the same extender as was used before
101
freezing.
102
Assessment of Sperm Variables
103
Concentration and objective kinetic sperm parameters were evaluated by a computer-
104
aided sperm analysis (CASA) system coupled to a phase-contrast microscope (Nikon Eclipse
105
model 50i; negative contrast, Nikon Instruments Europe B.V., Izasa S.A., Barcelona, Spain).
106
Sperm concentration was measured loading the extended semen onto a warmed (37 ºC) 20 µm
107
Leja® 8-chamber slide (Leja Products B.V., Nieuw-Vennep, The Netherlands). The percentage
108
of motile sperm, progressive motile sperm and kinematic values were recorded with the Sperm
109
Class Analyzer SCA® v.4.0. Software (Microptic S.L., Barcelona, Spain). A minimum of three
110
fields and 200 sperm tracks were evaluated at a magnification of 100× for each sample (image
111
acquisition rate 25 frames/s).
112
7
Staining with propidium iodide and SYBR-14 were used to measure sperm viability, a
113
total of 200 spermatozoa were counted by a fluorescence microscope (Eclipse E200, Nikon,
114
Tokyo, Japan) at 400× (wavelength: 450490 nm).
115
Statistical Analysis
116
To assess whether the data can be modeled by a normal distribution, Kolmogorov
117
Smirnov, Lilliefors, and ShapiroWilk tests were performed. If needed, the arc-sin
118
transformation, log-transformation, or the box-cox transformation of data was used. A first study
119
was conducted as a 2×2 factorial arrangement in completely randomized design, including two
120
factors: two methods of adding the cryoprotectant (Direct or Diluted) and two cryoprotectants
121
(glycerol and DMA). A second study was randomly assigned according to a 2×2 factorial
122
arrangement to effect of BSA on two different extenders (Lake and ASG). Data were analyzed
123
by GLM-ANOVA using the GLM procedure of SAS (Statistical Analysis System) 9.1 software
124
(SAS Institute Inc., Cary, NC, USA). The level of significance was set at P<0.05, and Tukey’s
125
test was performed for the mean comparison. When interaction effects were significant, the
126
SLICE command in SAS was accomplished and significant letters were capitalized.
127
Results and discussion
128
Experiment 1:
129
Post-thaw sperm variables in two different methods of adding cryoprotectants
130
There were no significant differences in post-thaw sperm viability and sperm motility
131
variables between the two addition methods (Direct vs. Diluted) for either glycerol or DMA
132
(Table 1). The interaction between the methods of adding and type of cryoprotectant were not
133
significant for any of the measured parameters. Overall, glycerol showed significantly better
134
8
results that DMA for all measured parameters (P<0.05). This indicates that addition of straight,
135
undiluted cryoprotectants does not appreciably aggravates the (potential) chemical and osmotic
136
stress associated with addition of cryoprotectants. In fact, post-thaw results for both addition
137
methods obtained with glycerol seem almost identical. However, for DMA, diluted addition
138
resulted in numerically higher values for all post-thaw sperm variables compared with direct
139
addition. While these differences were not significant, the observations are in line with the notion
140
that DMA is a more damaging compound for poultry sperm than glycerol, as just the addition of
141
DMA before freezing resulted in significantly more sperm damage, compared with glycerol
142
(Tselutin et al., 1999). We speculate that this may be related to the higher partition coefficient for
143
octanol/water (Kow), with Log (Kow) values being −0.77 and −1.76 for DMA and glycerol,
144
respectively (https://pubchem.ncbi.nlm.nih.gov). These values indicate that a substantial fraction
145
of DMA partitions in octane ([DMA] in octane = 17% of [DMA] in water), i.e. DMA is quite
146
lipophilic. If we assume that partitioning of DMA into lipid is similar, a medium DMA
147
concentration of 4% (v/v) would mean that the sperm membranes contain approximately 0.7 %
148
(v/v) of DMA, which could affect membrane stability. It seems not unlikely that this would be
149
aggravated by brief exposure to locally elevated concentrations of DMA in the direct (undiluted)
150
addition method.
