Volume 8
Issue 1
Animal Husbandry
JOURNAL OF
POLISH
AGRICULTURAL
UNIVERSITIES
Available Online: http://www.ejpau.media.pl/volume8/issue1/art-23.html
EFFECT OF SPERMATOZOA CONCENTRATION IN INSEMINATION DOSE ON REPRODUCTIVE PERFORMANCE OF RABBIT DOES AFTER ARTIFICIAL INSEMINATION
David Zapletal1, Jan Kuchtík1, Václav Adamec2, Piotr Nowakowski3
1 Department of Animal Breeding,
Mendel University of Agriculture and Forestry in Brno
2 Department of Statistics and Operations Research,
Mendel University of Agriculture and Forestry in Brno
3 Institut of Animal Husbandry,
Agricultural University of Wrocław
The aim of the study was to evaluate the effect of different concentrations of spermatozoa in insemination dose (ID) on fertility rate of inseminated does, and on the number of all and live-born young per litter in the breed of crossbred rabbits by using certain rates of dilution. The crossbred rabbits of female strain HYPLUS PS 19 and male strain HYPLUS PS 39 were used in the experiment. In the first part of the experiment does were inseminated with ID 0.5 cm3 with the concentrations of spermatozoa from 115 to 550 x 106 in 1 cm3. In the second part of the experiment does were inseminated with the same volume of ID (0.5 cm3), however with the lower concentration, namely from 55 to 150 x 106 in 1 cm3. The particular concentrations of spermatozoa in ID had no statistically significant effect on fertility rate and number of all and live born young per litter in both parts of experiment.
Key words: rabbit, insemination, concentration of spermatozoa, performance.
INTRODUCTION
The principal aspect of insemination success in crossbred rabbits is to dispose of insemi-nation doses (ID) with the adequate number of motile spermatozoa that cannot negatively affect the reproductive performance of does.
The volume of ejaculate is quite variable and it depends on the individuality, age, breed and body size of male. The volume of ejaculate is also affected by breeding conditions, nutrition, ambient temperature and by photoperiod. Morrell [9] obtained 0.5-2.0 cm3 liquid fraction at periodical collections of one male of the medium size breed. Brun et al. [3] found average volume of ejaculates liquid fraction of 0.62 cm3 in crossbred males.
Boussit [2] stated an average concentration of spermatozoa as 300 x 106 in 1 cm3, but in extreme cases it could be as much as 750 x 106 spermatozoa in 1 cm3. According to Gamčík [6] the average number of spermatozoa was 750 x 106 in 1 cm3, whereas the concentration of spermatozoa was greatly varying during the year. Brun et al. [3] detected the average concen-tration of spermatozoa of 492 x 106 in 1 cm3 in crossbred males.
Boussit [2] claims, that minimal number of spermatozoa could be 1 million in one inseminate dose, in agreement with findings of Wales and O´Shea [13]. At the average volume of ejaculate of 0.7 cm3 and the concentration of 250 millions spermatozoa in 1 cm3 an average ejaculate contains 175 millions of spermatozoa. Hypothetically it would be possible to obtain 175 inseminating doses with the volume 0.5 cm3, then the dilution ratio would be 1:125. However, most authors contradict this dogma and claim considerably lower dilution ratio. Adams [1], Kuttner et al. [7], Theau-Clement and Roustan [12], De Castro et al. [5], Castellini et al. [4] and Roca et al. [11] dealt with the problems concerning the dilution ratio of rabbit semen.
The aim of the study was to evaluate the effect of different concentrations of spermatozoa in insemination dose on fertility rate of inseminated does, and on the number of all and live-born young per litter in crossbred rabbits by using different rates of dilution.
MATERIALS AND METHODS
The experiment was performed in population of crossbred rabbits in Velké Němčice farm during the years 2002-2003 in Czech Republic. The basic stud was formed with 200 does of strain PS HYPLUS 19 and 7 bucks of strain PS HYPLUS 39. The temperature on the farm was varying from 12 to 24 degrees of Celsius during the year - extra heating was not used. From the viewpoint of photoperiod combination of natural and artificial lightning was used. Three days before the insemination the photoperiod was 16 hours of light and 8 hours of dark. During the day of insemination and in other days photoperiod was 13 hours of light.
