Research on some Biocharacteristics and Preservation Technology of Rabbit Semen

Do Van Thu¹ and Nguyen Ba Mui²

Hanoi, Vietnam

nbmui@yahoo.com

1. Biotechnology Institute

2. Faculty of Animal and Aquiculture,  Hanoi University of Agriculture

Abstract

A research was conducted to determine biocharacteristrics and conservation technology of rabbit semen. 125 buck-rabbits of New Zealand, California, Panon, grey rabbit and black rabbit were selected to inspect semen quality, to dilute and to conserve semen. Roe-rabbits were strong, normal reproduction and later first litter used for artificial insemination.  

 It was shown that semen volume was low from 0.65 to 0.91 ml; Percent of the swimming spermatozoa was 61.34 – 64.95%; Concentration of sperm was 263.46 – 291.03 million/ml; Total number of spermatozoa per the ejaculation was  263.46 – 291.03 million;  Percent of the alive spermatozoa was 69.82 – 72.52%; Rate of abnormality sperm was 18.47 – 19.09%; Resistance of sperm to NaCl 1% was 2584.09 – 2720.52. The interval between two ejaculations was three or four days for many good quality of semen. Semen diluted (1:5) got better result than diluted rate (1:10). Semen diluted in the Tris solution conserving at 15^oC had percent of the alive spermatozoa better than 5^oC. The proportions of pregnant of conserving semen were 75.00% in the first day, 71.43% in the second day and 50.00% in the fourth day.

Key words: Rabbit, Conservation, Semen, Sperm, Artificial Insemination

Introduction

The rabbit is a species of domestic animal with many good points. In particular, they do not compete with man and other livestock’s for food, it can exploit agricultural wastes. In addition, it can turn plant protein into animal protein with high value. Its protein transformation rate from food absorbed to animal protein was 20% (that of chicken, pig and cattle was 22-23%, 18%, 8- 12% respectively) (Nguyen Quang Suc, Đinh Văn Binh, 1998).

 Rabbit-products are highly economically valuable; its meat is tasty, nutritious, rich in protein, balanced in nutrients, and especially its cholesterol capacity is quite low with 1.36mg/100g raw material. Rabbit meat is used to treat cardiovascular problem stricken victims, aged and obese people (Đinh Văn Binh, Ngo Tien Dung, 2005). In medicine, rabbits are used as experimental subjects to test medicine and produce vaccine. 

 To satisfy the need of economical development programs and serve research, in 1977 our country imported some species of rabbits (New Zealand, California, Pon) and raised them in Goat-Rabbit Researching Centre, Son Tay.  Along with importing breeding animals, after many years of research on cross-breeding, now we have had 5 rabbit species in production including Vietnam New Zealand White, Panon, California, grey rabbits and black rabbits.

 In artificial insemination, the biological criteria of semen are the basis of evaluating their quality, it is closely related to the pregnant rate, productivity and the quality of the next generations. The biological features of semen reflect the breeding ability of each individuals and their adaptability to the environment. Doing research on biological features of rabbit semen is the scientific basis of evaluating the quality of male gender, identifying the components and the rate of substances in the medium and the suitable medium amount to dilute and conserve semen. Researching biological features of semen enables us to select good and qualified samples to put in the dilution, conservation to serve artificial insemination, thenceforth, the management, caring and breeding regimes as well as proper masculine gender exploitation system. 

 Now, in Vietnam artificial insemination of domestic animals and poultry  has had many achievements, the insemination net of frozen semen and diluted semen has covered all over the country. Besides, the frozen semen bank has been built up to serve artificial insemination and conservation of gene sources. Although rabbit cross-breeding and artificial insemination are urgent, this issue has not been well attended to, there has not been any works researching the biological features of rabbit semen systematically. The conservation of rabbit semen is quite a new issue in Vietnam.

Objectives

- Identifying (systematically) the biological figures of rabbit semen to ground the evaluation of the quality of semen and selection of buck rabbits. 

- Identifying technical procedure of diluting and conserving rabbit semen.

- Doing experiment on rabbit artificial insemination with diluted semen

Materials and Methods

Location and duration

The research was carried out in Goat and Rabbit Research Centre, Son Tay district, Hatay province and technology department of reproductive cell, Biotechnology Institute. The research was conducted for 12 months from December, 2006 to November, 2007.   

