1Department of Animal Sciences, Agricultural University – Plovdiv, Bulgaria Wydział Nauk o Zwierzętach, Uniwersytet Rolniczy w Płowdiw, Bułgaria
2 National Diagnostic and Research Veterinary Medical Institute – Sofia, Bulgaria Krajowy Instytut Diagnostyki i Badań Medycyny Weterynaryjnej w Sofii, Bułgaria
3 Department of Cryobiology, Institute of Biology and Immunology of Reproduction – Sofia, Bulgaria
Zakład Kriobiologii, Instytut Biologii i Immunologii Rozrodu w Sofii, Bułgaria
In this study the effect of six cryoprotectans – glycerol, dimethyl sulfoxide, polyethylene gly-col, thiodiethylene glygly-col, ethylene glycol and diethyl glycol on the cryopreservation of Muscovy drake semen was investigated.
The semen was collected twice a week from 9 one-year-old Muscovy drakes, with artificial vagina and a female as teaser. Semen samples were diluted 1:1 (semen:diluent) with HIA-1 diluent enriched with 15% egg yolk (v/v), equilibrated at 4°C for 30 min without cryoprotectant and once again equilibrated in the same conditions with one of the six tested cryoprotectants in final con-centration of 5%, dispensed into plastic straws, prefrozen to -140°C at 60°C/min and plunged into liquid nitrogen container. The thawing procedure was conducted in a water bath at 42°C.
Cryopreservation of Muscovy drake semen affected the morphological integrity and motility of spermatozoa. The use of glycerol was more successful in contrast to other cryoprotective agents, in relation to sperm mobility and morphological integrity. The sperm mobility were 36.43 ±2.66% with glycerol, 24.40 ±1.68% with dimethyl sulfoxide, 12.33 ±4.55% with ethylene glycol, 8.33 ±2.04% with polyethylene glycol, 1.85 ±1.25% with diethylene glycol and 0% with thiodiethylene glycol, i.e. 100% dead sperm cells in unfrozen semen. Muscovy spermatozoa were sensitive to
For citation – Do cytowania: GerzilovV., Kazachka D., Jeleva S., Petrov P., Sabev M., Nikolov I.,
2011. Effect of cryoprotectants on the mobility and the morphological integrity of muscovy spermatozoa. Zesz. Nauk. UP Wroc., Biol. Hod. Zwierz., LXII, 580: 157–165.
158 Vasko Gerzilov et al.
cryopreservation. The greatest damages were observed in the perforatorium, the midpiece and the mitochondrial helix.
KEY WORDS: Muscovy ducks, semen, cryopreservation, cryoprotectant, morphological integrity
iNTROdUCTiON
Species differences in spermatozoa sensitivity to freezing i.e. to cryogenic conditions, the choice of cryo-technology and the effectiveness of the cryoprotective agent are the foundation of a successful cryopreservation. The preservation of animal semen, including poultry, in the frozen state has been the subject of intense scientific interest beginning ap- beginning ap-beginning ap-proximately 60 years ago by Polge et al. (1949) and Polge (1951) with the discovery of glycerol as a good cryoprotective medium. Despite fact that this scientific breakthrough was accomplished with rooster semen (Polge 1951), the overall fertility rates with frozen/ thawed poultry semen are highly variable and not reliable enough for use in commercial production or preservation of genetic stocks (Long 2006).
Considerably more comprehensive studies and more remarkable success have been conducted on freezing the rooster and turkey sperm (Schramm, Hubner 1988, Hammerstedt 1995, Chalah et al. 1999, Blesbois et al. 2008) as well as on gander sperm (Tai et al. 2001, Łukaszewicz 2002, Łukaszewicz, Kruszynski 2003, Łukaszewicz et al. 2004) compared with the drake sperm (Maeda et al. 1984, Kasyanenko, Kurbatov 1986, Tselutin et al. 1995).
One of the most critical steps in successful cryopreservation of avian and mammalian semen is the choice of the cryoprotectant and its use during the process (Tselutin et al. 1999, Holt 2000, Han et al. 2005, Herrera et al. 2005). Many compound have been tested for their efficacy as sperm cryoprotectants, but most extensively the glycerol, dimethyl sulfoxide, dimethyl acetamide, dimethyl formamide, diethyl formamide, ethylene glycol, propylene glycol (Lake, Ravie 1982, Hammerstedt, Graham 1992, Surai, Wishart 1996, Tselutin et al. 1999, Holt 2000, Tai et al. 2001, Łukaszewicz 2001).
The aim of this study was to compare the effect of six cryoprotectans – glycerol, di-methyl sulfoxide, ethylene glycol, diethylene glycol, polyethylene glycol, thiodiethylene glycol on the cryopreservation of Muscovy semen.