Does and bucks were fed twice a day with complete granulated feeding mixture. Primiparous does were inseminated from the age of 18 weeks, and bucks were used for reproduction from the age of 23 weeks. At each insemination session 15-18% of primiparous, 24-27% of non-lactating does and 57-60% of lactating does were inseminated. Glass inseminating pipette in horizontal position was used. The fertility rate has been determined between 12-16 days after insemination by palpation. The number of all and live-born young per litter has been determi-ned till 24 hours post partum.
The concentration of spermatozoa was evaluated by haematocytometry. After basic exami-nation ejaculate was diluted with preparation KARE I (PLEBO BRNO, a.s., Czech Republic) that is used for dilution of boar sperm in Czech. Powder KARE I (254.75 grams) containing 220.00 g glucose, 18.50 g sodium citrate, 10.00 g EDTA, 6.00 g sodium carbonate, 0.125 g N-acetyl-L-cysteine was solved in 5 litre of distilled sterile water. The temperatures of the diluent and ejaculate were kept even in the first and in the second phase of dilution (difference +/- 0.5 degrees of Celsius) - the range was between 28-30 degrees of Celsius. The insemination was performed 4 hours after the dilution.
The experiment was divided into two parts. In the first part of the experiment 171 does PS HYPLUS 19 were divided into 7 experimental groups and they were inseminated with ID 0.5 cm3 in March and April of the 2002 year. The concentrations of spermatozoa were from 115 x 106 to 550 x 106 in 1 cm3. Forty-nine hours before insemination the does were synchronized by subcutaneous application of PMSG (SERGON inj.) of 25 IU per doe. At the moment of insemination injection was applied of 10 µg of hormone Gonadoreline (Dirigestran) per doe to induce ovulation.
The second part of the experiment was performed during March and April of 2003 year. In total 96 does were divided into 4 experimental groups. They were always inseminated in the same day by 0.5 cm3 of ID with lower and higher concentration of spermatozoa. The range of the concentrations of spermatozoa was from 55 x 106 to 150 x 106 in 1 cm3. Forty-eight hours before insemination the does were synchronized by subcutaneous application of PMSG (SERGON inj.) of 25 IU per doe. Hormone Lecireline (Supergestran inj.) 4.2 µg per doe was applied at the moment of insemination to induce ovulation.
The fertility rate was statistically evaluated by logistic recourse function [8]. The subsequent testing was done by goodness-of Ht test. The number of all and live-born young per litter was statistically evaluated by Poisson´s generalized model. Statistical estimation of significance between monitored levels was made by deviance analysis.
RESULTS AND DISCUSSION
In the first part of the experiment the fertility rate varied from 60.70% after ID concentration 375 x 106 to 80.00% at ID concentration 270 x 106 (Table 1). The number of spermatozoa in ID had no statistically significant (p>0.05) effect on the fertility rate (Fig. 1). The average number of all-born young for particular concentration of spermatozoa in ID is shown in Table 2 and varied from 6.90 at ID concentration of 550 x 106 to 9.30 at ID concentration of 170 x 106 spermatozoa in 1 cm3. The number of spermatozoa in ID had not statistically significant (p>0.05) effect on the all-born young rabbits per litter (Fig. 2). The average number of live-born young for particular concentrations of spermatozoa in ID (Table 2) varied from 6.30 at ID concentration 550 x 106 to 9.10 at concentration 170 x 106 spermatozoa in 1 cm3. The number of spermatozoa in ID had not statistically significant (p>0.05) effect on the number of live-born young rabbits per litter.
