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Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.

Volume 8
Issue 4
Topic:
ELECTRONIC
JOURNAL OF
POLISH
AGRICULTURAL
UNIVERSITIES
. , EJPAU 8(4), #54.
Available Online: http://www.ejpau.media.pl/volume8/issue4/art-54.html


 

ABSTRACT

The aim of the present study was to characterise the ultrastructural changes present in canine spermatozoa after freezing-thawing with the use of extender with addition of surface active agent Equex STM. The experiment was carried out on 10 ejaculates collected from 5 dogs of various breeds. Semen was extended in Tris–based diluent with addition of Equex STM. Glycerolisation started at 5°C. Semen was frozen in 0.5 ml French straws. Results of microscopical examination of frozen-thawed spermatozoa seemed to be excellent. Microscopically assessed post-thaw percentage of progressively motile spermatozoa, percentage of spermatozoa with normal morphology, percentage of cells with intact acrosome and proportion of live spermatozoa after thawing were 60.5 ±4.4, 62.1 ±6.8, 67.7 ±4.8 and 62.6 ±7.7, respectively. Transmission electron microscopic examination of frozen–thawed semen revealed major changes in the morphology of spermatozoa localised predominantly within the acrosome and postacrosomal region of a head. In many cells the acrosome and equatorial segment of a spermatozoal head were damaged. The most common alterations seen in frozen–thawed dog spermatozoa were due to the process of false acrosome reaction. Several degrees of false acrosome reaction were observed: swelling of acrosomes, vesiculation of outer acrosomal membrane and plasma membrane, and complete detachment of the acrosome. The degree and character of deterioration of the acrosome structure of cryopreserved dog spermatozoa extended in Tris–based and Equex STM–supplemented diluent is similar to ultrastructural changes observed in spermatozoa cryopreserved in other Tris extenders without surface–active agent addition.

Key words: .

INTRODUCTION

It is generally assumed that freezability of the dog spermatozoa is high [5, 8, 10, 23]. Routinely performed microscopic evaluation of semen revealed, that the proportion of spermatozoa with damaged acrosomes and percentage of cells with deteriorated membrane integrity after thawing are relatively low [11, 13, 16, 23]. The post-thaw motility of the dog spermatozoa is high, i.e. approximately 50-60% of them are progressively motile [5, 11, 16]. In spite of this, longevity of dog spermatozoa after thawing is considerably short [1, 21, 22]. According to many workers [9, 12, 18] variable and usually low conception rates obtained after artificial inseminations of bitches with frozen-thawed semen are partly due to a poor livability of thawed spermatozoa. The abrupt decrease of post-thaw motility of dog spermatozoa in first hours after thawing may be due to the latent changes in spermatozoal structure. This latent damage is not detectable by microscopic evaluation of the dog semen. Recently investigated method of protection and stabilisation of membranes of dog spermatozoa uses the surface-active ingredients, such as Equex STM, as the additives to semen extenders [10, 18, 21, 22]. Such substances prolong livability of spermatozoa after thawing [11, 22]. Moreover, to reduce the toxic effect of cryoprotectants on spermatozoal metabolism, the addition of the glycerol to extended semen at 5°C, rather that at 33°C is used. It is difficult to identify all post–thaw morphological abnormalities of spermatozoal structure using microscopical techniques of semen evaluation. These methods of semen assessment allow only for observation of the surface and edges of cells. Transmission electron microscopy (TEM) examination is more reliable method, which allows to observe the changes in cell membranes and other inner structures of cells [14, 17, 19]. TEM seems to be the proper method of observation of the character of changes in spermatozoa cryopreserved with the use of recently investigated modifications of semen extension and cryoprotection.

The aim of the present study was to characterise the ultrastructural changes present in canine spermatozoa after freezing-thawing with the use of extender with addition of surface-active agent Equex STM. Glycerolisation process of the semen before freezing-thawing started at 5°C.

MATERIAL AND METHODS

Cryopreservation and microscopical evaluation
The experiment was carried out on 10 ejaculates collected from 5 privately owned dogs of proven fertility of various breeds (2 German shepherd, 1 Great Dane, 1 elkhound and 1 golden retriever). Semen was extended in Tris–based diluent with 1% (volume:volume) addition of Equex STM (active ingredient SDS–sodium dodecyle sulphate, Minitüb GmbH, Germany) [11]. The extended semen was cooled to 5°C for 90 minutes. Glycerol was added at 5°C and equilibration of spermatozoa lasted for additional 90 minutes. Semen was frozen in 0.5 ml French straws (IMV Technologies, France). Semen was thawed at 70°C for 5 seconds.