МАТЕRiAl ANd mEThOdS
Birds
The experiment was carried out in the Poultry Farm of the Agricultural University of Plovdiv with 9 one-old-year Muscovy drakes. During entire reproductive season the males were kept individually in cages (0.6/0.8/0.6 m in size) under natural daylight and ventilation. The birds were fed with mixed diet consisting ME – 11.5 MJ/kg and CP – 16.0% and daily ration from 200 to 250 g/bird.
Effect of cryoprotectants on the mobility ... 159 Semen collection
The semen was collected individually by placing the female into the cage of Muscovy drake, using an artificial vagina and a female as teaser, two times per week (Tan 1980, Gerzilov 2000). The artificial vagina consisted of a rubber muff and a graduated test-tube.
Semen evaluation
Only good quality ejaculates (color – pearly-white; purity – free of any contamination with cloacal products; volume – above 0.3 ml; sperm mobility – above 70%, sperm con-bility – above 70%, sperm con-ility – above 70%, sperm con- sperm con-centration – above 1x109 sperm/mL) were use for cryopreservation.
The pooled semen was diluted at 1:1 ratio (semen : diluent) with HIA-1 extender and added to him egg yolk in a concentration of 15% (v/v). The HIA-1extender was created by Gerzilov (2003) and contains: 0.25 g D–glucose, 0.25 g D–fructose, 0.07 g saccharose, 0.50 g sodium citrate, 0.9 g sodium chloride, and 100 mL double distilled water. The osmolarity was 290 mOsmol/kg and pH – 7.00. Constituents of HIA-1 extender were supplied from Fluka-Riedel-de Haën.
The diluted semen was equilibrated in a refrigerator at 4°C for 30 min without a cryo-protectant. Then the semen was divided into six equal parts and again equilibrated under the same conditions by supplementing the glycerol, dimethyl sulfoxide, ethylene glycol, diethylene glycol, polyethylene glycol or thiodiethylene glycol in a 5% concentration. The semen samples were dispensed into plastic straws, prefrozen to -140°C at 60°C/ in using programmed biofreezer by method of Cassou (1964) and plunged into liquid nitrogen container. The thawing procedure was conducted in a 42°C water bath. In the freshly collected pooled semen, diluted and equilibrated semen the following parameters were evaluated:
Sperm mobility (%) – by visual determination of the spermatozoa with progressive •
motions using Nikon Alphaphot-2YS2 microscope (х 400 magnification),
Abnormal and dead spermatozoa (%) by supravital eosin/nigrosin staining and ob-bnormal and dead spermatozoa (%) by supravital eosin/nigrosin staining and ob- and dead spermatozoa (%) by supravital eosin/nigrosin staining and ob-ead spermatozoa (%) by supravital eosin/nigrosin staining and ob-and ob-•
servation by phase-condenser lens MCL 21102 (Nikon Alphaphot-2YS2 microscope) (Bakst, Cecil 1997).
The transmission electron microscopy (TEM) was conducted in National Diagnostic and Research Veterinary Medical Institute – Sofia. The semen samples were fixed in 5% glutaraldehyde solution in a cacodylate buffered salt for 1 h under low temperature (4°C), after that was centrifugated at 1000 g for 10 min and washed with a cacodylate buffered salt solution, followed by a postfixation in 1% osmium tetraoxide in a cacodylate buffered salt solution for 2 h under low temperature (4°C). The fixed material was dehydrated first in graded acetone series (30, 50, 70%) and then in graded ethanol series (70%, 96% and absolute ethanol) and embedded in Durcupan by method of Krustev et al. (1973). The ultrathin sections were cut on an LKB-NOVA Ultramicrotome, stained with uranylacetate and lead citrate and examined in a Tesla BS-500 electron microscope.
160 Vasko Gerzilov et al.
RESUlTS ANd diSCUSSiON
Light microscopy observation
The fresh undiluted semen and equilibrated diluted semen without cryoprotectant were characterized by good quality, regarding sperm mobility, percentage of abnormal and dead spermatozoa. These parameters changed significantly (P<0.001) after equilibration with addition of 5% cryoprotectant. The semen equilibrated with 5% diethylene glycol had most poor quality compared to other used cryoprotectants – the differences were significant in sperm mobility and dead spermatozoa (Tab. 1).
