Many problems in dog reproduction concern both dog male, its behaviour andsemen quality as well as the bitch which are connected with physiological factors asa time oestrus cycle, anatomical structure of reproductive organs, sexual behaviourand ovulation moment. The results of bitches’ artificial insemination (AI) with theuse of frozen semen are lower in comparison to raw semen. In connection with thisthe research work was performed with an idea of explanation of the problemconnected to low effect of the use of dog frozen semen for AI. It was found that it ispossible to receive more satisfactory results (about 75% of pregnancy rate) whendog semen is testified on the base of sperm concentration and motility and alkalinephosphatase activity (AP). On the other side it is necessary to perform bitchesexamination based on cytological and hormonal testes which allows establishing thepernicious time for AI

BITTMAR A.

KOSINIAK-KAMYSZ K.

PODSTAWSKI Z.

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 136Lucrări ştiinţifice Zootehnie şi Biotehnologii, vol. 40(1), (2007), Timişoara  THE RESULTS OF BITCH ARTIFICIAL INSEMINATION AFTER THE USE OF TESTED FROZEN SEMEN  REZULTATELE ÎNSĂMÂNŢĂRII ARTIFICIALE LA CĂŢEA DUPĂ FOLOSIREA SPERMEI CONGELATE TESTATE  KOSINIAK-KAMYSZ K., PODSTAWSKI Z., BITTMAR A. University of Agriculture, Department of Horse Breeding Al. Mickiewicza 24/28, Kraków 30-059, Poland  Many problems in dog reproduction concern both dog male, its behaviour and semen quality as well as the bitch which are connected with physiological factors as a time oestrus cycle, anatomical structure of reproductive organs, sexual behaviour and ovulation moment. The results of bitches’ artificial insemination (AI) with the use of frozen semen are lower in comparison to raw semen. In connection with this the research work was performed with an idea of explanation of the problem connected to low effect of the use of dog frozen semen for AI. It was found that it is possible to receive more satisfactory results (about 75% of pregnancy rate) when dog semen is testified on the base of sperm concentration and motility and alkaline phosphatase activity (AP). On the other side it is necessary to perform bitches examination based on cytological and hormonal testes which allows establishing the pernicious time for AI. Key words: bitch, semen quality, artificial insemination.  Introduction  The increase in breeding dogs’ population carries along the necessity of improvement in diagnostic and biotechnical methods of their reproduction. Trends of present-day breeding that include: (1) a proper mating of males and females which show rare and eligible appearance features, (2) international animals exchange, (3) the necessity of receiving a lot of litters and puppies and (4) the use of insemination and dogs’ semen conservation, require the usage of good and verified research methods that allow to achieve the intended goal (Jeffcoate, Lindsay 1989, Wright 1991, Goodman 1992, Niżański et al. 2004). Specific character of dogs’ breeding, emerging from the fact that oestrus occurs usually twice a year and shows individual variability from 5 to 20 days, and also anatomical build and topography of a sexual organ of a bitch, cause frequent difficulties not only in arrangement of optimal mating time but also in insemination, which results in the delay of the following try till the next ovarian cycle which usually starts after 5-7 months (Feldman, Nelson 1996, Wright 1991). All those conditionings indicate at the necessity to provide optimal times in bitches’ insemination. It is connected not only with male semen quality but also  137with the monitoring of the ovarian cycle regularity, the proper term of mating, diagnosis and therapy for sexual cycle disorders as well as looking for better and more efficient artificial insemination methods. Despite the fact that it is 37 years from the first successful bitch insemination with the use of semen preserved in low temperatures, made in the USA by Seager in 1969, these procedures are unfortunately still incidentally carried out in our country. Insemination of the bitch with frozen semen was done in a big way in the North America at the turn of 60’s and 70’s of the XX century, and then at the turn of 70’s and 80’s in the Western Europe. It should be remarked that, as far as in the American Cannel Club after the insemination of bitches with frozen semen 99 litters were born in 1992 (Linde-Forsberg 1991,1995), as Niżański et al. wrote (2004), in Poland the first documented insemination with the use of frozen semen took place just in 1997. However, until today the results of females’ insemination all over the world in the field conditions are often unsatisfactory. It seems that 3 factors are mainly responsible for that, i.e. short survival rate of sperms after thawing, difficult to establish optimal time for bitch insemination which needs the usage of laboratory tests and characteristically formed and uncomfortable in semen deposition the cervix uterus barrier (England 1993, Olar et al. 1989, Wright 1991, Niżański et al. 2004, Niżański, Klimowicz 2005). There was proved many times that the factor which decides about effectiveness of these procedures is the quality of semen used for the insemination. However, a great individual diversity in the dogs’ semen freezability and the lack of effective laboratory methods of its assessment contributed to conducting the further studies over the trials to explain those reasons.  Materials and Methods  The semen taken from 12 2–7-year-old dogs, belonging to 6 races: Beagle, Cane Corso, Dog de Bordox, Doberman, Labrador and Schnauzer was used to inseminate 16 bitches representing the same races. The inseminated bitches were 3-6-year-old and gave birth to at least one litter before.  