The investigation was performed to evaluate the dog semen freezability and itsquality after thawing allowing its use for artificial insemination (AI). On the basis ofsperm motility, concentration and alkaline phosphatase (AP) activity in semenplasma it was possible to establish that AP activity corresponds with the basic factorof semen examination. Significant statistical differences occurred between thequality of ejaculates which were qualified or disqualified to deep freezing and AI.These results show that AP activity in raw dog semen plasma can be used as amarker for the dog semen qualification for deep freezing and AI with 95%probability of the prognosis of the results

BITTMAR A.

KOSINIAK-KAMYSZ K.

PODSTAWSKI Z.

K. KOSINIAK-KAMYSZ

A. BITTMAR

Z. PODSTAWSKI

Directory of Open Access Journals

  131Lucrări ştiinţifice Zootehnie şi Biotehnologii, vol. 40(1), (2007), Timişoara  ALKALINE PHOSPHATASE ACTIVITY AS A MARKER OF DOG SEMEN FREEZABILITY  ACTIVITATEA FOSFATAZEI ALCALINE CA MARKER PENTRU CAPACITATEA DE CONGELARE A SPERMEI DE CÂINE  KOSINIAK-KAMYSZ K., BITTMAR A., PODSTAWSKI Z. Department of Horse Breeding, University of Agriculture, Al. Mickiewicza 24/28, Kraków 30-059, Poland  The investigation was performed to evaluate the dog semen freezability and its quality after thawing allowing its use for artificial insemination (AI). On the basis of sperm motility, concentration and alkaline phosphatase (AP) activity in semen plasma it was possible to establish that AP activity corresponds with the basic factor of semen examination. Significant statistical differences occurred between the quality of ejaculates which were qualified or disqualified to deep freezing and AI. These results show that AP activity in raw dog semen plasma can be used as a marker for the dog semen qualification for deep freezing and AI with 95% probability of the prognosis of the results. Key words: dog semen, freezability, AP.  Introduction  The increase in the dog artificial insemination with the use of frozen semen has been observed in recent years in the world. It was due to the needs of the dog breeders, who wanted to raise the genetic value of their dogs which is especially important in rare dog breeds. Biotechnical and biotechnological solutions in dogs’ reproduction can give satisfactory results and artificial insemination with the use of the frozen semen is a key to success in breeding work. On the other hand, it is known that the fertility rate after insemination with the use of the frozen semen is lower in comparison to the raw semen. For that reason, it is necessary to recognize relations which occurr between raw semen quality and sperm membrane integration, which determines semen freezability and its biological quality after thawing. The research work performed by Strzeżek (1988), Kosiniak et al. (1992), Kosiniak and Bittmar (1994), Niżański (2004) indicated that the high correlation occurred between aspartate aminotranspherase (AspAT), lactate dehydrogenase (LDH) activity and basic factors of semen examination. On the basis of these observations it was found that these enzymes can be used as markers in the semen selection for freezing and artificial insemination (AI) with very high probability of results prognosis reaching 80%. Based on the results which were published by   132Glogowski and Strzeżek (1979), Kutzler et al. (2003), Turner et al. (2003) concerning the role of alkaline phosphatase (AP) activity in male reproductive function, the research work was performed on the dog semen with an idea of the use of this enzyme as a factor determining dog semen quality and its ability for deep freezing and artificial insemination.   