Immediately after thawing, subjective progressive mobility was relatively the best in glycerol comparing to all other cryoprotectants (P<0.001). The sperm mobility in the thawed semen ranged from 0% using thiodiethylene glycol to 36.43 ±2.66% using gly-cerol, and the dead spermatozoa from 38.71 ±2.08% to 100%, respectively. The cryopro-tecntants thiodiethylene glycol, diethylene glycol, polyethylene glycol, ethylene glycol proved to be extremely inappropriate and the most toxic. The cryopreservation process caused significant decrease in sperm mobility and number of normal living spermatozoa. Han et al. (2005) in a series of sequental experiments, obtaining semen from Jinding drakes by abdominal massage, founded that crioprotectant containing 10% dimethyl sulfoxide was better than glycerol, dimethyl acetamide and dimethyl formamide. This cryoprotectants with 4%, 6% and 8% contents had worse effect on the postthaw sperm mobility. According to this authors, the optimum equilibration time was 15 min vs. 0, 30, 60 and 120 min, and the optimum thawning temperature was 40°C. In our previously study, the postthaw sperm mobility was lower using cryoprotectants with 3% and 7% vs. 5% contents including glicerol and dimethyl sulfoxide (Gerzilov 2010).
Electron microscopy observation
Frozen-thawed sperm had higher incidences of ultrastructural abnormalities of the perforatorium, midpiece and mitochondrial helix. The fully destroyed treated plasma- treated plasma-treated plasma-lemma with partial damage of the axonemal complex integrity was observed. The ripples of fibrous shield were well preserved and the microtubules in the principal piece of the tail (Phot. 1–2). Previously Xia et al. (1988) established similar observations in rooster sperm. According to Maeda et al. (1984) the most radical change in Muscovy sperm cells were the complete separation of the acrosome from the apical part of the nucleus. In our previously study a high percent of dead and abnormal spermatozoa in unfrozen semen was also established (Gerzilov 2010). Generally, avian spermatozoa are more sensitive to the freezing-thawing process and fertility rates of cryopreserved poultry sperm are dra-matically lower than any of the domestic mammalian species (Donoghue, Wishart 2000, Long 2006). According to Tselutin et al. (1995) the cryopreservation of Muscovy drake semen is more difficult than Pekin drake semen. However, the experiments indicate that spermatozoa of commercial lines of these two species of duck are able to be frozen with reasonable success of fertility of frozen-thawed semen (Blesbois 2007).) The cryobanking for other species than the chicken remains extremely limited (Blesbois, Brillard 2007).
Effect of cryoprotectants on the mobility ... 161
Table 1 Tabela 1 Characteristics of fresh, equilibrated and frozen-thawed Muscovy drake semen with different
cryoprotectants (n = 8; means ± SХ)
Charakterystyka świeżego, ekwilibrowanego i mrożonego–rozmrożonego nasienia kaczorów piżmowych z różnym krioprotektantem (n=8, średnia ± Sx)
Semen samples Próbki nasienia Sperm mobility (%) Ruchliwość plemników Viable abnormal spermatozoa (%) Plemniki żywe Dead spermatozoa (%) Plemniki martwe Fresh undiluted Świeże nierozrzedzone 78.17 ± 0.46 9.83 ± 1.15 8.33 ± 0.97
Diluted semen quilibrated for 30 min without cryoprotectants
Rozrzedzone i ekwilibrowane przez 30 min bez środka osłaniającego
75.67 ± 0.46 0.67 ± 1.01 9.17 ± 0.82
Equilibrated semen for 30 min with 5% cryoprotectant Ekwilibrowane przez 30 min ze środkiem osłaniającym Glycerol Glicerolem 62.17 ± 1.11A1 15.50 ± 0.74n.s 16.83 ± 1.11B1 Dimethyl sulfoxide Dwumetylo sulfotlenkiem 60.50 ± 1.76A2 16.67 ± 0.54n.s. 17.88 ± 0.82B2 Polyethylene glycol Glikolem polietylenowym 60.67 ± 1.62A3 17.33 ± 0.83n.s. 18.50 ± 1.49C1 Thiodiethylene glycol Glikolem tiodietylenowym 58.33 ± 3.06C1 17.83 ± 2.79 19.33 ± 0.94C2 Ethylene glycol Glikolem etylenowym 62.50 ± 2.35A4 14.67 ± 1.32n.s. 18.33 ± 1.25C3 Diethylene glycol Glikolem dietylenowym A47.50± 2.351; A2; A3; A4; C1 20.17± 2.70n.s. B24.33± 2.051; B2; C1; C2; C3
Thawed semen with 5% content of: – Nasienie rozmrożone z 5% dodatkiem: Glycerol Glicerolu 36.43 ± 2.66a1; a2; a3; a4 23.57 ± 1.35a1; a2; 38.71 ± 2.08a1; a2; c1; c2 Dimethyl sulfoxide Dwumetylo sulfotlenku 24.40 ± 1.68a1; a5; a6; c1 27.60 ± 1.44 a3; a4; 44.60 ± 2.17a3; a4 Polyethylene glycol Glikolu polietylenowego 8.33 ± 2.04a2; a5; c2 37.33 ± 1.47a1; a3; a5; c1 54.33 ± 6.87a5; c1 Thiodiethylene glycol Glikolu tiodietylenowego 0 0 100 Ethylene glycol Glikolu etylenowego 12.33 ± 4.55a3; c1 13.67 ± 8.55c1 75.67 ± 14.94c2 Diethylene glycol Glikolu dietylenowego 1.85 ± 1.25a4; a6; c2 5.00 ± 3.54a2; a4; a5; 93.20 ± 4.71a2; a3; a4; a5
Note: Means in columns with equal subscript differ significantly: Średnie w kolumnach z jednakowymi przypisami różnią się istotnie:
for equilibrated semen with different cryoprotectants at P<0.001 A1–A1 … A4–A4; at P<0.01 B1–B1; B2–B2; at P<0.05 C1–C1… C3–C3
dla nasienia ekwilibrowanego z dodatkiem różnych środków osłaniających przy: P<0,001 A1–A1 … A4–A4; przy P<0,01 B1–B1; B2–B2; przy P<0,05 C1–C1… C3–C3
for thawed semen with different cryoprotectanst at P<0.001 a1–a1 … a6–a6; at P<0.05 c1–c1; c2–c2