Rabbit selection

Semen of 125 male rabbits of New Zealand, California, Panon, grey and black rabbit species raised in Goat and Rabbit Research Centre, Son Tay. They weighed between 2,5 and 4,5kg, aging from 8 months to 40 months, being healthy, matting skillfully. Additionally, they all were in sperm provision period to serve breeding and under the diet of Goat and Rabbit Research Centre, Son Tay including concentration with 16% protein, 80gram per day and 1 kg of roughage.

 Female rabbits used in the artificial insemination were in the species of New Zealand, California, Panon, grey and black rabbits, they are healthy, being able to give normal birth and having already given birth once.

Research methodology and focus

The volume of semen V (ml/ejaculation), motility A (%), Concentration of semen C (million/ml) were determined following Milovanov (1962). The rate of survival spermatozoa LS (%) was determined following Chemineau et all (1991). Abnormality of spermatozoa K (%) was determined following Villiam (1921), total of motility spermatozoa was V.A.C (million/ejaculation). pH of semen was determined  meter (pH: 0 - 14 - model METTLER DELTA 320. Osmotic pressure of semen (Miliosmol/kg ) was determined by osmometre). The change of biological figures of rabbits throughout a year’s month. The influence of the interval between two ejaculations on biological figures.

 Diluting solutions of rabbit semen were Tris (Tris 3.80gram; acid Citric 2.1659gram; glucose 0.6gram; penicillin 60mg, streptomycin 60mg; distilled water 100ml) and  Citrate (Trisodium citrate 1.323gram; glucose 0.59466gram; fructose 0.59466gram; penicillin 60mg, streptomycin 60mg; distilled water 100ml).

 The influence of the preservation temperature on quality of rabbit semen and  the influence of the dilution rate on quality of rabbit semen. Artificial insemination for rabbits was used following method of Chemineau (1991)  

Statistical analysis   

Data collected were analyzed by analysis of variance (ANOVA) using the General Linear Model procedure of MINITAB software (Version 13.31 2000).

Results and discussion

Biological characteristics of rabbits' semen

 Some biological criteria of rabbits' semen

We have studied on 125 buck rabbits of New Zealand, California, Panon, Grey and Black genotypes which were kept at Goat and Rabbit Research Center, Son Tay. With 434 semen samples, biological criteria of rabbits' semen were shown in Table 1.

It can be seen from Table 1 that:

- The average volume of semen/ejaculation of rabbits was low, varying from 0.65  to 0.91 ml. Our finding confirmed results reported by Viudes – de- Castro, et al. (1996) with 0.80ml/time (varying from 0.5 to 1.8 ml). Đao Đuc Tha and Nguyen Tan Anh (1989) reported that local rabbits could produce 0.35ml/ejaculation; and 0.38 ml for exotic goats. Their findings were lower than ours.

- Motility of spermatozoa was differed among rabbit genotypes (P<0.05). Motility of spermatozoa of studied genotypes was rather high (61.34 – 64.95%), which meets standards for dilution and AI.

Our findings were lower than that reported by Viudes – de - Castro, et al., (1996) with motility of 80% (varying from 62 to 93%).  Đo Van Thu et al. (2004) reported that the motility (A) of New Zealand, California, Panon, Grey and Black rabbits were 70%; 72%; 74%; 65%; and 69%, respectively. Đao Đuc Tha and Nguyen Ten Anh (1989) reported that the motility of local and exotic rabbits was 65%; and 61%, respectively. Nguyen Quang Suc and Đinh Van Binh (1998) reported the motility of rabbit semen was varying from 55  to 65%.

- Concentration ( million/ml)

Concentration of rabbits' semen was rather high, varying from 263.46 – 291.03 million/ml. Semen concentration has negative relationship with semen volume. Genotypes differed in terms of semen concentration (P<0.05) (highest with Panon 291.03 million/ml; Grey 285.21 million/ml; Black 281.81 million/ml; California 276.39 million/ml  and lowest with New Zealand with, 263.46 million/ml).              

 Our findings on rabbit semen concentration were in agreement with findings of Viudes – de- Castro, et al. (1996) with C = 267 million/ml (varying from 104 – 570 million) and findings of Đao Đuc Tha, Nguyen Tan Anh (1989) with C = 320 million/ml and 247 million/ml, respectively for local and exotic rabbits.

