Study on Semen Freezing Preservation of German Shepherd Dogs

Huang He, Tian Ya-guang, Zheng Peng, and Zhang Gui-xue

College of Animal Sciences and Technology, Northeast Agricultural University, Harbin 150030, China

Introduction

German shepherd dog is known for termagancy, welltrained and natural police dogs. China has set up the breeding system of German shepherd dog which completely adapted to need of the public security,troops and the national defense on the preservation,breeding and utilization (Wang et al., 2008). Italian physiologist, Spallannani fi rst used the dogs on the AI test in 1780 (Zhang, 2004). Seager (1969)reported the success of AI of dogs with the freezing semen for the first time. Then he determined a series of parameter of semen quality, freezing and AI (Seager, 1975). Wu(1976)also reported the same result. Pan et al. (1986)fi rstly researched the semen freezing and AI of German shepherd dogs and succeeded. They result showed that the pregnancy rate was 22% and the average litter size was two. Wang et al. (1991)succeeded with semen freezing of German shepherd dog too and obtained higher pregnancy rate and litter size. This experiment researched the semen freezing preservation of German shepherd dog in order to improve breeding eff i ciency of male dogs, prolong semen preservation and increase pregnancy rate and litter size.

Materials and Methods

Experimental animals

We selected four male dogs and 20 rutting female dogs from Tianshang Farm. They weighed 15 to 25 kg and aged 2 to 5 years old. The dogs were fed by compound feed, moved and drinked freely everyday. The female dogs were in two equal groups (AI group and natural matting group).

Experimental methods

Comparison of dilution and formulation

The experiment used sperm motility of frozenthawed sperm as evaluation standard. Meanwhile, we

compared sperm motility of four kinds of dilution and two kinds of formulation, respectively (0.1 mL pellet and 0.5 mL tuble). In this case, we could electe the best dilution formula and semen formulation. The semen of four dogs was mixed together to reduce difference (Wu,1976; Pan et al., 1986). The experiment repeated 10 times.

Methods of semen freezing

The semen was collected from four dogs, each was collected 40 times. Centrifuged, wiped off seminal plasma and added dilution to the equal volume after examination. Sealed, packed with gauze and put into plastic bag, sealed again. Then, the semen was put in refrigerator for 4 h at 4℃. Removed the semen, added equal volume dilution and replaced for 30 min.

(1)Tuble semen with program freeze method

(2)Pellet semen with fried smoked method

Dripped 0.1 mL semen into liquid nitrogen for 5 to 8 s, took out pellet and fumigated for 8 min over the liquid nitrogen.

(3)Semen thaw method

Put the semen into warm water (40℃)for 10 s rapidly after the semen had been taken out from the liquid nitrogen. Then, we checked the sperm motility.

Table 1 Formula of dilution

Insemination method

We chose the pellet semen which diluted by dilution 2 and froze with fried smoked method. AI group were inseminated on the fi rst day and third day after oestrus.Semen (1.5 mL)was brought to the cervix with tailormade apparatus. The natural matting group was matted at the same time. The experiment was repeated three times.

Results

Comparison of semen index

Semen volume, sperm motility and sperm density of four dogs all reached to the standard level(Table 2). There was no signif i cant difference among four examined groups. The effect of individual difference on result could be eliminated.

Effect of dilution and formulation on sperm motility

The sperm motility of pellet semen of dilution 2 (0.44±0.02)was significantly higher than others (P<0.05),there was no signif i cant difference among dilution 1,dilution 3 and dilution 4 (P>0.05); the sperm motility of tuble semen of dilution 4 (0.25±0.01)was significantly lower than others (P<0.05), there was no signif i cant difference among dilution 1, dilution 2 and dilution 3 (P>0.05); sperm motility of particle semen was signif i cant higher than tuble semen except dilution 1(P<0.05). The result showed that sperm motility was higher with dilution 2 and fried smoked method (Table 3).

Table 2 Semen index of four male dogs

Table 3 Effect on sperm motility of dilution and formulation

Effect on sperm motility with freezing method

We froze semen of four dogs with fried smoked method and program freezing method, respectively,sperm motility of the fried smoked method was signif i -cantly higher than that of program freezing method(P<0.05).

Table 4 indicated that fried smoke method was superior to program freezing method.

Effect of hybridization method on conception rate and litter size

We inseminated female dogs with the pellet semen which contained dilution 2 and froze with fried smoked method. The result indicated that the conception rate of natural matting group was signif i cantly higher than that of AI group (P<0.05)and the litter size was the same (P<0.05).

