ULTRASTRUCTURAL CHANGES IN DOG SPERMATOZOA AFTER FREEZING–THAWING OF SEMEN EXTENDED IN TRIS–BASED DILUENT SUPPLEMENTED WITH EQUEX STM

Wojciech Niżański¹, Piotr Kuropka^2
1 Department and Clinic of Obstetrics, Ruminant Diseases and Animal Health Care, Wrocław University of Environmental and Life Sciences, Poland
² Department of Anatomy and Histology, Institute of Histology and Embryology, Agricultural University of Wrocław, Poland

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: semen, spermatozoon, dog, ultrastructure, cryopreservation.

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 ą4.4. 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).

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.

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.

REFERENCES

1. Battista M., Parks J., Concannon P.: Canine sperm post-thaw survival following freezing in straws or pellets using PIPES, lactose, TRIS or TEST extenders. Proc. 11^th Int. Congr. Anim. Reprod. Art. Insem., Dublin, 1988, vol.3, 229-230.

2. Bochenek M., Smoršg Z., Pilch J.: Sperm chromatin structure assay of bulls qualified for artificial insemination. Theriogenology 2001, 56, 557-567.

3. Froman D.P., Amann R.P., Riek P.M., Olar T.T.: Acrosin activity of canine spermatozoa as an index of cellular damage. J. Reprod. Fert. 1984, 70, 301-308.

4. Goodman M. Ovulation timing. Concepts and controversies. Vet Clin North Am Small Anim Pract 2001, 31, 219-235.

5. Günzel-Apel A.R.: Fertilitätskontrolle und Samenübertragung beim Hund. Gustav Fischer Verlag, Jena 1994.

6. Hashizume T., Tanimura I., Kanematsu S.: Ultrastructures of the acrosome in frozen-thawed boar spermatozoa by the pellet freezing method. Jpn. J. Anim. Reprod. 1990, 36, 195-202.

7. Healey P.: Effect of freezing on the ultrastructure of the spermatozoon of some domestic animals. J. reprod. Fert. 1969, 18, 21-27.

8. Johnston S.D., Root Kustritz M.V., Olson P.N.S.: Canine and Feline Theriogenology. W.B. Saunders Comp., Philadelphia 2001.

9. Linde-Forsberg C., Ström Holst B., Govette G.: Comparison of fertility data from vaginal vs. intrauterine insemination of frozen-thawed dog semen: a retrospective study. Theriogenology 1999, 52, 11-23.

10. Niżański W., Bielas W.: Wpływ Equex STM na wybrane własciwosci plemników psa w nasieniu poddanym konserwacji w niskich temperaturach [Effect of Equex STM on the quality of frozen-thawed dog semen]. Medycyna Wet. 2003, 59, 691-695 [in Polish].

11. Niżański W., Dubiel A., Bielas W., Dejneka G.J.: Effect of three cryopreservation methods and two semen extenders on the quality of dog semen after thawing. J. Reprod. Fert., Suppl. 2001, 57, 365-369.

12. Niżański W., Klimowicz M., Dubiel A.: Technika inseminacji domacicznej, dawka inseminacyjna i wyznaczanie optymalnego terminu unasienniania suk [Artificial insemination in bitches: intrauterine insemination technique, insemination dose and determining the optimal time of insemination]. Medycyna Wet. 2004, 60, 915-919 [in Polish].

13. Oettlé E.E.: Changes in acrosome morphology during cooling and freezing of dog semen. Anim. Reprod. Sci. 1986, 12, 145-150.

14. Oettle E.E., Soley J.T.: Sperm abnormalities in the dog: a light and electron microscopic study. Vet. Med. Rev. 1988, 1, 28-70.

15. Pedersen H., Lebech P.E.: Ultrastructural changes in the human spermatozoon after freezing for artificial insemination. Fert. Steril. 1971, 22, 125-133.

16. Peña A.I., Lugilde L.L., Barrio M., Herradón P.G., Quintela L.A..: Effects of Equex from different sources on post-thaw survival, longevity and intracellular Ca⁺² concentration of dog spermatozoa. Theriogenology 2003, 59, 1725-1739.

17. Rodriguez-Martinez H., Ekwall H., Linde-Forsberg C.: Fine structure and elemental composition of fresh and frozen dog spermatozoa. J. Reprod. Fert., Suppl. 47, 1993, 279-285.

18. Rota A., Iguer-Ouada M., Verstegen J., Linde-Forberg C.: Fertility after vaginal or uterine deposition of dog semen frozen in a TRIS extender with of without Equex STM Paste. Theriogenology 1999, 51, 1045-1058.

19. Ström-Holst B., Rota A., Andersen Berg K., Linde-Forsberg C., Rodriguez-Martinez H.: Canine sperm head damage after freezing-thawing: ultrastructural evaluation and content of selected elements. Reprod. Dom. Anim. 1998, 33, 77-82.

20. Strzeżek J., Głogowski J., Magierska E., Luberda Z., Jabłonowska Cz.: Some aspects of cryobiochemistry of boar semen. Proc. X Int. Congr. Anim. Reprod. Art. Insem., Urbana-Champaign, 244, 1984.

21. Thomas P.G.A., Surman V., Myers-Wallen V.N., Concannon P.W., Ball B.A.: Addition of sodium dodecyl sulphate to Tris-citrate extender improves motility and longevity of frozen-thawed canine spermatozoa. Proc. 12^th Int. Congr. Anim. Reprod. Art. Insem., Hague, 1992, vol. 4, 1823-1825.

22. Tsutsui T., Hase M., Hori T., Komoriya K., Shimizu N., Nagakubo K., Kawakami W.: Effect of addition of Orvus ES Paste to frozen canine semen extender on sperm acrosomes. J. Vet. Med. Sci. 2000, 62, 537-538.

23. Watson P.F.: Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reprod. Fert. Dev. 1995, 7, 871-891.

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

Responses to this article, comments are invited and should be submitted within three months of the publication of the article. If accepted for publication, they will be published in the chapter headed 'Discussions' and hyperlinked to the article.