 Although semen volume of rabbits is not much, semen concentration is rather high that can be diluted to increase does for AI. This helps to increase reproductive performance of nucleus sires.

-  V.A.C (million/time)

The higher V.A.C is, the better semen quality and the higher AI efficiency are. Different rabbit genotypes produce semen with different V.A.C. Our findings on V.A.C of New Zealand, California, Panon, Grey and Black rabbits were 152.69, 164.69, 147.84, 111.75, and 113.65 million/ejaculation, respectively.

 Our findings on V.A.C of rabbit semen were in agreement with findings reported by Đo Van Thu, et al. (2004) with  V.A.C of New Zealand, California, Panon, Grey and Black rabbits of 159.96, 167.95, 158.94, 111.06 and 117.86 million/time, respectively. Our findings were higher than those reported by Đào Đao Đuc Tha, Nguyen Tan Anh (1989) who declared that V.A.C of local and exotic rabbits were 94, and 82 million/ejaculation, respectively. It can also be seen from Table 1 that V.A.C varied a lot, which requires to be checked to determine a relevant dilution ration.

 Changes of biological characteristics of rabbits' semen over time

Volume of rabbits' semen significantly varied according to months of the year (P<0.05). V of rabbit semen was high in Jan, Feb and Mar with 0.92ml; 0.92ml and 0.95ml/time, respectively. V was gradually lower in May and Jun (hot season) with 0.73ml and 0.59ml/ejaculation, respectively. 

 Motility of rabbits' semen changed according to months, lowest in May and Jun (hot season) with 61.39% and 54.17%, respectively; for other months motility of rabbit semen changed little, high in Jan and Feb. and gradually decreased from March onwards.
 

 Our findings on A of rabbit semen from Jan to Jun were lower than those reported by Đo Van Thu (2004) with 73.9%; 71.7%; 77.1%; 74.0%; 73.6% and 73.9%, respectively. C of rabbit semen changed over the months, high from Jan to Mar with C of 265.88; 274.80 and 267.63 million/ml, respectively; low from April to Jun with 261.47; 256.45 and 243.68 million/ml, respectively.

V.A.C of rabbit semen significantly differed between months of the year (P<0.05). V.A.C was high in Jan, Feb and March with 164.16; 170.50 and 176.42 million/ejaculation, and lowest in Jun with 72.58 million/ejaculation.

Effects of semen collecting time on semen quality implicates that rabbit semen should be collected when the weather is cool. During the hot season, the semen quality is decreased. To increase semen quality in the hot season, heat stress should be prevented and relevant feed and feeding should be provided for rabbits. In addition semen collection should be done in the morning when the weather is still cool.

Effects of semen collection interval

Semen quality is affected by many factors. One of the factors is semen collection interval. To identify appropriate semen collection interval, we studied biological characteristics of New Zealand rabbit semen with collection interval of 1, 2, 3 and 4 days The results are shown in Table 3.

It can be seen in Table 3 that semen collection interval affected V, C, A, living proportion, abnormality proportion of semen. Semen collection interval significantly affected V (P <0.05). One day collection interval produced less semen than 2, 3 and 4 day collection interval. The V of 1, 2, 3 and 4 day collection interval was V = 0.75; 0.90 ml; 0.96 ml and 1.03ml, respectively.

Our findings were consistent with findings reported by Đo Van Thu, et al. (2004), in which V of 1, 2, 3 and 4 day collection interval was 0.806ml, 0.886ml; 1.006ml and 1.045ml, respectively.

Collection interval significantly affected motility and abnormality proportion of rabbit semen (P<0.05). If the collection interval is too short, the motility in living sperms is decreased. This is probably due to stems of young sperms attach each other making them unable to move straightly.

Collection interval affected C and V.A.C. With one day collection interval, C and V.A.C was 259.68 million/ml and 128.72 million/ejaculation, respectively. With two day collection interval, these values were 266.12 million/ml and 164.44 million/time, respectively. With 3 day collection interval, these values were 268.60 million/ml and 179.51 million/ejaculation, respectively. With 4 day collection interval, these values were 275.72 million/ml and 191.90 million/ejaculation.      Our findings showed that 03 and 04 day collection interval produced better semen quality than 01 or 02 days.

