8/13/2019 1-s2.0-S0007193505800223-main

1/12

Br. vet.J. (1995). 151,477

R E V I E W

A R T I F I C I A L I N S E M I N A T I O N I N R A B B I T S

j M M O R R E L LEurop ean Molecular Biology Laboratory P ostfach 10.22.09 D-69 012 Heidelberg G ermany

S U M M A R YArtificial insemination (AI) in rabbits can be a useful aid to colonymanagement. In this review, simple non-invasive techniques for semencollection and M are described. Concep tion rates following M can beequivalent to, or better than, those achieved by natural mating, with theadded advantage that contact between animals is avoided. Ovulation canbe induced reliably by the administration of a gonadotrophin releasinghormone analogue, buserelin, as an alternative to the use of a vasectom-ized buck or the injection of luteinizing hormone. The use of enzyme-linked immunoassay kits for progesterone assay can assist colony manage-ment by rapidly identifying non-pregnant animals for re-insemination.Frozen-thawed sperm have been inseminated but careful attention to thecryopreservation technique is required to ensure good conception rates.I~.vwopd~s: Rabbits; artificial ins emination; sperm; pregnancy detect ion ;cryopreservation.

I N T R O D U C T I O NArtificial insemination (M) has been employed in rabbits since the 1920s (Adams,1961) and gives similar or bette r pr egnancy rates than natural b reed ing(Harkness & Wagner, 1983). Most breeding units rely on natural mating, with aconception rate of approximately 85% in healthy individuals (Harkness &Wagner, 1983) but M permits more controlled mana gem ent and better planningthan conventional means, e.g. in batch parturition and batch weaning (Adams,1987). In addition, M offers the same benefits for rabbit breedi ng as in ot herspecies in the conta'ol of genetic diversity, rapid upg radi ng of stock, establishmentof pregnancies in females which refuse to mate, and avoidance of the spread ofdiseases, such as pasteurellosis and T r e p o n e m a c u n i m d i . The number of malesnee ded is reduc ed compa red with natural mating, where one male is required forapproximately 8-10 females (Hafez, 1970): an ejaculate from one male can beused to inseminate up to 20 females, depending on the original sperm concen-0007-1935/95/050477-12/ 12.00/0 1995 Bailli&eTindall

8/13/2019 1-s2.0-S0007193505800223-main

2/12

478 B R ITISH V ETER IN A RY J O U R N A L 151 5

tration and required insemination dose. Semen donors from other colonies canbe used, provided that proper quarantih e proce dures are carried out. The latter isparticularly important where diseases such as rabbit haemorrhagic disease areconcerned, since it is not known at present whether this virus can be transmittedin semen (Dr H. Fuller, personal communication).

This paper outlines simple techniques for semen collection and insemination inthe rabbit an d summarizes info rmati on available on re lated topics such as trainingmales to use an artificial vagina (AV), semen assessment, freezing semen and preg-nancy detection. Where personal observations are recorded, they refer to theauth or's experiences with sandy halflop rabbits over a 7 year period.

R E P R O D U C T I V E I O L O G YRabbits become sexually mature at 4-7 month s de pen ding on the mature bodyweight of the strain. Small strains, e.g. Dutch a nd Polish, reach puberty at arou nd4 months; medium-sized strains, e.g. Sandy lop and New Zealand White, between4 and 6 months; while larger breeds, e.g. Flemish, are not sexually matu re until 7months of age. Rabbits are capable of breeding for several years under naturalmating conditions but litter size tends to decrease in older females (Adams, 1970)and ejaculate quality may also decrease with age (personal observation). Moun tingbehaviour has been observed between litter mates before weaning but usually thefirst matu re sper matozoa (sperm) are not seen in the epididymis until approxi-mately 4 months of age (Thefford e t a l . 1992).

Females do not have regular oestrous cycles but may show variation in sexualreceptivity, linked to waves of follicular growth and regression within the ovary.Seasonal patterns of reproducti ve behaviour have been reporte d (Farrell e t a l .1968), although this may be influenced by the husbandry system under which theanimals are kept, such as controlled day length.