Material and methods  The experiment was performed on 122 ejaculates obtained from 65 breeding and non breeding dogs. The semen was collected by a glands penis’s masage, the preejaculatory and sperm reach fractions were collected together. After a routine macro- and microscopic semen examination and sperm morphology, AP activity of raw semen plasma was estimated (IU/cm3). On the basis of the semen examination results (sperm motility, concentration and morphology) ejaculates were qualified for freezing with the use of Morton (1989) extender. After centrifugation the semen was diluted in samples of 100 millions motile sperms contained in the extender volume of 1.5 ml. The semen was frozen in mini-straws (0.25 ml) and thawed at 70ºC for 5 sec. Semen quality examination after thawing was based on sperm motility and survival time at 4 ºC. The semen, which represented minimum 30% progressive motility and survived min. 84 hours, was qualified for AI. The remaining ejaculates which represented lower quality were not used for AI. Do not live up to above expectations were eliminated from freezing.  Results and Discussions  The performed analysis showed that the mean AP activity in dog semen plasma was 30.6 IU/cm3 with very high variations (0.5 – 257 IU/cm3). The semen examination revealed that 90 ejaculates (group I) represented the high quality (sperm motility – mean 85%, sperm concentration – mean 0.24 x 109/cm3 (and these ejaculates were qualified for freezing). But in 42 ejaculates (group II) mean sperm motility was 23.3% and sperm concentration – mean 0.24 x 109/cm3 and these ejaculates were not qualified for freezing. It was also found that AP activity in ejaculates qualified for freezing (group I) was significantly higher (23.9 IU/cm3) than in ejaculates unqualified for freezing, in which this enzyme activity was significantly lower (1.2 IU/cm3) (Table 1). A very high correlation occurred also between AP activity and sperm concentration (r=0.75, p≤0.01) and between the number of sperm with progressive motility (r=0.40, p≤0.05) and negative correlation occurred between ejaculate volume (r=-0.38, p≤0.05).  The obtained results correspond with the observation performed by Glogowski and Strzeżek (1979), Glogowski (1988) in boar semen and Kutzler et al. (2003) in dogs’ semen. The research work performed by Turner (1996) and Estrada et al. (2003) showed that the low AP activity characterised the stallion semen with   133oligospermia. These observations are consistent with our results, in which very low AP activity was observed in ejaculates with low sperm concentration and motility while this enzyme activity was 20 times higher in ejaculates qualified for freezing.  It is necessary to stress that a very high AP activity in semen plasma can be sometimes a result of inflammatory process in accessory sex glands or another part of male reproductive system [Turner and McDonnel (2003); Estrada et al. (2003)]. The similar results were also received in the presented observations in the case of 4 ejaculates in which the mean AP activity was very high (150 IU/cm3) while the semen quality was very low.  On the basis of the performed observations, we can state that from 90 ejaculates qualified for freezing, 70 were qualified for AI (group A). It was found that all parameters of this semen examination were very high (sperm concentration – mean 0.26 x 109/cm3, more than 70% morphologically normal sperms and AP activity – 24.5 IU/cm3) while in 20 ejaculates unqualified for AI (group B) these parameters were significantly lower (sperm concentration 0.15 109/cm3, 50% morphologically normal sperms and AP activity 15.6 IU/cm3) (Table 2). A statistical analysis, performed on the basis of AP activity in 70 ejaculates qualified for AI after thawing (24.5 IU/cm3), in 20 ejaculates unqualified for AI (15.6 IU/cm3) and in 32 ejaculates unqualified for freezing (1.2 IU/cm3), showed significant differences between these three groups. Table 1 Parameters of dog semen quality of ejaculates qualified (group I) and unqualified (group II) for freezing  Semen quality parameters  Group I Group II n 90  32 Ejaculate volume (cm3)  AV SD 2.3 0.9 1.4 1.0 Sperm motility Progressive (scale 1-4) AV SD 3.4 0.8 0.7 1.0 Total (%)  AV SD 85.0 7.0 23.3 36.1 Sperm concentration (cm3x109)  AV SD 