Br. vet.J. (1995).

151,477 REVIEW ARTIFICIAL INSEMINATION IN RABBITS

j. M. MORRELL European Molecular Biology Laboratory, Postfach 10.22.09, D-69012 Heidelberg, Germany

SUMMARY
Artificial insemination (AI) in rabbits can be a useful aid to colony management. In this review, simple non-invasive techniques for semen collection and M are described. Conception rates following M can be equivalent to, or better than, those achieved by natural mating, with the added advantage that contact between animals is avoided. Ovulation can be induced reliably by the administration of a gonadotrophin releasing hormone analogue, buserelin, as an alternative to the use of a vasectomized buck or the injection of luteinizing hormone. The use of enzymelinked immunoassay kits for progesterone assay can assist colony management by rapidly identifying non-pregnant animals for re-insemination. Frozen-thawed sperm have been inseminated but careful attention to the cryopreservation technique is required to ensure good conception rates. I~.vwopd~s: Rabbits; artificial insemination; sperm; pregnancy detection; cryopreservation.

INTRODUCTION
Artificial insemination (M) has been employed in rabbits since the 1920s (Adams, 1961) and gives similar or better pregnancy rates than natural breeding (Harkness & Wagner, 1983). Most breeding units rely on natural mating, with a conception rate of approximately 85% in healthy individuals (Harkness & Wagner, 1983) but M permits more controlled management and better planning than conventional means, e.g. in batch parturition and batch weaning (Adams, 1987). In addition, M offers the same benefits for rabbit breeding as in other species in the conta'ol of genetic diversity, rapid upgrading of stock, establishment of pregnancies in females which refuse to mate, and avoidance of the spread of diseases, such as pasteurellosis and Treponema cunimdi. The number of males needed is reduced compared with natural mating, where one male is required for approximately 8-10 females (Hafez, 1970): an ejaculate from one male can be used to inseminate up to 20 females, depending on the original sperm concen0007-1935/95/050477-12/$12.00/0 1995 Bailli&eTindall

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tration and required insemination dose. Semen donors from other colonies can be used, provided that proper quarantihe procedures are carried out. The latter is particularly important where diseases such as rabbit haemorrhagic disease are concerned, since it is not known at present whether this virus can be transmitted in semen (Dr H. Fuller, personal communication). This paper outlines simple techniques for semen collection and insemination in the rabbit and summarizes information available on related topics such as training males to use an artificial vagina (AV), semen assessment, freezing semen and pregnancy detection. Where personal observations are recorded, they refer to the author's experiences with sandy halflop rabbits over a 7 year period.

REPRODUCTIVE BIOLOGY

Rabbits become sexually mature at 4-7 months depending on the mature body weight of the strain. Small strains, e.g. Dutch and Polish, reach puberty at around 4 months; medium-sized strains, e.g. Sandy lop and New Zealand White, between 4 and 6 months; while larger breeds, e.g. Flemish, are not sexually mature until 7 months of age. Rabbits are capable of breeding for several years under natural mating conditions but litter size tends to decrease in older females (Adams, 1970) and ejaculate quality may also decrease with age (personal observation). Mounting behaviour has been observed between litter mates before weaning but usually the first mature spermatozoa (sperm) are not seen in the epididymis until approximately 4 months of age (Thefford et al., 1992). Females do not have regular oestrous cycles but may show variation in sexual receptivity, linked to waves of follicular growth and regression within the ovary. Seasonal patterns of reproductive behaviour have been reported (Farrell et al., 1968), although this may be influenced by the husbandry system under which the animals are kept, such as controlled day length. Rabbits are classified as induced ovulators, with ovulation occurring 9-13 h after coitus, although spontaneous ovulation is also possible. In our colony, spontaneous ovulation occurred at a rate of approximately 3%, as assessed by measurement of progesterone levels in females housed singly for at least 3 weeks before collection of blood samples. The precise mechanism of induction of ovulation is not known in this species but appears to be different from that of other induced ovulators. The inducing stimulus in cats is thought to be mechanical stimulation of the cervix, while the prolonged, sometimes violent, courtship behaviour of ferrets may play a role in initiating ovulation (Porter & Brown, 1987). Since it is possible for female rabbits to induce ovulation in other females (Staples, 1967), vaginal penetration is not a prerequisite for ovulation induction in this species. Furthermore, vaginal stimulation during AI does not usually cause ovulation: only one out of 72 females (1.4%) ovulated after being inseminated without an accompanying ovulatory stimulus, as detected by a raised plasma progesterone concentration within 2 h of insemination (Morrell, 1990) and the appearance of a litter 30 days later. A sterile mating or spontaneous ovulation is followed by pseudopregnancy lasting 1%19 days (Napier, 1963). Females are most likely to conceive if re-mated 18