Rabbits are classified as induced ovulators, with ovulation occurring 9-13 h aftercoitus, although spontaneous ovulation is also possible. In our colony, spon-taneou s ovulation occu rred at a rate of approx imately 3 , as assessed by measure-ment of progesterone levels in females housed singly for at least 3 weeks beforecollection of blood samples. The precise mechanism of induction of ovulation isnot known in this species but appears to be d ifferent from that of other in ducedovulators. The inducing stimulus in cats is thought to be mechanical stimulationof the cervix, while the prolonged, sometimes violent, courtship behaviour of fer-rets may play a role in initi ating ovulation (Porter & Brown, 1987). Since it is poss-ible for female rabbits to induce ovulation in other females (Staples, 1967),vaginal penetration is not a prerequisite for ovulation induction in this species.Fur the rmor e, vaginal stimulation during AI does not usually cause ovulation: onlyone out of 72 females (1.4 ) ovulated after being insemina ted without anaccompanying ovulatory stimulus, as detected by a raised plasma progesteroneconc ent rati on within 2 h of inseminat ion (Morrell, 1990) and the appearance of alitter 30 days later.

A sterile mating or spon tane ous ovulation is followed by pseudo pregna ncy last-ing 1 19 days (Napier, 1963). Females are most likely to conceive if re-mated 18

8/13/2019 1-s2.0-S0007193505800223-main

3/12

ARTIFICIAL INSEMINATION IN RABBITS 79

days after a sterile mating, counting the day of mating as day 0 (Fischer et aL1986). The e nd o f pseud opregn ancy s marked by hair-plucking in some animalsdue to the changes in progesterone levels causing loosening of the hair (Sawin etal. 1960) but there is considerable variation between individuals in the exte nt ofthe hair loss. In some animals all the hair from the chest, abdomen and hind legsmay be pulled out, while in other animals little more hair is lost than occursthrough natural moulting.Pregnancy lasts 30-33 days with an average litter size of eight (Hafez, 1970).Prol onged gestation may occur if the litter size is small; gestation lengths of up to40 days were observed for females producing singletons (Morrell Dresser, 1989)and the youn g in such cases were abnormally large and stillbor'n.

SEMEN COLLECTIONThe equipment required for semen collection (Fig. 1) comprises a rabbit AV andrubber liners (Holborn Surgical, Kent, UK), and a small collecting tube of anappropriate size to fit the AV. An alternative design for an AV was reported byBredderman et al. (1964) to avoid loss of sperm th rou gh a dhe rence to the rubberliner. Rinsing the AV liner with 2-3 ml medium (see later) also reduces semenloss. 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 femalerabbit.

8/13/2019 1-s2.0-S0007193505800223-main

4/12

480 BRITISH VETERINARYJOURNAL, 151, 5A rabbit skin can be prepare d easily from a culled doe: it is cons idered essential touse the skin from a female for this purpose (K. D. Keller, personal communi-cation). The skin is preserved by covering the underside with borax for up to 1week an d then softening with lanolin.The AV is assembled by inserting the rubber liner into the rigid outer case andturning the ends of the liner over the top and bottom edges. The collecting tubeis placed in the bottom hole and the space between the outer case and the liner isfilled with hot water (40-45C, depending on the individual rabbit s preference).A the rmo met er should be used to measure the temperatu re o f the water in the AVrather than guessing, as the sample may be contaminated with urine if the tem-pera ture is too higli (Hafez, 1970). If the water is not warm eno ugh , there will beinsufficient stimulation for the buck to ejaculate. The liner is lubricated with asuitable (non-spermicidal) obstetrical jelly.

The assembled AV is held in the collector s han d with the rabbit skin coveringthe han d an d forearm. The arm is intro duce d into the buck s cage and the male isallowed to mo un t the skin (Fig. 2), which occurs readily with exper ienc ed males.After mounting the buck gives several vigorous pelvic thrusts and ejaculatesimmedia tely the penis enters the vagina, before falling off the skin sideways due tothe vigour of the copulatory thrusting (Hafez, 1970). Ejaculation is accompaniedby a characteristic noise in some animals. To the u ninitia ted, the su dden collapseof the male appears alarming, bu t is in fact typical of rabbits du ring mating.