Physical and chemical characteristics of rabbit semen

To provide a basis for making diluting medium which meets the demands osmotic pressure, viscosity and density of semen living outside the body, we studied physical and chemical characteristics of semen of some rabbit genotypes keeping in Vietnam. Results are shown in Table 4. It can be seen from the table that different rabbit genotypes had different physical and chemical characteristics of semen. Osmotic pressure of rabbit semen was in equilibrated zone. New Zealand rabbits had highest osmotic pressure with 366.09 miliosmol/kg; lowest was for California rabbits with 318.95 miliosmol/kg. Osmotic pressure of semen is a basic to make medium preserving semen outside living bodies.

pH of rabbit semen varied 6.69 – 6.89. pH of semen depends on V, metabolic activities, hormone. pH of semen is different among semen collections. When semen is diluted with medium with lightly acid, it creates a favorable condition for sperms, because sperms are less active that saves reserved energy. This makes sperms live longer. Living ability of sperms during preservation depends very much on dilution medium. Dilution medium must have a buffering ability to maintain favorable pH for sperms. Watson (1990) cited by Roca (2000) reported that sperms had higher living ability in pH =6.9 of dilution medium than pH = 7.05. Roca (2000) reported that rabbit sperms can survive in dilution medium with osmotic pressure between 333 - 336 mosm/l. Dalimata (1997) diluted rabbit semen in medium with osmotic pressure of 300 mosm/l.

Diluting and preserving rabbit semen

Preserving rabbit semen in  Tris and Citrate environments

The quality of diluted semen can be evaluated via living ability of sperms. Living ability of sperms depends on not only semen quality but also dilution medium and preserving temperature. After studying physical and chemical characteristics of rabbit semen and dilution medium, we diluted semen in two types of environment and preserved semen at 15 0C. We evaluated motility, living proportion, abnormality proportion at 0, 24, 48, 72 hours after dilution. When evaluating diluted semen quality, semen samples were stored at 37⁰C. Results showed in Table 5.

Table 5 was shown that semen diluted with Tris solution had motility of spermatozoa higher than Citrate solution (P<0.05). Motility of spermatozoa reduced following conserving times, at 0 hour (after diluting semen with solution), motility of spermatozoa were different in two solutions (P<0.05), A = 65.73% in Tris solution; A = 63.17% in Citrate solution. After conserving 24 hours, motility of spermatozoa were 58.29% in Tris solution, and were 54.15% in Citrate solution, it was enough criteria for insemination. After conserving 72 hours, motility of spermatozoa reduced rapid, it was not enough criteria for insemination.

Our results were lower than Do Van Thu et al (2004) reported in Tris – Citrate – Glucose solution, at 15^oC, The motility of spermatozoa of 0, 24, 48, 72 and 96 conserving hour  were 70.71%, 65.29%, 54.38%, 31.25% and 21.75%, respectively.  The rate of survival spermatozoa had higher in Tris solution comparison with Citrate solution (P<0.05).  

We thought that Tris solution had vitality of rabbit spermatozoa better than Citrate solution in conserving and maintaining. It can explain, Tris solution preserved acrosome of  sperms, pH of Tris solution (6.90) was equivalent with pH of rabbit semen (6.69-6.89). Buffer ability of Tris solution (1583.16) had higher than it of rabbit semen, It had signification in conserving semen, because it maintained stable pH of solution during conserving times. 

Effect of conserving temperature on quality of rabbit semen

 (Conserving solution of Tris, n = 29)

It can be seen in Table 6 that the vitality of rabbit spermatozoa effected by conserving temperature. The vitality of rabbit spermatozoa reduced gradually following conserving time. The semen diluted with Tris solution at 15^oC had motility of sperms higher than it at 5^oC calculation until 72 conserving hours (P<0.05).

Results above were shown that the semen diluted with Tris solution at 15^oC had motility of sperms higher than it at 5^oC. Because diluting semen conserved at 5^oC, the spermatozoa were in a state of shock, the rabbit spermatozoa were very sensitive to temperature, chemical… So motility of sperms reduced quicker than it at 15^oC.  Our results were corresponding  with Roca (2000) reported that rabbit semen diluted with Tris solution maintaining at 15^oC was more suitable than at 5^oC.  