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days after a sterile mating, counting the day of mating as day 0 (Fischer et aL, 1986). The end of pseudopregnancy is marked by hair-plucking in some animals due to the changes in progesterone levels causing loosening of the hair (Sawin et al., 1960) but there is considerable variation between individuals in the extent of the hair loss. In some animals all the hair from the chest, abdomen and hind legs may be pulled out, while in other animals little more hair is lost than occurs through natural moulting. Pregnancy lasts 30-33 days with an average litter size of eight (Hafez, 1970). Prolonged gestation may occur if the litter size is small; gestation lengths of up to 40 days were observed for females producing singletons (Morrell & Dresser, 1989) and the young in such cases were abnormally large and stillbor'n.

SEMEN COLLECTION The equipment required for semen collection (Fig. 1) comprises a rabbit AV and rubber liners (Holborn Surgical, Kent, UK), and a small collecting tube of an appropriate size to fit the AV. An alternative design for an AV was reported by Bredderman et al. (1964) to avoid loss of sperm through adherence to the rubber liner. Rinsing the AV liner with 2-3 ml medium (see later) also reduces semen loss. In addition to the AV, either a rabbit 'skin' is needed for the male to mount, or a female teaser can be introduced into the male's cage for this purpose.

Fig. 1. Equipment required for semen collection. The artificial vagina (right) has a rubber liner and a small collecting tube for the ejaculate. The 'skin' (left) is prepared from a female rabbit.

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A rabbit skin can be prepared easily from a culled doe: it is considered essential to use the skin from a female for this purpose (K. D. Keller, personal communication). The skin is preserved by covering the underside with borax for up to 1 week a n d then softening with lanolin. The AV is assembled by inserting the rubber liner into the rigid outer case and turning the ends of the liner over the top and bottom edges. The collecting tube is placed in the bottom hole and the space between the outer case and the liner is filled with hot water (40-45C, depending on the individual rabbit's preference). A thermometer should be used to measure the temperature of the water in the AV rather than guessing, as the sample may be contaminated with urine if the temperature is too higli (Hafez, 1970). If the water is not warm enough, there will be insufficient stimulation for the buck to ejaculate. The liner is lubricated with a suitable (non-spermicidal) obstetrical jelly. The assembled AV is held in the collector's hand with the rabbit skin covering the hand and forearm. The arm is introduced into the buck's cage and the male is allowed to m o u n t the skin (Fig. 2), which occurs readily with experienced males. After mounting the buck gives several vigorous pelvic thrusts and ejaculates immediately the penis enters the vagina, before falling off the skin sideways due to the vigour of the copulatory thrusting (Hafez, 1970). Ejaculation is accompanied by a characteristic noise in some animals. To the uninitiated, the sudden 'collapse' of the male appears alarming, but is in fact typical of rabbits during mating. Alternatively, a teaser female can be used, particularly if the male is accustomed to natural mating and has never been trained with a rabbit skin. The doe is intro-

Fig. 2. Buck starting to mount the 'skin' which is covering the hand and forearm of the semen collector. The artificial vagina is held in the collector's hand.

ARTIFICIAL INSEMINATION IN RABBITS

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duced into the buck's cage and, as the male mounts, the AV is quickly inserted between the animals to prevent the penis entering the female's vagina. Since there is very little courtship behaviour in rabbits it is essential to position the AV swiftly to avoid missing the sample completely and causing undesired pregnancies. Use of a 'skin' instead of a teaser avoids these problems and it is relatively easy to train the male to m o u n t the 'skin'.
TRAINING MALES

Sandy halflop bucks can be trained to m o u n t a skin and ejaculate into an AV at approximately 5.5 months old. When the skin and AV are introduced into the animal's cage as described previously, a young male will usually approach the skin cautiously and sniff at it. Within a few minutes he will attempt to mount and start thrusting, although sometimes he tries to m o u n t at the wrong end. Once the penis has entered the AV, ejaculation occurs straight away. At the next training session the animal associates the appearance of the skin in his cage with a 'pleasurable' experience and therefore mounts readily. It is important that the interval between training sessions is not too long so that the association between the skin and ejaculation is not forgotten. Training sessions every day or every other day are desirable for the first week. Occasionally an animal will be reluctant to approach the skin at first and the process of introducing the skin into the cage will have to be repeated several times. It is important not to rush this process, since moving the skin to the rabbit instead of allowing the rabbit to approach the skin may intimidate tlae animal and inhibit him from mounting. This training method has also been used successfully with older males which have had prior mating experience, although the initial conditioning phase with the skin may need longer than with younger inexperienced males.
SEMEN