151
Experiment 2:
152
Pre-freeze sperm variables in BSA-supplemented extenders
153
Semen extended in media without BSA supplementation showed better pre-freeze sperm
154
viability and VSL in Lake than in ASG medium (Table 2; P<0.05). However, supplementation of
155
BSA and the interaction effect between BSA and ASG extender improved sperm viability in
156
ASG extender and made no difference between two BSA-supplemented extenders. BSA
157
9
improved most sperm motility parameters in both extenders and reduced non-progressive sperm
158
in fresh semen (P<0.05).
159
Post-thaw sperm variables in BSA-supplemented extenders
160
Supplementation with BSA resulted in better values in post-thaw semen for most sperm
161
motility parameters (Table 2) in both extenders after thawing (P<0.05) and it only had no effect
162
on ALH and BCF. The interaction between extender type and treatments (with or without BSA)
163
was significant (P<0.05) for sperm viability, VCL, VSL, VAP, and ALH. While the % viable
164
sperm was not different in the two non-supplemented extenders, % viable sperm was higher in
165
ASG-BSA compared with Lake-BSA (P<0.05). Also, supplementation of the extenders with
166
BSA annulled the differences in VCL, VSL, VAP, and ALH, which were higher in non-
167
supplemented ASG extender than non-supplemented Lake medium.
168
The somewhat more pronounced effect of the presence of BSA in ASG than in Lake may
169
be due to the fact that Lake contains 0.3% polyvinylpyrrolidone. This is a macromolecule that is
170
used as a blood plasma substitute as it shares a number of properties with BSA. Woelders (2021)
171
reported that the addition of BSA after freezing and thawing is also positive for the motility. It is
172
mentioned that BSA adsorb on the membranes of rabbit sperm cells and coats the surface area of
173
sperm (Blank et al., 1976). The coating causes the sperm to be less hindered in their motility by
174
physical obstacles (Bakst and Cecil, 1992). Chakarov and Mollova, (1980) suggested that
175
adsorption of BSA to the sperm plasma membrane rendered ram sperm more resistant to the
176
otherwise harmful effect of a high rate of semen dilution. BSA could augment the elimination of
177
free radicals generated by oxidative stress, and protect the sperm membrane from cold shock
178
during freeze-thaw in canine sperm (Uysal et al., 2005). It has been reported the antioxidant
179
property of BSA (5 mg/mL) as it is able to increase the activity of catalase and protect the sperm
180
10
morphology, and preserve the fertilizing potential of bull sperm after the freeze-thawing process
181
(Sariözkan et al., 2009). Since BSA is a mammal-derived protein and may cause immune
182
responses in hens' reproductive system, its effectiveness in improving fertility rates by artificial
183
insemination should be considered in future studies.
184
Conflict of interest
185
None of the authors have any conflict of interest to declare.
186
187
ACKNOWLEDGMENTS
188
This research is part of a project that received funding from the European Union's
189
Horizon 2020 Research and Innovation Programme under grant agreement Nº 677353 IMAGE.
190
Part of this research was funded by the project RZP2015-00002-00-00 and Zoitechlab S.L-INIA
191
contract CON 18-141.
192
REFERENCES
193
Bakst, M. R., and H. C. Cecil. 1992. Effect of bovine serum albumin on motility and fecundity of
194
turkey spermatozoa before and after storage. Reprod. 94: 287-293.
195
Blank, M., L. Soo, and J. S. Britten. 1976. Adsorption of albumin on rabbit sperm membranes. J.
196
Membr. Biol. 29: 401-409.
197
Chakarov, E. L., and M. V. Mollova. 1980. Influence of bovine serum albumin (BSA) on the
198
motility of highly diluted spermatozoa studied by photokinesiography. Dokl. Bolg. Akad. Nauk.
199
33: 655-658.