Table 4 Effect on sperm motility with freezing method

Table 5 Effect of hybridization method on conception rate and litter size

Discussion

Semen freezing method

The principle of semen freezing was that semen changed energy with cold source, and the semen turned into solidoid after it released much energy (Zhao et al., 2008). The tempreture from 0℃ to –60℃ harmed to sperm during this process. Cooling rate was highly correlation with sperm motility and cell integrity.Rapid cooling cells formed ice crystal inside the cell which led to sperm dead and cell damage; slow cooling caused solute concentration rise outside the cell so that sperm died (Lian and Li, 1997). In addition, magnitude of changes varied with osmotic pressure and pH value of different dilution under the same condition. So different freezing fl uid had different requirements on freezing program. Fried smoked method which Yu et al. (1997)set up for dog semen freezing obtained higher sperm motility than fluorine board method.The experiment researched freezing method of pellet semen and tuble semen of German shepherd dogs preliminarily. The result showed that fried smoked method was superior to program freezing method.

Freezing formulation

Semen formulation included pellet, tuble and ampoules, each had advantages and disadvantages, and the views of researchers were not uniform either. Olar et al. (1989)considered that tuble semen was easy to mark, preserve and unfreeze, it was more practical than that of pellet semen and ampoules semen. Seager(1969)thought that tuble semen and ampoules semen should replace pellet semen. Pen et al. (1986)deemed that ampoules semen was superior to pellet semen on the viewpoint of production. The result of Yu et al.(1997)showed that sperm motility of pellet semen was signif i cantly higher than that of tuble semen and ampoules semen (P<0.01). Although, there were less valid sperm in the pellet semen, pellet semen froze expediently had higher sperm motility and utilization.We collected couple pellets when dogs were inseminated, it could not only overcome less valid sperm, but also enhance the semen utilization. Pellte semen was worth applying and recommending in the dog semen preservation and AI.

In the comparative experiment of conception rate and litter size, the conception rate of natural matting group was signif i cantly higher than that of AI group.We didn't take into account the effective sperm count in the trial, so much work remained to be further research. Oettle (1988)thought sperm motility of frozen semen was significantly lower than that of fresh semen. Therefore, the conception rate was lower too. The litter size related with individual, nutrition,heredity and management. Meanwhile, sterilization of instrument would affect conception rate and litter size.We only compared different hybridization methods which affected the conception rate and litter size in the experiment, it remained to be further research in the later trials. The conception rate of AI was lower than that of natural matting and litter size was not higher either, but AI technique could improve efficiency of male dogs, change introduction and conservation manner, improve dog quality, reduce raising costs,inf i nitize utilization of frozen semen in time and space,overcome matting diff i culties and prevented spread of disease, therefore, it has the broad application prospect in the dog breeding.

Conclusions

The dilution which contained yolk-Tris mainly was the best; the pellet semen frozen with fried smoked method was superior to tuble semen with program freezing method; sperm motility of pellet semen was higher than that of tuble semen in the same dilution,and the conception rate and litter size of natural matting were higher than that of AI.

Lian Z X, Li W. 1997. Biological effects of deer semen on development of testes in mice. Journal of Northeast Agricultural University, 4(1):52-54.

Oettle E E.1988. Sperm abnormalities in the dog. A light and electronmicroscopic study. Vet Med Rev, 59: 28-70.

Olar, Bowen, Pickett. 1989. Influence of extender, cryoperservative and seminal processing procedures on postthaw motility of canine spermatozoa frozen in straws. Theriogenology, 31(2): 451.

Pan S W, Liu H, Ma D J. 1986. Semen freezing technique of dog.Animal Husbandy And Veterinary Medicine, 2(3): 105-106.

Seager. 1969. Storage of boar semen. AI Digest, 27(3): 6.

Seager. 1975. Relation of semen quality tosperm transport, fertilization and embryo quality in ruminants. Reprod J Fertil, 45: 189-192.

Wang L B, Yang D J, Zhou X Y. 1991. Study on semen freezing of dog and artificial insemination. Heilongjiang Animal Science And Veterinary Medicine, 5: 35-36.

Wang X L, Liu Yan, Zeng S M. 2008. Advance in international development of canine semen cryopreservation. Chinese Journal of Animal Science, 2(15): 12-16.

Wu S C J. 1976. Artificial insemination technique of dog. Journal of Animal Reproduction, 22(1): 28-32.

Yu X L, Pang Y Z, Li Y H, et al. 1997. Study on freezing canine pellet semen by the new method of frying in liquid nitrogen. Chinese Journal of Veterinary Science, 3: 289-291.

Zhao X J, Li Y T, Sun S, et al. 2008. Study on the pellet freezing technique of boar semen. Journal of Northeast Agricultural University, 39(3): 70-75.

Zhang Z C. 2004. Animal reproduction science. China Agriculture Press, Beijing. pp. 221.