Sandy halflops produce 0.5-2.0 ml semen which may contain a gel component. The average sperm concentration for medium-sized breeds is around 500 million sperm ml -~ (Napier, 1963), although breed differences occur with regard to volume of ejaculate and daily sperm output (Amann, 1966). Semen can be collected up to four times per week from each buck without reducing the sperm concentration (Desjardins et al., 1968). There are reports of collections being made more frequently without affecting the animal's libido or the quality of the ejaculate (Hafez, 1970; Adams, 1983). However, Wicher et al. (1987) reported a rise in antibodies to sperm antigens in the seminal fluid of rabbits used very frequently as semen donors: their collection schedule was two or three times daily, several days a week for 9 months. Such intensive use is not necessary for breeding under norreal circumstances.
SEMEN PROCESSING

Laboratory assessment of semen quality can include sophisticated techniques such as those used routinely for other species (Moss et al., 1979), or may only involve

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microscopical examination of an aliquot on a wanned slide for the subjective estimation of sperm viability and motility." Computerized sperm motility analysis can be used to provide an objective assessment of sperm quality (Stephens et al., 1988; Young et al., 1992) but this technology is not widely available yet. Moreover, there have been conflicting reports on its usefulness as a means of predicting sperm fertilizing capacity for in vitro or in vivo fertilization in man (Holt el aL, 1985, 1989; Graczykowski & Siegel, 1991). So far, no relationship has been established between sperm motility patterns and fertilizing potential in other species. If the sperm are to be inseminated fresh, i.e. on the day of collection, a subjective assessment of dae proportion of sperm which are motile and the motility pattern exhibited will be sufficient to enable ejaculates of poor quality to be identified and excluded. Usually at least 70% of the sperm show forward progressive motility in ejaculates from fertile males (personal obsel-vation). After quality assessment, the ejaculate is diluted with a suitable semen extender according to its proposed use. Wales el al. (1965) reported that insemination doses of not less than one million sperm should be used for maximum fertility. Other authors suggest using higher sperm dose, for example 20 million sperm (Adams, 1961) or 20-50 million sperm (Hafez, 1970). In insemination studies with 'sexed' sperm where only low numbers of sperm (1-10 million) were available for insemination, the conception rate was seen to increase with increasing sperm dose, with double the number of pregnancies being obtained following the use of 7-10 million sperm as for 1-6.5 million sperm (unpublished observation). Semen for 'fi'esh' insemination requires the use of very simple extenders such as physiological saline or Krebs-Ringer solution, to which antibiotics have been added (Adams, 1961). The author prefers to use an egg yolk-ciu'ate medium, modified by reducing the egg yolk content of Willett and Salisbury's original medium fi'om 20 to 2% (Willett & Salisbury, 1942). In a small study, reducing the egg yolk content of the medium was associated with an increase in conception rate among inseminated rabbits (Morrell & Dresser, 1989); the conception rate doubled where only 5% egg yolk was included in the medium, instead of 10 or 20%. Decreasing the proportion of egg yolk still further was associated with another increase in the number of pregnancies. A similar observation was made with bull sperm in Caprogen diluent (Shannon, 1965): decreasing the proportion of egg yolk from 20 to 5% was associated with an increase in conception rate in inseminated cows (Shannon, 1972). A more complex medium containing cryoprotectants is required for freezing rabbit semen, e.g. Tris-yolk-12.5% dimethylsulphoxide (DMSO) medium (Stranzinger el al., 1971). Conflicting results have been obtained from the insemination of frozen rabbit semen. Su'anzinger et al. (1971) reported that the postthaw fertilizing ability of rabbit sperm was no different from that of fi'esh sperm, but the method of freezing, for example in pellets, ampoules or tubing, had a marked effect on sperm viability. Parrish and Foote (1986) found that it was necessary to reduce the time between insemination and ovulation where frozen sperm were used in order to achieve comparable fertilization rates to fresh sperm. Chen et al. (1989) also achieved a lower conception rate with frozen sperm than with fresh sperm.

