The sperm morphology is one of the factors determining semen quality besidessperm motility and concentration. An important role in this aspect plays someenzymes which are estimated in raw semen plasma. The examination of numerouspopulations of stallions of different breeds and age performed by Kosiniak-Kamyszet al. (2005) showed that significant differences occurred between stallion semenquality concerning both macro- and microscopic examination and some enzymesactivity. It was found that aspartate aminotranspherase (AspAT), lactatedehydrogenase (LDH) and alkaline phosphatase (AP) activity and total proteinamount (TP) in raw seminal plasma decreased when the percent of sperms withcytoplasmatic droplets increased. The increase of these enzymes activity is observedwith the increase of the number of loose heads. These observations showed thatmany examined factors of the semen and semen plasma decided on its quality and onthis reason that these factors need to be applied for seminological diagnosi

BITTMAR A.

KOSINIAK-KAMYSZ K.

PODSTAWSKI Z.

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 152Lucrări ştiinţifice Zootehnie şi Biotehnologii, vol. 40(1), (2007), Timişoara  RELATIONSHIP BETWEEN SOME ENZYMES ACTIVITY, SPERM MORPHOLOGY AND STALLION SEMEN QUALITY  RELAŢIA DINTRE ACTIVITATEA UNOR ENZIME, MORFOLOGIA SPERMATOZOIZILOR ŞI CALITATEA MATERIALULUI SEMINAL LA ARMĂSARI  PODSTAWSKI Z., KOSINIAK-KAMYSZ K., BITTMAR A. University of Agriculture, Department of Horse Breeding, Al. Mickiewicza 24/28, Kraków 30-059, Poland  The sperm morphology is one of the factors determining semen quality besides sperm motility and concentration.  An important role in this aspect plays some enzymes which are estimated in raw semen plasma. The examination of numerous populations of stallions of different breeds and age performed by Kosiniak-Kamysz et al. (2005) showed that significant differences occurred between stallion semen quality concerning both macro- and microscopic examination and some enzymes activity. It was found that aspartate aminotranspherase (AspAT), lactate dehydrogenase (LDH) and alkaline phosphatase (AP) activity and total protein amount (TP) in raw seminal plasma decreased when the percent of sperms with cytoplasmatic droplets increased. The increase of these enzymes activity is observed with the increase of the number of loose heads. These observations showed that many examined factors of the semen and semen plasma decided on its quality and on this reason that these factors need to be applied for seminological diagnosis. Key words: stallion, semen plasma, sperm morphology, enzymes activity, biochemical components  Introduction  Many observations of veterinarians and practitioners showed that the fertility of some stallions is very low independent of the positive results of its semen macro- and microscopic examination based on sperm motility and concentration. On the basis of research work of Varner (2001), Low (2000, 2002), Mayers (2002), Bittmar and Kosiniak (1992) and Kosiniak –Kamysz et al. (2000) is known that the sperm enzymatic apparatus play a significant role in this aspect and its damaged resulting of lower stallion fertility. In connection to this the comparison between sperm motility, concentration, sperms morphological status and some enzymes activity (AspAT, LDH, AP, TP) which increase in seminal plasma after sperm membrane damage, allows to recognize the causes of these problems and finally to precise real biological stallion reproductive quality. Voss et al. (1981), Gravance et al. (1996), Magistrini et al. (1996) stressed in their publications that the lower  153number of morphological sperms abnormalities were registered in the semen representing higher fertility. In relationship with these results it is interesting to recognize some undefined till now reasons resulting in lower stallion fertility. The purpose of this study was to inquire into these factors which influence on sperm membrane damages which finally decreased stallion fertility.  Materials and Methods  The semen was collected from 803 stallions 2-20 year of age representing six different breeds which were localized in ten stallion depots. Stallion groups’ characteristics are presented in Table 1.  