200
11
Mazur, P., and N. Rigopoulos. 1983. Contributions of unfrozen fraction and of salt concentration
201
to the survival of slowly frozen human erythrocytes: influence of warming rate. Cryobiology. 20:
202
274-289.
203
Santiago-Moreno, J., and E. Blesbois. 2020. Functional Aspects of Seminal Plasma in Bird
204
Reproduction. Int J Mol Sci. 21: 5664.
205
Sariözkan, S., P. B Tuncer, M. N. Bucak, and P. A. Ulutaş. 2009. Influence of various
206
antioxidants on microscopic-oxidative stress indicators and fertilizing ability of frozen-thawed
207
bull semen. Acta Vet. Brno. 78: 463-469.
208
Tselutin, K., F. Seigneurin, and E. Blesbois. 1999. Comparison of cryoprotectants and methods
209
of cryopreservation of fowl spermatozoa. Poult. Sci. 78: 586-590.
210
Uysal, O., T. Korkmaz, and H. Tosun. 2005. Effect of bovine serum albumine on freezing of
211
canine semen. Indian Vet. J. 82: 97-98.
212
Woelders, H. 2021. Cryopreservation of avian semen. Pages 379-399 in: Cryopreservation and
213
Freeze-Drying Protocols, Lab protocol series ‘Methods in Molecular Biology’, W. F. Wolkers
214
and H. Oldenhof, ed. Humana, New York, NY.
215
Woelders, H., C. A. Zuidberg, and S. J. Hiemstra. 2006. Animal genetic resources conservation
216
in the Netherlands and Europe: poultry perspective. Poult. Sci. 85: 216-222.
217
12
Table 1. Effect of adding diluted or direct cryoprotectant on frozen-thawed semen characteristics
Method:
Direct
Method
Cryoprotectant
SEM
Significance
Cryoprotectant:
GLY
DMA
GLY
DMA
Diluted
Direct
GLY
DMA
Method
CR
Method×CR
Viable spermatozoa (%)
40.20
17.40
40.25
13.20
28.80
26.72
40.22a
15.30b
1.84
NS
*
NS
Motility quality score (1-5)1
3.10
0.95
3.07
0.85
2.02
1.96
3.08a
0.90b
0.14
NS
*
NS
Non-progressive sperm (%)
31.26
13.20
33.28
11.04
22.23
22.16
32.27a
12.12b
1.59
NS
*
NS
Progressive sperm (%)
18.90
2.44
20.90
1.45
10.67
11.17
19.90a
1.95b
1.27
NS
*
NS
Total motility (%)
50.16
15.65
54.19
12.50
32.91
33.34
52.17a
14.08b
1.55
NS
*
NS
VCL (µm/s)
63.03
34.80
62.94
30.61
48.91
46.77
62.98a
32.70b
2.97
NS
*
NS
VSL (µm/s)
40.34
19.52
40.76
13.58
29.93
27.17
40.55a
16.55b
2.63
NS
*
NS
VAP (µm/s)
48.72
24.06
49.13
20.20
36.39
34.66
48.93a
22.13b
2.74
NS
*
NS
LIN (%)
62.49
53.07
63.45
44.29
57.78
53.87
62.97a
48.68b
2.76
NS
*
NS
STR (%)
81.59
78.60
81.48
68.33
80.09
74.91
81.54a
73.46b
2.75
NS
*
NS
WOB (%)
76.21
66.68
77.25
65.17
71.45
71.21
76.73a
65.92b
1.78
NS
*
NS
ALH (µm)
2.80
1.96
2.80
1.76
2.38
2.28
2.80a
1.86b
0.20
NS
*
NS
BCF (Hz)
8.17
6.28
8.27
6.20
7.23
7.24
8.22a
6.24b
0.69
NS
*
NS
GLY: glycerol; DMA: dimethylacetamide; CR: Cryoprotectant
SEM: standard error of the mean; NS: non-significant differences
a,b Means within a row with no common superscript differ significantly (*P<0.05)
VCL: curvilinear velocity; VSL: straight-line velocity; VAP: average path velocity; LIN: linearity; STR: straightness; WOB: wobble; ALH: amplitude of lateral head
displacement; BCF: beat-cross frequency
1Subjective measurement: The quality of motility was scored on a scale of 0 (lowest) to 5 (highest); 0 = no movement, 1 = tail movements but no sperm progression, 2 = only
circular sperm movements, 3 = a large percentage of spermatozoa showed progressive but no rectilinear movement, 4 = a large percentage of spermatozoa showed rectilinear but
not very vigorous movement, and 5 = a large percentage of spermatozoa showed vigorous, rectilinear, progressive movement.