Effect of dilute multiple (semen: solution) on quality of rabbit semen

Vitality of rabbit spermatozoa influence on dilute multiple was influenced by dilute multiple between semen and solutions. The dilute multiple depended on concentration and motility of spermatozoa. If dilute multiple was high, it would reduce pregnant rate and vitality of spermatozoa. If dilute multiple was low, it would increase the cost of semen. To determine suitable dilute multiple, we carried out to dilute the semen and the Tris solutions following multiples 1:5 and 1:10, conserving at 15 ^oC. The results were described in table 7. 

 Table 7 shown that rabbit semen were diluted 1:5 (semen : solution) had motility of spermatozoa higher than 1:10 (p<0.05). After 72 conserving hours, the motility of spermatozoa with diluting 1:5 (31.11%) were higher than diluting1:10 (21.94%) (p<0.05). Rate of survival sperm diluted with 1:5 had higher than 1:10 (P<0.05). So rabbit semen diluted with 1:5 (semen: solution) was more suitable than 1:10.

Artificial insemination for rabbit by dilute semen 

The results of artificial insemination for rabbit by dilute semen conserving Tris environment

Tris solution is mixed simple, low cost and no effect on The proportions of pregnant. Therefore, we took dilute semen with Tris solution conserving at 15^oC to inseminate for the bucks of New Zealand and black rabbit by artificial insemination method in repeated two times. The first time was in the morning, the second was in the afternoon after 6 – 8 hours.

 Table 8 showed that rabbit semen diluted with Tris solution conserving at 15^oC after 0, 24, 48 hours maintained the alive and insemination abilities. Total of inseminated females were 21 heads, number of pregnant buck-rabbits were 14. The proportions of pregnant of conserving semen were 75.00% in the first day, 71.43% in the second day and 50.00% in the fourth day.

According to Nguyen Quang Suc and Dinh Van Binh (1998) showed that the proportions of pregnant of direct insemination were 55 – 75%. Roca (2000) used semen diluting with solution of Tris – acid Citric – glucose conserving at 15^oC inseminated buck-rabbits to attain 77.1 % of  the proportions of pregnant.

In research of Lopez et al., (2005) as shown the proportion of pregnant of rabbits inseminated with semen supplementing gelatin conserving at 15^oC were 88% after 48 hours, 83% after 72 hours. The author concluded that rabbit semen conserving at 15^oC until 5 days of semen storage still get pregnancy.

Monitoring young rabbits born by artificial insemination

 Table 8 and table 9 shown that total of pregnant roe-rabbits were 14 heads. Total of young rabbits at birth were 91, average 7.58 head/litter, number of live baby rabbits at 1 month were 76 (rate of live was 73.63%). Live weight at birth was 51.73 gram/head, 566.54 gram/head at 1 month.

 According to Nguyen Quang Suc and Dinh Van Binh (1998) informed that the roe-rabbits mated for 50-60 gram per head at birth, 500-600 gram per head at 1 month and average number of live rabbits were 4.5 – 7.5 head/litter. Local rabbit got 5 – 6 head/litter, rabbit of New Zealand had 6 – 8 head/litter, 55 – 60 gram/head at birth and 650 – 700 gram/head at weaning.

Conclusions

- The volume of rabbit semen per ejaculation was low 0.65 – 0.91 ml. Percent of the swimming spermatozoa was 61.34 – 64.95%. Concentration of sperm was 263.46 – 291.03 million/ml. Total number of spermatozoa per the ejaculation was  263.46 – 291.03 million.  Percent of the alive spermatozoa was 69.82 – 72.52%. Rate of sperm of abnormality was 18.47 – 19.09%. Resistance of sperm to NaCl 1% was 2584.09 – 2720.52.

- The osmotic pressure of rabbit semen was 318.95 – 366.09 miliosmol/kg. Buffer ability of rabbit semen was 1138.93 – 1314.72. The viscosity of semen was 2.78 – 2.96. The density of semen was 1.023 – 1.025. The pH of semen was 6.69 – 6.89. 

- The rabbit semen should be ejaculated in the cool months for better quality than the hot months. The interval between two ejaculations was three or four days for many good quality of semen.

 - Semen diluted (1:5) got better result than diluted rate (1:10). Semen diluted in the Tris solution conserving at 15^oC had percent of the alive spermatozoa better than 5^oC.

- The proportions of pregnant of conserving semen was 75.00% in the first day, 71.43% in the second day and 50.00% in the fourth day.

References 

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