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Research Article
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Sperm Abnormalities in Post-thawed Semen of Tunisian Arab Stallions |
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A. Najjar,
S. Ben Said,
B. Benaoun,
C. Chetoui,
M. Ezzaouia
and
M. Ben Mrad
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ABSTRACT
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The study was undertaken in order to evaluate sperm morphology
features of post-thawed semen of Tunisian Arab stallions. Forty two ejaculates
was collected and frozen, during years 2009 and 2010, from 9 stallions aged
between 9 to 24 years. After thawing, sperm morphology was studied after eosin-nigrosin
stain. The percentages of abnormal head, mid piece, flagella, sperm with droplets
and the total abnormal sperm were determined. Analysis of variance was carried
out using SAS software. The results showed that all sperm morphology features
varied among ejaculates within stallion and among stallions (p<0.01). The
percentage of abnormal flagella and total abnormal sperm varied between young
and old stallions (p<0.01). We concluded that freezing and thawing processes
increased abnormal sperm that is due to cell alteration for old and young stallions,
and consequently, the decrease of the quality of the thawed semen of Tunisian
Arab stallions.
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Received: December 07, 2012;
Accepted: February 19, 2013;
Published: April 16, 2013
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INTRODUCTION
In all species, the evaluation of sperm morphology is an important step to
understand reasons for decreasing fertility (Garavance
et al., 1997). In human, abnormal sperm morphology increases low
fertility (Chan et al., 1989). In stallions,
controversially, some studies reported no relation between sperm morphology
features and fertility (Voss et al., 1981; Love
et al., 2000), others however showed that an increase in sperm abnormality
affected negatively stallions fertility (Jasko et
al., 1990). For example, it was reported that presence of proximal droplets
affected negatively pregnancy rate per cycle (Jasko et
al., 1990). Besides, Katila (2001) showed that
low number of alive and normal post-thaw sperm could decrease the pregnancy
rate. The objective of this study was to investigate the sperm morphology features
of post-thawed semen of Tunisian Arab Stallion and to determine factors that
could affect this morphologic quality.
MATERIAL AND METHODS
General: The study took place in the National Stud Farm of Sidi Thabet
located in the north of Tunisia. Forty two ejaculates from 9 Tunisian Arab stallions
aged between 9 to 24 years (young stallions: age <15 years, n = 5; old stallions:
age = 15 years, n = 4) were used (2 to 10 ejaculates from each stallion). Semen
of these stallions were collected and frozen during the month of December 2009
and December 2010. The freezing semen was performed when the percentage of mobile
spermatozoa, determined under light microscope, and sperm concentration were
respectively greater than 70% and 120 millions spermatozoa mL. The freezing
semen was carried out according to the French method (Haras
Nationaux, 2004): Once filtered, the semen was immediately diluted in the
UHT skimmed milk extender supplemented with antibiotics gentamicin (50 mg L-1)
and penicillin (50000 IU L-1) (¼ of semen for ¾ of
the extender) in a water bath at 37°C. Then, the semen was placed in a water
bath at 22°C for 10 min. After that, the semen was centrifuged during 10
min at 3000 rpm (600 g). The supernatant was removed and the pellet of sperm
was re-suspended in the INRA 96® (IMV, LAigle, France)
supplemented with 2% chicken egg yolk clarified and 2.5% glycerol to obtain
a final concentration of 100 millions sperm mL-1. The diluted semen
was cooled to + 4°C in the refrigeration showcase for one hour and 20 min.
During this period, we prepared the straws (0.5 mL) which had been already identified
in the refrigerated display case. Finally, the straws were automatically filled
at + 4°C. The freezing of straws was performed using a programmable freezer
which provided the decrease of temperature from+4-140°C at the velocity
of-20-60°C min-1 during 2 min and 30 sec. At the end of the freezing,
the straws were stored in a tank of liquid nitrogen at-196°C.
Sperm morphology features: Two straws per ejaculate were thawed in a
water bath at 37°C for 30 sec. The semen straw (0.5 mL) were diluted in
2 mL of INRA 96® which was previously placed in a water bath
at 37°C and then incubated for 10 min at the same temperature. Sperm morphology
was determined using eosin-nigrosin stain. From each thawed straw, two droplets
of stain and semen (2 μL each one) was mixed and smeared on a prewarmed
glass slide (37°C). The slide was examined under light microscope at x400
magnification (Brito, 2007). At least 150 sperm were
examined and classified into 4 different classes according to their features:
percentages of abnormal head, abnormal mid piece, abnormal flagella and percentage
of sperm with droplets. After that, the percentage of total abnormal sperm was
estimated.
Statistical analysis: Data were analyzed by Statistical Analysis System
(SAS Institute Inc., Cary, NC, USA). General differences between means were
tested using factorial analysis for unbalanced data (GLM procedure of SAS).
We took into consideration the effect of stallions, ejaculates, age, years of
the freezing. The comparison of sperm morphology parameters between variables
cited above was carried out using the DUNCAN test. Significance was considered
at p<0.05.
RESULTS
Our results showed that all sperm abnormalities varied both between stallions
and between ejaculates within stallion (p = 0.0001). A significant effect of
stallions age on the percentage
of abnormal flagella (p = 0.0001) and total abnormal sperm (p = 0.0397) was
observed (Fig. 1).