13
Table 2. Effect of supplementing BSA to Lake and ASG extenders on rooster semen quality
BSA:
-
+
BSA
Extender
SEM
Significance
Extender:
Lake
ASG
Lake
ASG
-
+
Lake
ASG
BSA
(A)
Extender
(B)
A×B
FRESH SEMEN
Viable spermatozoa (%)
80.50A(2)
71.50B(2)
78.50
77.60
76.00
78.05
79.50a
74.55b
1.34
NS
*
*(2)
Motility quality score (1-5)1
2.72
2.85
3.97
3.95
2.78b
3.96a
3.35
3.40
0.10
*
NS
NS
Non-progressive sperm (%)
37.62
34.69
16.74
27.92
36.15a
22.33b
27.18
31.30
2.47
*
NS
NS
Progressive sperm (%)
31.52
23.66
55.92
46.50
27.59b
51.21a
43.72
35.08
3.15
*
NS
NS
Total motility (%)
69.14
58.35
72.67
74.42
63.75
73.54
70.90
66.39
3.61
NS
NS
NS
VCL (µm/s)
75.39
68.61
116.69
100.88
72.00b
108.78a
96.04
84.74
4.41
*
NS
NS
VSL (µm/s)
45.20
35.25
92.59
75.80
40.23b
84.20a
68.89a
55.53b
4.39
*
*
NS
VAP (µm/s)
55.45
47.00
102.11
85.91
51.22b
94.01a
78.78
66.45
4.29
*
NS
NS
LIN (%)
57.27
50.73
78.80
74.24
54.00b
76.52a
68.03
62.48
2.37
*
NS
NS
STR (%)
78.88
73.51
90.36
87.46
76.20b
88.91a
84.62
80.49
1.62
*
NS
NS
WOB (%)
72.01
68.18
87.03
84.67
70.09b
85.85a
79.52
76.42
1.46
*
NS
NS
ALH (µm)
2.97
2.92
3.06
3.28
2.95
3.17
3.02
3.10
0.11
NS
NS
NS
BCF (Hz)
9.60
8.78
10.28
9.82
9.19b
10.05a
9.94
9.30
0.24
*
NS
NS
POST-THAW SEMEN
Viable spermatozoa (%)
36.90
30.30
33.90B(3)
43.40A(3)
33.60b
38.65a
35.40
36.85
1.71
*
NS
*(3)
Motility quality score (1-5)1
1.47
1.95
2.70
2.72
1.71b
2.71a
2.08
2.33
0.14
*
NS
NS
Non-progressive sperm (%)
12.49
14.04
15.65
20.50
13.26b
18.08a
14.07
17.27
1.37
*
NS
NS
Progressive sperm (%)
5.39
9.48
16.58
15.28
7.43b
15.93a
10.99
12.38
1.23
*
NS
NS
Total motility (%)
17.88
23.52
32.24
35.78
20.70b
34.01a
25.06
29.65
2.24
*
NS
NS
VCL (µm/s)
48.22B(4)
61.76A(4)
67.90
61.84
54.99b
64.87a
58.06
61.80
2.61
*
NS
*(4)
VSL (µm/s)
28.42B(5)
39.07A(5)
50.19
45.83
33.74b
48.01a
39.30
42.45
2.45
*
NS
*(5)
VAP (µm/s)
34.68B(6)
47.08A(6)
56.32
51.70
40.88b
54.01a
45.50
49.39
2.55
*
NS
*(6)
LIN (%)
58.13
62.62
73.18
73.64
60.38b
73.41a
65.66
68.13
1.79
*
NS
NS
STR (%)
81.07
82.45
88.68
88.43
81.76b
88.55a
84.88
85.44
1.11
*
NS
NS
WOB (%)
71.42
75.68
82.30
83.15
73.55b
82.73a
76.86
79.42
1.25
*
NS
NS
ALH (µm)
2.16B(7)
2.74A(7)
2.59
2.39
2.45
2.49
2.38
2.57
0.09
NS
NS
*(7)
BCF (Hz)
8.84
9.14
9.55
9.20
8.99
9.37
9.19
9.17
0.26
NS
NS
NS
(-) Absence of BSA, (+) Supplementation with BSA
Lake: Lake and Ravie 1984 medium; ASG: Animal Sciences Group (Wageningen University, Lelystad, The Netherlands) medium
SEM: standard error of the mean; NS: non-significant differences
a,b Means within a row with no common superscript differ significantly (*P<0.05)