| Table 1. The effect of concentration of spermatozoa in ID on fertility rate of does in the first part of experiment |
|
Concentration of spermatozoa |
Total number of spermatozoa in ID |
Dilution ratio (ejaculate/ diluent) |
Number of inseminated does |
Fertility rate |
|
115 |
57 500 000 |
1/9 |
26 |
61.50 |
|
130 |
65 000 000 |
1/6 |
22 |
63.60 |
|
170 |
85 000 000 |
1/5 |
29 |
65.50 |
|
270 |
135 000 000 |
1/3 |
20 |
80.00 |
|
370 |
185 000 000 |
1/2 |
25 |
64.00 |
|
375 |
187 500 000 |
1/2 |
28 |
60.70 |
|
550 |
275 000 000 |
1/1 |
21 |
71.40 |
| Table 2. The effect of concentration of spermatozoa in ID on average number of all and live born young rabbits per litter in the first part of experiment |
|
Concentration of spermatozoa |
All born young |
Live born young |
||
|
average |
standard error |
average |
standard error |
|
|
115 |
8.30 |
0.544 |
8.30 |
0.544 |
|
130 |
7.10 |
0.569 |
7.10 |
0.569 |
|
170 |
9.30 |
0.506 |
9.10 |
0.455 |
|
270 |
8.60 |
0.473 |
8.60 |
0.473 |
|
370 |
7.50 |
0.605 |
6.90 |
0.623 |
|
375 |
9.00 |
0.469 |
8.40 |
0.444 |
|
550 |
6.90 |
0.687 |
6.30 |
0.617 |
| Fig. 1. The effect of number spermatozoa in ID on fertility rate in does |
 |
| Fig. 2. The number of all born young rabbits per litter by using various number of spermatozoa in ID |
 |
The fertility rate for particular concentration of spermatozoa in ID in the second part of experiment is stated in (Table 3). The difference in fertility rates of does regarding number of spermatozoa in ID was not statistically significant (p>0.05) (Fig. 3). There were no losses in litters due to periparturient mortality so the average number of all-born and live-born young was the same within all treatments (Table 4). Litter size varied from 8.89 at ID concentration of 150 x 106 to 10.44 at concentration 55 x 106 spermatozoa in 1 cm3. The stated differences among the numbers of all and live-born young rabbits per litter were not statistically significant (p>0.05) regarding the number of spermatozoa in ID (Fig. 4).
| Table 3. The effect of concentration of spermatozoa in ID on fertility rate of does in the second part of experiment |
|
Concentration of spermatozoa |
Total number of spermatozoa in ID |
Dilution ratio (ejaculate/ diluent) |
Number of inseminated does |
Fertility rate |
|
55 |
27 500 000 |
1/5 |
24 |
75.00 |
|
60 |
30 000 000 |
1/5 |
24 |
70.83 |
|
145 |
72 500 000 |
1/16 |
24 |
70.83 |
|
150 |
75 000 000 |
1/16 |
24 |
75.00 |
| Table 4. The effect of concentration of spermatozoa in ID on average number of all and live born young rabbits per litter in the second part of experiment |
|
Concentration of spermatozoa |
All born young |
Live born young |
||
|
average |
standard error |
average |
standard error |
|
|
55 |
10.44 |
0.673 |
10.44 |
0.673 |
|
60 |
9.47 |
0.430 |
9.47 |
0.430 |
|
145 |
9.94 |
0.680 |
9.94 |
0.680 |
|
150 |
8.89 |
0.676 |
8.89 |
0.676 |
| Fig. 3. The effect of number of spermatozoa in ID on fertility rate in does |
 |
| Fig. 4. The number of all and live born young rabbits per litter by using lower number of speratozoa in ID |
 |
From the results of our experiment follow, that lower concentration of spermatozoa (55 x 106 in 1 cm3) did not evoke the decline of pregnancy rate and consecutively the numbers of all and live-born young per litter. The concentration of spermatozoa 55 x 106 in cm3 coresponds to the dilution of 1:16 and in 0.5 cm3 of ID is deposited in total 27.5 million of spermatozoa. Adams [1] claims that the 20 millions of spermatozoa in ID is the minimal number of spermatozoa that does not affect the fertility rate. On the contrary De Castro et al. [5] did not found the significant effect of 4 hours storage of ID (6 million spermatozoa in ID) on the fertility rate and the number of born young. Boussit [2] determined that the minimum number of spermatozoa, which did not affect the fertility rate and the number of born young per litter, was 1 million spermatozoa in ID. Kuttner et al. [7] stated that the rate of dilution should be from 1:2 to 1:10. Theau-Clement and Roustan [12] found that the rate of dilution 1:25, 1:50, and 1:100 had no significant effect on pregnancy rate when they used frozen spermatozoa. However they stated that increasing proportion of diluent led to decreasing number of young per litter. Boussit [2] suggested that the rate of dilution depend mainly on individual qualities of ejaculate. If the motility of spermatozoa is scored 5-7 points, according to Petitjean [10], the rate of dilution should be 1:5, but if the motility of spermatozoa is classified higher then the rate of dilution may be 1:10 or more.