We also showed that the percentage of total abnormal sperm varied within freezing
year, and it was about 34±2.2 and 37±1.8 %, respectively for 2009
and 2010 (p = 0.0518; Table 1).
DISCUSSION
Our results showed that all abnormal sperm, except proximal and distal droplets
after semen thawing, were higher in young stallions than in old ones. Previous
study in our laboratory (Najjar et al., 2010),
using fresh semen of the same stallions, showed that abnormal sperm was higher
in old stallions than the young ones (31.4 vs. 25.7%). Thus, and according to
these findings, it seems that young stallions sperm support less the freezing
process when compared to the old ones. Besides, and always according to our
previously findings on fresh semen, we noted that the freezing process increased
the percentage of total abnormal sperm in both classes of age. The increase
of sperm abnormalities could be attributed to cryopreservation. In fact, it
was shown that crypreservation may generate morphological alterations (Bailey
et al., 2000) and membrane destabilization (Holt
and North, 1986) of mammalian sperm. Christensen et
al. (1995) observed ultrastructural changes in the acrosome, the outer
fibres of the midpiece, and the axoneme of the principal piece after freezing
and thawing processes. Besides, Varner (2008) reported
that cytoplasmic droplets seems to have a minor effect on fertility of stallions.
However, detached head, abnormal mid pieces and premature germ cell had noxious
effect on fertility.
The important variations between stallions and between ejaculates within stallions
were shown by many others authors (Blach et al.,
1989; Katila, 2001). In fact, Blach
et al. (1989) reported that morphological sperm variation is due
to the freezing and thawing processes.
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Fig. 1: |
Sperm morphology features in young and old stallions (Means±sem) |
Table 1: |
Sperm morphology features according to years of thawing (Means±sem) |
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a, bSignificant at: p = 0.0518 |
Katila (2001) found that these variations were assigned
to sperm ability to support the freezing and thawing processes.
CONCLUSION
It is clear that freezing and thawing processes increased sperm abnormalities
that are due to cell alteration of young and old stallions. Consequently, we
can say that abnormal spermatozoa affects the quality of the thawed semen of
Tunisian Arab stallions.
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REFERENCES |
1: Bailey, J.L., J.F. Bilaodean and N. Cormier, 2000. Semen cryopreservation in domestic animals: A damaging capacitating phenomenon. J. Androl., 20: 1-7. PubMed |
2: Blach, E.L, R.P. Amann, R.A. Bowen and D. Frantz, 1989. Changes in quality of stallion spermatozoa during cryopreservation: Plasma membrane integrity and motion characteristics. Theriogenology, 31: 283-298. CrossRef | PubMed | Direct Link |
3: Brito, L.F.C., 2007. Evaluation of stallion sperm morphology. Clin. Tech. Equine Pract., 6: 249-264. CrossRef | Direct Link |
4: Chan, S.Y.W., C. Wang, S.T.H. Chan, P.C. Ho, W.W.K. So. Y.F. Chan and H.K. Ma, 1989. Predictive value of sperm morphology and movement characteristics in the outcome of in vitro fertilisation of human oocyte. J. In Vitro Fertil. Emb. Trans., 6: 142-148. CrossRef | Direct Link |
5: Christensen, P., J.M. Parlevliet, A. Van Buiten, P. Hyttel and B. Colenbrander, 1995. Ultrastructure of fresh and frozen-thawed stallion spermatozoa. Biol. Rep., 1: 769-777. Direct Link |
6: Garavance, C.G., Z. Champion, I.K.M. Liu and P.J. Casey, 1997. Sperm head morphometry analysis of ejaculate and dismount stallion semen samples. Anim. Rep. Sci., 47: 149-155. CrossRef | Direct Link |
7: Haras Nationaux, 2004. Equine artificial insemination. Practical Guide Pratique, 3rd Edition, Direction of Knowledge, ENPH 61310, Le Pin au Haras. http://www.haras-nationaux.fr/.
8: Holt, W.V. and R.D. North, 1986. Thermotropic phase transitions in the plasma membrane of ram spermatozoa. J. Reprod. Fertil., 78: 447-457. CrossRef |
9: Jasko, D.J., D.H. Lein and R.H. Foote, 1990. Determination of the relationship between sperm morphologic classifications and fertility in stallions: 66 cases (1987-1988). J. Am. Vet. Med. Assoc., 197: 389-393. Direct Link |
10: Katila, T., 2001. In vitro evaluation of frozen-thawed stallion semen: A review. Acta Vet. Scand., 42: 199-217. PubMed |
11: Love, C.C., D.D. Varner and J.A. Thompson, 2000. Intra and inter-variation in sperm morphology and their relationship with fertility. J. Rep. Fertil., 56: 93-100. PubMed | Direct Link |
12: Najjar, A., B. Benaoun, M. Ezzaouia, A.B. Maatoug, M. Magistrini and M.B. Mrad, 2010. Determination of semen and sexual behavir parameters of Arabian stallions to be selected for an Artificial Insemination program under Tunisian conditions. Am. Eurasian J. Agric. Environ. Sci., 8: 173-177. Direct Link |
13: Varner, D.D., 2008. Developments in stallion semen evaluation. Theriogenology, 70: 448-462. CrossRef | Direct Link |
14: Voss, J.L., B.W. Pickett and E.L. Squires, 1981. Stallion spermatozoal morphology and motility and their relationships to fertility. J. Am. Vet. Med. Assoc., 178: 287-289. PubMed | Direct Link |
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