162 Vasko Gerzilov et al.
Phot. 1. Swelling and destruction of plasmalemma – longitudinal section x 10 000 Fot. 1. Zgrubienie i zniszczenie plasmalemma – sekcje podłużna x 10 000
Phot. 2. Destroyed mitochondrial helix and plasmalemma – x 10 000 Fot. 2. Zniszczone mitochondrialny helix i plasmalemma x 10 000
CONClUSiONS
Cryopreservation of Muscovy drake semen affected the spermatozoa morphological integrity and mobility. The use of glycerol was the most successful in contrast to other cryoprotectants.
Effect of cryoprotectants on the mobility ... 163
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WPŁYW śRODKóW KRIOOCHRONNYCH NA RUCHLIWOść I INTEGRALNOść mORFOLOGICZNĄ PLEmNIKóW
KACZEK PIŻmOWYCH S t r e s z c z e n i e
W pracy badano wpływ sześciu środków kriochronnych – glicerolu, dwumetylo sulfotlenku, glikolu polietylenowego, glikolu tiodietylenowego, glikolu etylenowego oraz glikolu dwuetylowe-go na proces kriokonserwacji nasienia kaczorów piżmowych.
Nasienie pobierano dwa razy w tygodniu od 9 jednorocznych kaczorów piżmowych z wyko-rzystaniem sztucznej pochwy i samic do stymulacji płciowej samców. Próbki nasienia rozrzedzano w proporcji 1:1 (nasienie:rozcieńczalnik) rozcieńczalnikiem HIA-1 wzbogaconym 15% dodatkiem żółtka jaja (v/v) ekwilibrowano w temp. 4°C przez 30 min bez dodatku środka krioochronnego oraz ponownie w tych samych warunkach po dodaniu jednego z sześciu środków krioochronnych w ilości 5% w stosunku do łącznej objętości nasienia z rozcieńczalnikiem. Tak przygotowane próbki wciągano do plastikowych pajetek, przemrażano do temp. -140°C z prędkością 60°C/min i wrzuca-no do kontenera z ciekłym azotem. Rozmrażanie próbek przeprowadzawrzuca-no w łaźni wodnej o temp. 42°C.
Proces kriokonserwacji nasienia kaczorów piżmowych wpłynął na integrację morfologiczną i ruchliwość plemników. Zastosowanie glicerolu miało najkorzystniejszy wpływ na ruchliwość i integrację morfologiczną plemników w porównaniu z pozostałymi środkami krioochronnymi.
Effect of cryoprotectants on the mobility ... 165
Ruchliwość plemników wynosiła 36.43 ±2.66% przy użyciu glicerolu, 24.40 ±1.68% z dwumetylo sulfotlenkiem, 12.33 ±4.55% z glikolem etylenowym, 8.33 ±2.04% po dodaniu glikolu poliethle-nowego, 1.85 ±1.25% glikolu dwuetylenowego i 0% po użyciu glikolu tiodietylepoliethle-nowego, tj. w nie-mrożonym nasieniu 100% plemników było martwych. Plemniki kaczorów piżmowych były bardzo wrażliwe na proces mrożenia. Największe zmiany obserwowano w perforatorium, wstawce oraz spirali mitochondrialnej.
Reviewer – Recenzent: Ewa Łukaszewicz, Prof. Dr. Sci., Wrocław University of Envi-ronmental and Life Sciences
ZESZYTY NAUKOWE
UNIWERSYTETU PRZYRODNICZEGO WE WROCŁAWIU 2011 BIOLOGIA I HODOWLA ZWIERZĄT LXII NR 580