Alternatively, a teaser female can be used, particularly if the male is accustome dto 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 thesemen collector. The artificial vagina is held in the collector s hand.

8/13/2019 1-s2.0-S0007193505800223-main

5/12

ARTIFICIAL INSEMINATI ON IN RABBITS 48

duc ed into the buck s cage and, as the male mounts, the AV is quickly insertedbetween the animals to prevent the penis en terin g the female s vagina. Sincethere is very little courtship behaviour in rabbits it is essential to position the AVswiftly 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 totrain the male to mo un t the skin .

T R I N I N G M L E SSandy halflop bucks can be trained to mount a skin and ejaculate into an AV atapproxi mately 5.5 mon ths old. When the skin and AV are int roduce d into the ani-mal s cage as described previously, a young male will usually app roach the skincautiously and sniff at it. Within a few minutes he will attempt to mou nt and startthrusting, although sometimes he tries to mount at the wrong end. Once thepenis has entered the AV, ejaculation occurs straight away. At the next trainingsession the animal associates the appe arance of the skin in his cage with a pleasur-able exper ience a nd therefo re moun ts readily. It is import ant that the intervalbetween training sessions is not too long so that the association between the skinand ejaculation is not forgot ten. Tra ining sessions every day or every other day aredesirable for the first week. Occasionally an animal will be reluctant to approachthe skin at first and the process of introducing the skin into the cage will have tobe repeated several times. It is important not to rush this process, since movingthe skin to the rabbit instead of allowing the rabbit to approach the skin mayintimidate tlae animal and inhibit him from mounting.

This t raining me tho d has also been used successfully with older males whichhave had prior mating experience, although the initial conditioning phase withthe skin may need longer than with young er inexperienced males.

S E M E NSandy 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 millionsperm ml -~ (Napier, 1963), alt hough breed differences occur with regard to vol-ume of ejaculate and daily sperm outp ut (Amann, 1966). Semen can be collectedup to four times per week from each buck without reducing the sperm concen-tration (Desjardins e t a l . 1968). Ther e are reports o f collections being made morefrequently without affecting the animal s libido or the quality of the ejaculate(Hafez, 1970; Adams, 1983). However, Wicher e t a l . (1987) reported a rise in anti-bodies to sperm antigens in the seminal fluid of rabbits used very frequently assemen donors: their collection schedule was two or three times daily, several daysa week for 9 months. Such intensive use is not necessary for breeding under nor-real circumstances.

S E M E N P R O C E S S I N G

Labora tory assessment of semen quality can inc lude sophisticated techniques suchas those used routinely for other species (Moss e t a l . 1979), or may only involve

8/13/2019 1-s2.0-S0007193505800223-main

6/12

482 BRITISH VETERINARY JOURNAL 151 5

microscopical examinatio n o f an aliquot on a wanned slide for the subjective esti-mation of sperm viability and motility. Computerized sperm motility analysis canbe used to provide an objective assessment of sperm quality (Stephens et al . , 1988;Young et al . , 1992) but this technol ogy is not widely available yet. Moreover, therehave been conflicting reports on its usefulness as a means of predicting sperm fert-ilizing capacity for in v i t ro or i n v i v o fertilization in man (Holt e l aL , 1985, 1989;Graczykowski & Siegel, 1991). So far, no relationship has been establishedbetween sperm motility patterns and fertilizing potential in ot her species.

If the sperm are to be inseminated fresh, i.e. on the day of collection, a subjec-tive assessment of dae proportion of sperm which are motile and the motility pat-tern exhibited will be sufficient to enable ejaculates of poor quality to be ident-ified and excluded. Usually at least 70% of the sperm show forward progressivemotility in ejaculates from fertile males (personal obsel-vation).