The post–thaw progressive motility was assessed using Computer Assisted Sperm Analyser IVOS ver. 12.2l (Hamilton Thorne Biosciences, USA). Proportion of live spermatozoa was evaluated with the use of eosin–nigrosin staining procedure [8]. The morphology of spermatozoa was assessed microscopically after staining of semen smears with Giemsa stain [11].

Transmission electron microscopy
For transmission electron microscopy, immediately after thawing the aliquots of spermatozoa from each sample were prefixed in 3% glutaraldehyde in 0.1 M cacodylate buffer and postfixed with 2% OsO4 in 0.1 M cacodylate buffer for 2 hours. The samples were centrifuged and pellets were dehydrated in increasing acetone concentrations and embedded in Polarbed 812 epoxy resin. The blocks were cut on an ultramicrotome and ultrathin serial sections picked up on copper grids. The sections were counterstained with uranyl acetate and lead citrate and examined in Tesla BS 613 TEM microscope (Tesla, Czech) at 60 kV. Longitudinal, oblique and transverse sections of spermatozoa were included in the evaluation [14, 15, 19].

RESULTS

The percentage of progressively motile spermatozoa after thawing was 60.5 ±4.4. Microscopically assessed percentage of spermatozoa with normal morphology, percentage of cells with intact acrosome and proportion of live spermatozoa after thawing were 62.1 ±6.8, 67.7 ±4.8 and 62.6 ±7.7, respectively (Tab. 1).

Table 1. Results of microscopic evaluation of the quality of frozen-thawed dog semen (n=10)

Spermatozoal property

Mean ± SD
(range)

Progressively motile spermatozoa (%)

60.5 ± 4.4
(55-65)

Spermatozoa with normal morphology (%)

62.1 ± 6.8
(53.5-74)

Spermatozoa with normal morphology of acrosome (%)

67.7 ± 4.8
(62-77)

Live spermatozoa (%)

62.6 ± 7.7
(54-75)

Transmission electron microscopic examination of frozen–thawed semen revealed major changes in the morphology of numerous spermatozoa localised predominantly within the acrosome and postacrosomal region of a sperm head. In many cells the acrosome and equatorial segment of a head were damaged. Variable forms of structural abnormalities of frozen–thawed spermatozoa were observed. In sagittal sections through the heads of thawed spermatozoa, the plasmalemma often was seen to be loosely attached in the region of acrosome (Fig. 1, 1a). Thee plasmalemma remained, in most cases, intact. The formation of large space between the plasma membrane and outer acrosomal membrane (OAM) was a typical alteration in frozen–thawed spermatozoa. Cysts and disintegration of plasmalemma were rarely observed over the acrosome (Fig. 2). The presence of space between inner acrosomal membrane (IAM) and nucleus was also seen. The most common alterations seen in frozen–thawed dog spermatozoa were due to the process of false acrosome reaction (FAR). Several degrees of FAR were observed: swelling of acrosomes with formation of irregular space between IAM and OAM and between OAM and plasma membrane (fuzzy appearance of acrosome with loss of unevenly distributed electron dense material) (Fig. 3), vesiculation of plasma membrane and OAM (Fig. 4), complete detachment of the acrosome. The cross sections through the middle piece revealed that the mitochondrial matrix is thinned and dense deposits of firmly granulated material were found regularly within mitochondrium (Fig. 5). Observations of numerous transverse sections through the spermatozoal tails revealed the normal axial 9+2 fiber pattern. The coarse and fine fibers of the axoneme have been clearly seen.