0.24 0.13 0.02 0.03 AP (U/cm3) AV SD 23.9 14.8 1.2 1.87 AV- mean SD- standard deviation   134On the basis of these observations, it can be concluded that AP activity measurement in dog semen plasma can be a useful marker for its quality examination and it can also be applied for semen selection for freezing and for AI after thawing with very high (95%) probability of the results’ prognosis. Table 2 Parameters of dog semen quality of ejaculates qualified (group A) and unqualified (group B) for artificial insemination  Semen quality parameters  Group A  Group B n 70  20 Ejaculate volume (cm3)  AV SD 2.1 0.8 2.7 0.9 Sperm motility Progressive (scale 1-4) AV SD 3.5 0.6 3.3 0.9 Total (%)  AV SD 86.0 11.9 83.5 8.4 Sperm concentration (cm3x109)  AV SD 0.26 0.11 0.15 0.09 AP U/cm3  AV SD 24.5 13.4 15.6 13.1 U/109pl.  AV SD 100.1 34.1 112.1 33.3  Bibliography  1.  Estrada A., Samper J. C., Lilich J. D., Rathi R. R., Brault L. S., Albrecht B. B., Imel M. M., Senne A. (2003) Azoospermia associated with bilateral segmental aplasia of the ductus deferens. J.Am.Vet.Med.Assoc. pg. 1740-1742  2.  Glogowski J., Strzeżek J. (1979) Fosfataza alkaliczna – niektóre właściwości i funkcja w żeńskim i męskim układzie rozrodczym. Med. Vet. 1, pg. 34-38 3.  Glogowski J. (1988) Fosfataza alkaliczna układu rozrodczego knura. Zeszyty Naukowe ART., No 31, Suppl. B., pg. 1-53 4.  Kosiniak K., Bittmar A., Sallwerk K. (1992) Relationship between some biochemical components of dog seminal plasma and semen freezability. Mat. Kongr. XII Int. Cong. on Animal Reprod. Vol. 4, pg.1791-1792 5.  Kosiniak K., Bittmar A. (1994) Prognosis on dog semen freezability: Biochemical markers in seminal plasma. ARTA Conference Spain, Vol.5. pg. 129-131 6.  Kutzler M.A., Solter P.F., Hoffman W.E., Volkmann D.H. (1986) Characterization and localization of alkaline phosphatase in canine seminal plasma  and gonadal tissues. Teriogenology 60 (2), pg. 299-306  7.  Morton D.B. (2004) Review on the use of frozen semen in dog breeding. Aim. Technology, Vol. 37, No. 1   1358.  Niżański W. (2004) Zależności pomiędzy wybranymi właściwościami plemników psa w nasieniu poddanym konserwacji w niskich temperaturach. Med. Wet., 60 (5), pg. 537-543 9.  Strzeżek J. (1988) Struktury morfologiczne plemnika i ich funkcje biochemiczne. Biul. Inf. 25, 67-96 ART – Olsztyn, 1988. 10. Turner R. M., McDonell S. M.(2003) Alkaline phosphatase in stallion semen: characterization and clinical applications. Theriogenology 60 (1) pg. 1-10 11. Turner R.M. (1996) Alkaline phosphatase activity in stallions semen ad clinical  implication. Proc Am Assoc Equine Pract 42, pg. 284-293  This work was supported by project 2PO6D 01426 of Ministry of Science and Education.     ACTIVITATEA FOSFATAZEI ALCALINE CA MARKER PENTRU CAPACITATEA DE CONGELARE A SPERMEI DE CÂINE  KOSINIAK-KAMYSZ K., BITTMAR A., PODSTAWSKI Z. Department of Horse Breeding, University of Agriculture, Al. Mickiewicza 24/28, Kraków 30-059, Poland   Investigaţia a fost efectuată pentru a evalua capacitatea de congelare şi calitatea după decongelare a spermei de câine în vederea folosirii acesteia pentru însămânţările artificiale (IA). Pe baza mobilităţii  şi concentraţiei spermatozoizilor şi a activităţii fosfatazei alcaline (FA) s-a stabilit faptul că activitatea FA corespunde cu factorul de bază a examinării spermei. S-a determinat diferenţe semnificative din punct de vedere statistic între calitatea ejaculatelor care s-au calificat şi care nu s-au calificat pentru congelare şi IA. Aceste rezultate arată că activitatea FA în plasma spermei neprelucrate de câine poate fi utilizată ca marker pentru calificarea spermei de câine la congelare şi IA cu o probabilitate de 95% de prognoza rezultatele. Cuvinte cheie: spermă de câine, capacitate de congelare, fosfataza alcalină 

ALKALINE PHOSPHATASE ACTIVITY AS A MARKER OF DOG SEMEN FREEZABILITY

Abstract

Similar works

Full text

Available Versions

Directory of Open Access Journals

Directory of Open Access Journals