CONCLUSIONS
Results of both parts of the experiment, dealing with the concentration of spermatozoa in the inseminating dose (0.5 cm3), shown that all tested concentrations of spermatozoa in ID (from 55 x 106 to 550 x 106 in 1 cm3) did not lead to changes in fertility rate of rabbit does. They had no effect on the number of all and live-born young per litter, either.
ACKNOWLEDGEMENTS
The study was supported by MSM 432100001.
REFERENCES
Adams C., 1961. Artificial insemination in the rabbit. J.Reprod. Fertil. 2, 521-522. Boussit D., 1989. Reproduction et insemination artificielle en cuniculture. France, I´AFC. Brun J.M., Theau-Clement M., Bolet G., 2002. Evidence for heterosis and maternal effects on rabbit semen characteristics. Anim. Res. 51(5), 433-442. Castellini C., Lattaioli P., Moroni M., Minelli A., 2000. Effect of seminal plasma on the characteristics and fertility of rabbit spermatozoa. Anim. Reprod. Sci. 63 (3-4), 275-282. De Castro M.P.V., Vicente J.S., Lavara R., 1999. Effect of conservation time and number of rabbit spermatozoa on fertility. Ann. Zootech. 48 (5), 407-412. Gamčík P., 1976. Umelá inseminácia a andrológia hospodárskych zvierat. Bratislava Príroda. Kuttner M., Löhle K., Schramm R., 1975. Artificial insemination of rabbits with special reference to extension and conservation of semen. Archiv für Tierzucht 18 (4), 247-254. Michálek J., 2002. Zobecněný lineární model - aplikace v biometrice. In Biometrické metody a modely v současné vědě a výzkumu, Brno. ÚKZÚZ, 15-40. Morrell J. M., 1995. Artificial insemination in rabbits. Br. Vet. J. 151, 477-488. Petitjean M., 1965. Recherches sur l´estimation du pouvoir fécondant des coqs. Thése d´ingénieur d´Etat. CNAM, Paris, France, 1-85. Roca J., Martinez S., Vazquez J.M., Lucas X., Parilla I., Martinez E.A., 2000. Viability and fertility of rabbit spermatozoa diluted in Tris-buffer extenders and stored at 15 degrees C. Anim. Reprod. Sci. 64 (1-2), 103-112. Theau-Clement M., Roustan A., 1982. Etude des possibilités de dilution de sperme de lapin congelé pour l´insemination artificielle. In 3 émes Journées de la Recherche Cunicole : Paris, France, Comunication numéro 19. Wales R., O´Shea T., 1968. Fertility in rabbits inseminated with diluted or mashed spermatozoa. Aust. J. Biol. Sci. 21, 181-183.
David Zapletal
Department of Animal Breeding,
Mendel University of Agriculture and Forestry in Brno
Zemědělská 1, 613 00 Brno, Czech Republic
Jan Kuchtík
Department of Animal Breeding,
Mendel University of Agriculture and Forestry in Brno
Zemědělská 1, 613 00 Brno, Czech Republic
email: kuchtik@mendelu.cz
Václav Adamec
Department of Statistics and Operations Research,
Mendel University of Agriculture and Forestry in Brno
Zemědělská 1, 613 00 Brno, Czech Republic
Piotr Nowakowski
Institut of Animal Husbandry,
Agricultural University of Wrocław
Chełmońskiego 38c, 51-630 Wrocław, Poland
Tel. (+48-71) 3205-778
email: peter@sheep.ar.wroc.pl
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