Table 1 Characteristics of examined stallions  Breed Age 2-4 5-9 10-14 15-20 Total Cold-blood 32 82 56 10 180 (22.4%) Half-bred 67 80 25 4 176 (21.9%) Wielkopolski 27 47 68 19 161 (20.0%) Małopolski 30 44 57 16 147 (18.4%) Silesian 29 32 35 18 114 (14.2%) Hucul 1 9 6 9 25 (3,1%) Total 186 (23.2%) 294 (36.6%) 247 (30.7%) 76 (9.5%) 803 (100%)  The semen was collected with the use of “open” artificial vagina (AV), and only sperm rich fractions were used for analysis. All collected ejaculates were examined using generally known macro- and microscopic methods. In the semen plasma the activity of AspAT, LDH, AP and TP level were determined after centrifugation. Sperm morphology examination was performed under light microscope in colouring smears by eosine-nigrosine Shore method using the system of sperm qualification in two groups of primary and secondary abnormalities.  Results and Discussions  On the basis of performed analysis all ejaculates were divided into two groups:   154 group I – ejaculates represented sperm motility > 2/70%, sperm concentration > 0.2 x 109 and sperm morphology - > 50% of normal sperms  group II – ejaculates represented sperm motility, concentration and morphology lower than in ejaculates of group I. Morphological analysis of the sperms from 197 stallions of 5 different breeds 5-9 years of age showed that very high negative correlation (p<0.001) occurred between the number of sperms with secondary abnormalities, sperms with loose heads, different forms of loop tail and sperm motility both total and progressive.  Table 2 Results of stallion semen examination  reed Sperm motility Sperm concentration [ml x 10 9 ] Morphology [%] TP LDH AspAT AP Progressive [scale 0-4] Total [%] Normal 2abc 4a/4b mg/109 mU/109 IU/ml IU/109 Mało-polski 2.7 76 1.05 75.9 4.8 12.6 38.3 1762.6 469.4 371.6 297.5 Silesian 2.9 79 0.7 77.8 8.7 5.8 61.2 955.6 1070.9 176.6 266.3 Half-bred 3.0 79 0.74 74.9 5.2 8.3 41.6 1884.9 1062.4 236.7 308.1 Wielko-polski 3.2 82 0.91 72.9 7.3 5.6 42.0 1492.2 775.1 264.1 290.0 Cold-blood 3.1 81 0.56 77.9 3.8 9.8 65.4 3286.4 1376.8 222.5 458.7 Group I mean 3.0 80 0.77 74.0 6.0 9.2 48.2 2258.2 999.3 236.7* 422.3 Mało-polski 1.2 51 0.48 48.1 14.0 19.6 129.4 4343.6 1869.5 235.9 567.1 Silesian 1.0 43 0.84 60.0 11.4 20.6 85.9 3028.8 1647.5 229.0 421.7 Half-bred 1.0 45 0.33 60.9 14.8 9.9 99.0 2036.9 2118.1 82.6 385.9 Wielko-polski. 1.8 60 0.45 52.1 16.3 18.2 113.0 3346.6 2365.0 198.3 683.4 Cold-blood 1.3 51 0.6 59.8 7.8 19.6 147.5 4031.2 2319.8 134.3 416.6 Group II mean 1.3 48 0.56 57.9 12.4 17.7 122.4** 3406.8* 2126.4* 170.3 378.8  On the basis of these results it is possible to state that the increase of secondary abnormal sperms in ejaculate corresponds with sperm motility  155decreasing in raw semen. Significant correlation (p<0.05) occurred between the number of sperm with cytoplasmatic droplets (2a) and ejaculate volume. It was found too that significant negative correlation (p<0.01) occurred between sperm concentration and the number of sperms with cytoplasmatic droplets, because of this in the semen plasma with lower sperm concentration the number of sperms with cytoplasmatic droplets is higher, which decided on the lower semen quality. The comparative analysis performed between ejaculates qualified to groups I and II on the basis of macro- and microscopic semen quality examination, sperm morphology, as well as biochemical components of semen plasma, allows to establish that the ejaculates qualified to group I represented higher sperm motility and higher number of normal spermatozoa both for different stallion breeds as well as mean value than ejaculates qualified to group II (Table 2). The relationship between estimated biochemical components of semen plasma and registered morphological abnormalities showed that AspAT, LDH and AP activity decreased with an increase of the number of sperms with cytoplasmatic droplets (abnormalities 2abc) (Table 3).  Table 3 Relationship between sperm morphological abnormalities and amount of biochemical components in semen plasma  Cytoplasmatic  droplets  2abc [%] LDH AspAT AP TP range mean mU/ml mU/109 mU/ml mU/109 mU/ml mU/109 mg/ml mg/109 0 -  15 4.1 1813.4 3170.4 700.8 1604.4 200.5 420.7 37.8 93.6 >15  27.2 1310.5* 2080.7** 545.2* 1773.8 192.7 386.2* 30.0 83.1  An increase of all examined biochemical components (in relationship to sperm concentration) was observed in ejaculates with the increasing number of abnormal sperms (4ab) (Table 3). Very high positive or negative correlation (coefficient 0.2-0.75) occurred between all biochemical components of semen plasma. Significant positive correlation was observed too between sperm concentration and biochemical components of semen plasma.  