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INSEMINATION

483

Insemination pipettes can be prepared from glass tubing with a slight b e n d approximately 8 cm fi'om one end. A rubber teat is attached to the other e n d of the pipette (Napier, 1963) or, alternatively, for greater control during semen deposition, a 1 ml syringe can be attached to the pipette with a short length of plastic tubing (personal observation). Fig. 3 shows an insemination pipette prepared from a standard laboratory 1 ml glass pipette. Rabbits for AI should be housed separately for at least 19 days prior to insemination to avoid the possibility of pseudopregnancy. Females are chosen which are t h o u g h t to be sexually receptive on the basis of general behaviour and vulval colour, which varies from pale pink to purple (Adams, 1983). A female which is sexually receptive may appear restless and excited, particularly if the person who normally performs the insemination is in the vicinity. Such behavioural changes may be obvious only to people who are familiar with the animals concerned. Behaviour during insemination is a useful guide to sexual receptivity (see below). Vulval colour alone is not a reliable indicator of receptivity since occasionally a doe with a pale pink vulva may mate, while one with a purple vulva may refuse to mate. Furthermore, purple vulval colouration can be observed in a proportion of females during pregnancy: when m o n i t o r i n g of vulval colouration was carried out three times weekly over a 30-day period, purple colouration was observed at least once in 15 out of 16 p r e g n a n t females and in all of 16 females during pseudopregnancy (unpublished observation). Vulval colour is assessed using a similar technique as for sexing rabbits: the animal is held firmly by the scruff with its back to the holder while the lips of the vulva are gently spread apart to reveal the mucous membranes. For insemination the female is placed in a shallow restraining box without neck restraint (Fig. 4). The bottom of the box should have a non-slip surface; a rubber bath mat can be used to line a conventional restraining box. A receptive female will exhibit lordosis when the tail is lifted gently, thus permitting the insertion of the lubricated insemination pipette into the vagina. It is necessary to insert the pipette at an angle of approximately 45 to negotiate the rim of the pelvis. Once the diluted semen is released into the upper vagina and the pipette is removed, the doe will sit down again. An unreceptive doe will not lift her hindquarters when

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Fig. 3. An insemination pipette prepared from a standard 1 ml glass laboratory pipette. A bend is produced by heating the pipette in a bunsen flame approximately 8 cm from one end. When the glass is soft, the pipette can be bent to the required angle.

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the tail is raised: instead she clamps her tail firmly against the perineal region and remains sitting. Alternatively, the doe "can be restrained in a supine position for insemination (Adams, 1961; Hafez, 1970). Intracervical insemination is not possible in sandy halflops using the type of insemination pipette shown in Fig. 3 because of.the length of the vagina in this su'ain (average length in 20 females was 20.8 _+ 2.4cm). It is debatable whether intracervical insemination can be accomplished with certainty in this species. Since there is no connection between the two uterine horns in rabbits, it would be necessary to deposit the sperm suspension in both cervices if intracervical insemination was desired.

INDUCTION OF OVULATION Three methods of inducing ovulation are available for this species: (1) mating with a vasectomized buck; (2) administration of h u m a n chorionic gonadotropin (hCG); or (3) administration of a gonadotropin releasing h o r m o n e (GnRH) analogue, e.g. buserelin (Receptal; Hoechst, UK) (Battista et al., 1987; Morrell, 1990). For preference the author uses 0.8 ptg buserelin, administered by subcutaneous injection. Although the surgical preparation of vasectomized males is not complicated, their use can negate some of the benefits of AI, for example in disease control and in reducing the number of animals kept. In addition, females exhibit preferences concerning which buck they will allow to mate with them. Thus several vasectomized bucks would have to be kept to provide an alternative if the first one does not meet with 'approval', presenting considerations for husbandry and management. Secondly, vasectomized males can develop anti-sperm antibodies in their seminal plasma because of the alteration of the blood-testis barrier after vasectomy (Singh & Yang, 1984). Therefore mating with such a male immediately after

Fig. 4. Restraining box for rabbits. The restraining piece which normally goes over the animal's neck has been removed since animals quickly become accustomed to sitting quietly in the box without restraint. This type of box allows easy access to the rabbit's hindquarters. The bottom of the box is lined with a non-slip mat.

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insenainafion could reduce the number of viable sperm available and therefore adversely affect fertility. However, allowing the mating to occur 1-4 h after insemination can circumvent the problem because of sperm migration away from the site of semen deposition. Thirdly, it is possible for a vasectomized buck to mount a doe and ejaculate without inducing ovulation (Hafez, 1970). Allowing the pair to mate more than once does not appear to affect the size of the subsequent litter. The female should always be taken to the male's cage for mating, not vice versa, to avoid fighting and inhibition of the male (Napier, 1963). The administration of hCG intravenously to induce ovulation also has several disadvantages. Since hCG is highly antigenic, repeated administration results in antibody formation and consequently there is no ovulatory response when hCG is administered on subsequent occasions (Adams, 1961). Moreover, training is required to perform the intravenous injection correctly. The recent development of the GnRH analogue, buserelin, appears to have solved the problem of induction of ovulation in rabbits, since the decapeptide molecule is non-antigenic. The preparation is injected subcutaneously at the time of insemination and causes ovulation approximately 10-12 h later. This method is simple and reliable and can be used in subsequent AI attempts in the same females without loss of efficacy.