VCL: curvilinear velocity; VSL: straight-line velocity; VAP: average path velocity; LIN: linearity; STR: straightness; WOB: wobble; ALH: amplitude of lateral head
displacement; BCF: beat-cross frequency
1Subjective measurement: The quality of motility was scored on a scale of 0 (lowest) to 5 (highest); 0 = no movement, 1 = tail movements but no sperm progression, 2 = only
circular sperm movements, 3 = a large percentage of spermatozoa showed progressive but no rectilinear movement, 4 = a large percentage of spermatozoa sho wed rectilinear but
not very vigorous movement, and 5 = a large percentage of spermatozoa showed vigorous, rectilinear, progressive movement.
2(A,B) Interaction effect (BSA×Extender) for viable spermatozoa (%) in fresh semen
3(A,B) Interaction effect (BSA×Extender) for viable spermatozoa (%) in post-thaw semen
14
4(A,B) Interaction effect (BSA×Extender) for VCL (µm/s) in post-thaw semen
5(A,B) Interaction effect (BSA×Extender) for VSL (µm/s) in post-thaw semen
6(A,B) Interaction effect (BSA×Extender) for VAP (µm/s) in post-thaw semen
7(A,B) Interaction effect (BSA×Extender) for ALH (µm) in post-thaw semen
... 1 000 mL 1 000 mL 1 000 mL 1 000 mL 1 000 mL TES and BES = zwitterion buffers. L&R-84: Lake and Ravie extender (Lake and Ravie 1984); EK (Łukaszewicz, 2001b); Lake 7.1 (Lake and Ravie, 1981); BPSE: Beltsville Poultry Semen Extender (Sexton, 1977); ASG: Lake and Animal Sciences Group extender (Behnamifar et al., 2021). ...
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The general belief is that slow freezing injury is either the result of exposure to high salt concentrations or the result of excessive cell shrinkage. Increased salt concentration arises as increasing amounts of pure ice precipitate out of solution during freezing and cause the liquid-filled channels in which the cells are sequestered to dwindle in size. Cell shrinkage is an osmotic response to the concentration of external solutes. The consensus has been that the injury is related to the composition of the solution in these channels and not to the amount of residual liquid.
Influence of bovine serum albumin (BSA) on the motility of highly diluted spermatozoa studied by photokinesiography
  • Chakarov
Chakarov, E. L., and M. V. Mollova. 1980. Influence of bovine serum albumin (BSA) on the 198 motility of highly diluted spermatozoa studied by photokinesiography. Dokl. Bolg. Akad. Nauk. 199 33: 655-658. to the survival of slowly frozen human erythrocytes: influence of warming rate. Cryobiology. 20: 202 274-289.