Conclusions  On the basis of performed comparative analysis we would like to state that it is necessary to apply for a practice of stallion fertility examination much wider  156seminological diagnostic program which allows establishing stallions’ biological quality.  Bibliography  1. Bittmar A.,  Kosiniak K. (1992). The role of selected biochemical components of equine seminal plasma in determining suitability for deep-freezing. Pol. Arch. Vet. 32, 1-2, 17-29.  2. Gravance C. G., Liu I. K. M., Davis R. O., Hughes J. P., Casey P. J. (1996) Quantification of normal head morphometry of stallion spermatozoa. Journal of Reproduction and Fertility, 108: 1, 41-46 3. Kosiniak-Kamysz K., Bittmar A., Podstawski Z. (2000) The influence of aspartate aminotransferase activity and sperm morphology on stallion and dog semen freezability. Naukowij Wisnik Nacionalnogo Agrarnogo Uniwesitetu 22,140-144, Kijów 4. Kosiniak-Kamysz K., Bittmar A.,  Podstawski Z.,  Gorazd M.: Characteristics of the breed structure and semen quality of stallions from National Stallion Depots.. 8th  Inter Symp.Modern Trends in Livestock Production Belgrad. Biotechnoligy in Animal Husbandry vol. 21, 5-6,73-75, 2005. 5. Love C. C. (2000) Intra- and inter-stallion variation in sperm morphology and their relationship with fertility. J Repro Fertil, supple 2000; 56:93-100 6. Love C. C. (2002) Stallion semen evaluation and interpretation. SFT & ACT Proceedings Annual Conference & Symposium, Colorado Springs, Colorado, 93-101 7. Magistrini M., Vidament M., Clement F., Palmer E. (1996) Fertility prediction in stallions. Animal-Reproduction-Science. 42: 1-4, 181-188 8. Meyers S. A. (2002) Non-traditional methods of diagnosing abnormal sperm function. SFT & ACT Proceedings Annual Conference & Symposium, Colorado Springs, Colorado, 483-489 9. Varner D. D. (2001) Subfertility in stallions associated with spermatozoal acrosome dysfunction. Proceedings American Association of Equine Practitioners; 47:227-228 10. Voss J. L, Pickett B. W., Squires E. L. (1981) Stallion spermatozoa morphology and motility and thier relationship to fertility. J. Am.Vet. Med. Assoc. 178; 287-289 This work was supported by project 0620/R-1/P01/07/02 of Ministry of National Education.     157RELAŢIA DINTRE ACTIVITATEA UNOR ENZIME, MORFOLOGIA SPERMATOZOIZILOR ŞI CALITATEA MATERIALULUI SEMINAL LA ARMĂSARI  PODSTAWSKI Z., KOSINIAK-KAMYSZ K., BITTMAR A. University of Agriculture, Department of Horse Breeding, Al. Mickiewicza 24/28, Kraków 30-059, Poland  Morfologia spermatozoizilor este unul dintre factorii care determină calitatea spermei, în afară de mobilitate şi concentraţie. Un rol important în acest aspect îl joacă unele enzime care se apreciază în plasma spermei proaspete. Examinarea numeroaselor populaţii de armăsari de rase şi vârste diferite efectuate de Kosiniak-Kamysz şi colab. (2005) au arătat că există diferenţe semnificative între calitatea materialului seminal al armăsarilor atât în ceea ce priveşte examinarea macro- şi microscopică cât şi activitatea anumitor enzime. S-a decoperit că activitatea enzimelor aspartat aminotransferază (AspAT), lactat dehidrogenază (LDH) şi fosfataza alcalină (AP) şi a cantităţii totale de proteină (TP) din plasma spermei proaspete s-au redus atunci când a crescutr procentul de spermatozoizi cu picături citoplasmatice. S-a constatat creşterea activităţii acestor enzime o dată cu creşterea numărului de spermatozoizi fără cap. Aceste observaţii au demonstrat că mulţi factori examinaţi ai spermatozoizilor şi a plasmei seminale au avut un rol decivis asupra calităţii spermei şi ca urmare aceşti factori trebuie să fie utilizaţi în disgnosticul semiologic. Cuvinte cheie: armăsar, plasma seminală, morfologia spermatozoizilor, activitatea enzimatică, componente biochimice. 

RELATIONSHIP BETWEEN SOME ENZYMES ACTIVITY, SPERM MORPHOLOGY AND STALLION SEMEN QUALITY

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RELATIONSHIP BETWEEN SOME ENZYMES ACTIVITY, SPERM MORPHOLOGY AND STALLION SEMEN QUALITY

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