Fig. 1. Major changes in ultrastructure of frozen-thawed dog spermatozoa are localised within acrosomal segment of sperm head – loosely attached plasma membrane in the region of acrosome and equatorial segment; particles of egg yolk on the background (x 14 000)

Fig. 1a. Formation of large space between plasma membrane and outer acrosomal membrane; in most of cells plasmalemma remained intact (x 24 000)

Fig. 2. Cyst and disintegration of plasmalemma at apical and lateral part of acrosome (x 14 000)

Fig. 3. Irregular space between inner and outer acrosomal membrane and between outer acrosomal membrane and plasma membrane – fuzzy appearance of acrosome; unevenly distributed electron dense material within the acrosome
(x 24 000)

Fig. 4. Vesiculation of outer acrosomal membrane and plasma membrane – false acrosome reaction (x 14 000)

Fig. 5. Firmly granulated material was found within mitochondrium; normal axial 9+2 fiber pattern in cross sections of sperm tails (x 28 000)

DISCUSSION

Many researchers suggest that percentage of progressively motile spermatozoa in frozen–thawed semen used for artificial insemination of bitches should exceed 40% [5, 12]. Moreover microscopic evaluation of frozen–thawed spermatozoa used for artificial insemination should revealed not less than 50% of cells with intact acrosome [5]. Thus the quality of frozen–thawed semen obtained in the present study seems to be excellent, when microscopic examination is the only one criterion of semen evaluation. However, examination using transmission electron microscopy revealed severe damage of spermatozoal ultrastructure, predominantly within the acrosome. It should be noted, that variable forms of cell deterioration were observed, in spite of the optimisation of cryopreservation procedures by means of addition of glycerol at 5°C and supplementation of extender with Equex STM. It seems therefore, that prolongation of post–thaw livability of spermatozoa extended in Equex STM–containing media are rather due to the changes in spermatozoal metabolism than stabilisation of plasmalemma and acrosomal membranes. Thus the direct action of surface–active agents on mitochondrial function may be supposed. The subtle changes of mitochondrial matrix and mitochondrial membranes support this hypothesis. Using microscopic and biochemical methods of examination of semen other researchers [20] found, that the introduction of surface–active substances into extender results in dispersing the yolk–plasmatic conglomerates which form directly after semen dilution. This phenomenon slightly increases the stabilising effect towards spermatozoal membranes by yolk lipoprotein.

In the present study transmission electron microscopy examination did not revealed any changes in the chromatin structure of the nucleus. In contrast, X–ray microanalysis of thawed dog spermatozoa showed the decrease of intracellular concentration of potassium, sulphur, chlorine and the increase of the concentration of intracellular calcium [17, 19]. It might be a reflection of DNA damage. The use of acridine–orange staining was shown that freezing and thawing alter the stability of spermatozoal chromatin in mammals [2]. The major defects of spermatozoa exerted by freezing–thawing procedures are seen within the acrosome [6, 7, 17, 19]. Acrosome contains hydrolytic enzymes, which are necessary for penetration of corona cells and zona pellucida of the ovum [3, 23]. Consequently the fertilizing ability of spermatozoa are determined by integrity of acrosomal structure. On the other hand, it is not known which types of acrosomal deterioration exclude the ability of spermatozoa to penetrate of ova and which type of them do not exclude the normal course of fertilisation process. Watson [23] concluded that frozen–thawed spermatozoa of mammals are in state resembling acrosome reaction (FAR–false acrosome reaction). Characteristic feature of these cells is the ability to fertilise the ova immediately after thawing. Due to the short livability, the frozen–thawed sperm should be deposited into genital tract several hours after ovulation, i.e. in a time corresponding with a complete maturation of oocytes. Defects of acrosomes observed using transmissible electron microscopy in the present study might be responsible for reduced fertilising ability and low conception rates obtained after artificial insemination of bitches with frozen–thawed semen, even when intrauterine insemination is performed [18]. The fact, that thawed dog spermatozoa are in a state of false acrosome reaction and that they are capable to fertilise the ovum for a short time immediately after thawing, necessitates the optimisation of the time of artificial insemination of bitches. Dog oocytes reach maturity in vivo within 2 days after the onset of ovulation and 4 days after luteinizing hormone (LH) peak [4]. Consequently the frozen–thawed semen should be deposited directly into the uterus between day 4 and 6 after LH peak. [4, 12].

In conclusion, the procedure of cryopreservation of the semen exerts major damage of the dog spermatozoa, especially in the region of acrosome. The degree and character of the deterioration of the acrosome structure of cryopreserved dog spermatozoa extended in Tris–based and Equex STM–supplemented diluent is similar to ultrastructural changes observed in spermatozoa cryopreserved with the use of other Tris-based extenders without surface–active agent addition. The prolongation of post–thaw livability of spermatozoa extended in Equex STM–supplemented diluent is of value, but cryopreservation procedures allowing for better protection of acrosomal structure need further investigation.

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Study conducted within the realization scope of project KBN 3PO6K 004 23.



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