Results and Discussions  The semen qualification for freezing The semen was qualified to be frozen when it contained ≥ 60% motile and morphologically normal sperms at concentration ≥50 x 106 sperms in ejaculate. Morton extender (1986) was used to the process of freezing. Centrifuged sperms were diluted in the way that every 1.5 ml of extender contained 100 mil. of motile sperms, counted from the concentration and semen activity. The semen was frozen in straws of 0.25 ml. Frozen semen was thawed at 70C during 5 sec.  Frozen semen examination and qualification for AI  138The frozen semen was examined just after thawing on the base of sperm motility in a sample and its survival time at 4C, as well as alkaline phosphatase activity estimation in the raw semen plasma. The semen with minimum 30% motile sperms, which survived not shorter than 84 hrs in 4C and in which the alkaline phosphatase activity was higher than 10 IU/ cm3 (taking into account the volume and the concentration of the sperms) was qualified for the insemination. The insemination dose was 5-6 ml of semen containing 150-200 x 106 sperms which presented progressive motility.  Determination of the time to inseminate the bitches The optimal time for the artificial insemination was determined on the basis of bitches’ cytological examination of the vaginal swab and determination of progesterone in peripheral blood. The cytological assessment of the vaginal swab in preparations dyed with Shorr’s method was made every second day, beginning from the second day of the heat. When 80% of eozynophilic epithelial cells were registered, the level of progesterone was measured in the blood serum in the next day, by use of Rapid Progesterone Kit. During the first examination the increase in progesterone concentration in plasma samples was found in over 80% samples and within the second examination after 48 hrs the level raised in the rest of the samples. All bitches were inseminated twice in the 4th and 6th day after the pre-ovulatory LH surge, determined by the progesterone measurement. Insemination technique The bitches were inseminated with the pipette „Osiris gun”, the semen was located in the vagina near the external orifice of the uterus cervix. The sealing balloon was filled with air by the use of a syringe (volume 20 or 40 ml). After the insemination the bitches’ backside was lifted with the catheter and it stayed in that position for about 10 minutes.  The results of bitches’ insemination Following the described method in treating the tested dogs’ semen as well as in determining the term of bitches’ insemination with the use of the pipette „Osiris gun”, 4 out of 16 inseminated bitches were not pregnant so the obtained insemination effectiveness was 75%.  Bibliography  1. England G.C.W.: (1993) Cryopreservation of dog semen: a review J. Reprod.. Fert., Suppl 47, pg. 243-255 2. Feldman E.C, Nelson R.W.: Canine and feline endocrinology and reproduction. W.B. Saunders Comp., Philadelphia, 1996. 3. Goodman M.F.: (1992) Canine ovulation timing. Probl. Vet. Med. 4, pg. 433-444 4. Jeffcoate I.A., Lindsay F.E.F.: (1989) Ovulation  detection and timing of insemination based on hormone concentrations, vaginal cytology and the  139endoscopic appearance of the vagina in domestic bitches. J. Reprod. Fert., Suppl. 39, pg. 277-287 5. Linde-Forsberg C.: (1991) Achieving canine pregnancy by using  frozen or chilled extended semen. Vet. Clin. North Am. Small Anim Pract., 21, pg. 467-485 6. Linde-Forsberg C.: (1995) Artificial insemination with fresh, chilled extended, and frozen-thawed semen in the dog. Sem. Vet. Med. Surg. Small Anim.  10,  pg. 48-58 7. Morton D.B.: (1986) Review on the use of frozen semen in dog breeding. Aim. Technology, Vol. 37, No. 1 8. Niżański W., Klimowicz M., Dubiel A.: (2004) Wskazania i czynniki skuteczności zabiegu oraz technika unasieniania dopochwowego suk. Medycyna Weterynaryjna 60 (7), pg. 682-686 9. Niżański W., Klimowicz M.: (2005) Skuteczność sztucznej inseminacji suk nasieniem świeżym przy zastosowaniu różnych metod wyznaczania terminu unasieniania . Med. Wet. 61 (1),  pg. 75-81 10. Olar T.T., Bowen R.A. and Pickett B.W.: (1989) Influence of extender, cryoperservative and seminal processing procedures on postthaw  motility of canine spermatozoa frozen in straws. Theriogenology Vol. 31, 2  pg. 451-461  11. Wright P.J.: (1991) Practical aspects of estimation of the time of ovulation and of insemination in the bitch. Austr. Vet. J. 68, pg. 10-13 This work was supported by project 2PO6D 01426 of Ministry of Science and Education  REZULTATELE ÎNSĂMÂNŢĂRII ARTIFICIALE LA CĂŢEA DUPĂ FOLOSIREA SPERMEI CONGELATE TESTATE  KOSINIAK-KAMYSZ K., PODSTAWSKI Z., BITTMAR A. University of Agriculture, Department of Horse Breeding Al. Mickiewicza 24/28, Kraków 30-059, Poland  Multe din problemele reproducţiei la câine interesează atât masculul (comportemantul şi calitatea spermei acestuia) cât şi femela, care este legată de factorii fiziologici cum ar fi momentul ciclului estral, structura anatomică a organelor de reproducere, comportamentul sexual şi momentul ovulaţiei. Rezultatele obţinute prin însămânţarea artificială a căţelelor folosind spermă congelată sunt mai reduse în comparaţie cu rezultatele obţinute cu sperma proaspătă. Legat de aceste rezultate cercetarea s-a efectuat în ideea explicării problemelor legate de efectul redus al folosirii sperma congelată de câine pentru însămânţările artificiale. S-a decoperit că este posibil să se obţină rezultate mai satisfăcătoare (rată de gestaţie de 75%) atunci când sperma de câine este testată pe baza concentraţiei şi motilităţii spermatozoizilor şi activitatea fosfatazei alcaline. Pe de altă parte este necesară examinarea căţelelor pe baza testelor citologice şi hoemonale care permit stabilirea momentului optim pentru însămânţare. Cuvinte cheie: căţea, calitatea spermei, însămânţare artificială. 

THE RESULTS OF BITCH ARTIFICIAL INSEMINATION AFTER THE USE OF TESTED FROZEN SEMEN

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THE RESULTS OF BITCH ARTIFICIAL INSEMINATION AFTER THE USE OF TESTED FROZEN SEMEN

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