PREGNANCY DIAGNOSIS

Until recently there was no simple reliable method for pregnancy diagnosis in rabbits. Sexual receptivity at a test mating does not indicate non-pregnancy because pregnant females may permit mating (Adams, 1983) and bucks are not deterred from mating a female by either pregnancy or pseudopregnancy. Likewise, the absence of hair plucking at around day 18 after insemination is not a reliable indicator that the female is pregnant and not pseudopregnant. Developing foetuses can be palpated in the uterus 10 days (Suitor, 1946) or 13-14 days (Hafez, 1970) after mating, but the technique can be risky because the foetuses may be damaged, causing abortion or toxaemia. Furthermore, the technique may not be effective where only one or two foetuses are present. Radiography is possible after the 11 th day of pregnancy but is not a practical means of pregnancy diagnosis for most breeding units. However, it has been shown that enzyme-linked immunoassay (ELISA) kits for the assay of plasma progesterone in other species can be used with rabbit plasma and serum (Morrell, 1990, 1993). The Ovucheck ELISA kit (Cambridge Life Sciences) for bovine progesterone has been used to measure rabbit plasma progesterone. A comparison between measurements of the same samples by ELISA and radioimmunoassay (RIA) showed that there was a good relationship between ELISA and RIA values (Fig. 5), although the absolute concentrations as assayed by ELISA were higher than RIA, as is also the case for bitch plasma progesterone (England et al., 1989). Using the Ovucheck ELISA kit it is possible to differentiate between pregnant and pseudopregnant animals reliably at around day 17-18 after insemination. Since the optimal time for re-mating pseudopregnant rabbits is at day 18 (Fischer et al., 1986) measurement of the progesterone level at this time provides a useful

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6-

7
r-

5-

4-

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 RIA progesterone ng m1-1 Fig. 5. The relationship between plasma progesterone concentrations measured by radioimmunoassay (RIA) and enz}qne-linked immunoassay (ELISA). The coefficient of regression is significant.

tool for colony m a n a g e m e n t . Thus p r e g n a n t females can be h o u s e d and fed appropriately d u r i n g late p r e g n a n c y while n o n - p r e g n a n t females can be insemin a t e d again. F u r t h e r m o r e , m e a s u r e m e n t o f p r o g e s t e r o n e in plasma or serum from samples taken 2 h after the administration o f the ovulatory stimulus can be used to predict o ~ d a t i o n since p r o g e s t e r o n e c o n c e n t r a t i o n reaches a peak a p p r o x i m a t e l y 10 h b e f o r e ovulation in rabbits (Mills & Geradot, 1984) and then declines sharply again.

ACKNOWLEDGEMENTS

I am grateful to Professor David Noakes, Royal Veterinary College, for a r r a n g i n g the RIA o f rabbit plasma samples.

REFERENCES

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Journal of Reproduction and Fe~ilitr 24, 111-13.

SLTN'OR, A. E. (1946). Palpating domestic rabbits to determine pregnancy. Leaflet 245, United States Department of Agriculture. TH~TrORO, C. R., MORRZLL,J. M. & COCKLE, S. M. (1992). TRH-related peptides in rabbit prostate during sexual maturation. Biochemica et Biophysica Aaa 1115, 252-58. WALES, R. G., IVL.~RTIN, L. & O'SHEA, T. (1965). Effect of dilution rate and of the n u m b e r of spermatozoa inseminated on the fertility of rabbits ovulated with chorionic gonadotrophin. Journal of Reproduction and Fertilitr 10, 69-78. WICI-IER,V., WITHER,K. & GRUI-IN,R. F. (1987"). Increased production of antibodies to spermatozoa and seminal fluid in rabbits used as semen donors. Inte~ational Archives of Alle~, and Applied hnmunology 83, 104-108. Wn.LETT, E. L. & SALtSBURV,G. W. (1942). The effect of various dilutors, cooling rate, temperature of storage, and some other factors on the livability of spermatozoa in stored samples of bull semen. Memoirs of the Cornell University Agricultum.l Expel~mental Station No. 249. YOt,NC,, R. J., BOOT, B. A., I'rURV, At.DE, T. G. & ST.am~t. W. C. (1992). Automated analysis of rabbit sperm motility and the effect of chemicals on sperm motion parameters. Molecular Reproduction and Development 33, 347-56.

(Accepted3 Februal7 1995)