After quality assessment, the ejaculate is diluted with a suitable semen ext end eraccording to its proposed use. Wales e l a l. 19 65 ) reported that insemination dosesof not less than one million sperm should be used for maximum fertility. Otherauthors 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 numbe rs of sperm (1-10 million) were available for insemi-nation, the conceptio n rate was seen to increase with increasing sperm dose, withdouble the number of pregnancies being obtained following the use of 7-10million sperm as for 1-6.5 million sperm (unpubli shed observation).Semen for 'fi'esh' insemina tion requires the use of very simple extende rs suchas physiological saline or Krebs-Ringer solution, to which antibiotics have beenadded (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 originalmedium fi'om 20 to 2% (Willett & Salisbury, 1942). In a small study, red uci ng theegg yolk con ten t of the med ium was associated with an increase in conceptio n rateamong inseminated rabbits (Morrell & Dresser, 1989); the conception ratedoubled where only 5% egg yolk was included in the medium, instead of 10 or20%. Decreasing the proportion of egg yolk still further was associated withanother increase in the number of pregnancies. A similar observation was madewith bull sperm in Caprogen diluent (Shannon, 1965): decreasing the proportionof egg yolk from 20 to 5% was associated with an increase in conception rate ininseminat ed cows (Sha nnon , 1972).A more complex medium containing cryoprotectants is required for freezingrabbit semen, e.g. Tris-yolk-12.5% dimethylsulphoxide (DMSO) medium(Stranzinger el al . , 1971). Conflicting results have been obta ined from the insemi-nation of frozen rabbit semen. Su'anzinger et al. (1971) rep or ted that the post-thaw fertilizing ability of rabbit spe rm was no differ ent from that of fi'esh sperm,but the method of freezing, for example in pellets, ampoules or tubing, had amar ked effect on sperm viability. Parrish an d Foote (1986) found that it was neces-sary to reduce the time between insemination and ovulation where frozen spermwere used in order to achieve comparable fertilization rates to fresh sperm. Chenet al. (1989) also achieved a lower conception rate with frozen sperm than withfresh sperm.

8/13/2019 1-s2.0-S0007193505800223-main

7/12

ARTIFICIAL INSEMINATION IN RABBITS 483I N S E M I N T I O N

I n s e m i n a t i o n p i p e t te s c a n b e p r e p a r e d f r o m g la ss t u b i n g w i th a s l ig h t b e n da p p r o x i m a t e l y 8 c m f i o m o n e e n d . A r u b b e r t e at is a t t a c h e d to t h e o t h e r e n d o ft h e p i p e t t e ( N a p i e r , 1 9 6 3) o r , a l te r n a ti v e ly , f o r g r e a t e r c o n t r o l d u r i n g s e m e nd e p o s i t io n , a 1 m l s y r in g e c a n b e a t t a c h e d t o t h e p i p e t t e w i th a s h o r t l e n g t h o fp l a s ti c t u b i n g ( p e r s o n a l o b s e r v a t i o n ) . F i g . 3 s h o w s a n i n s e m i n a t i o n p i p e t t e p r e -p a r e d f r o m a s t a n d a r d l a b o r a t o r y 1 m l g la ss p ip e t t e .

R a b b i t s f o r A I s h o u l d b e h o u s e d s e p a r a t e l y f o r a t l e a s t 19 d a y s p r i o r t o in s e m i -n a t i o n t o a v o i d th e p o s s ib i li ty o f p s e u d o p r e g n a n c y . F e m a l e s a r e c h o s e n w h i c h a r et h o u g h t t o b e s e x u a l ly r e c e p t iv e o n t h e b a si s o f g e n e r a l b e h a v i o u r a n d v u lv a lc o l o u r , w h i c h v a r ie s f r o m p a l e p i n k t o p u r p l e ( A d a m s , 1 9 8 3 ). A fe m a l e w h i c h i ss e x u a ll y re c e p t iv e m a y a p p e a r r e st le s s a n d e x c i t e d , p a r t ic u l a r l y if t h e p e r s o n w h on o r m a l l y p e r f o r m s t h e i n s e m i n a t i o n is i n t h e v ic in it y. S u c h b e h a v i o u r a l c h a n g e sm a y b e o b v i o u s o n l y to p e o p l e w h o a r e f a m i l ia r w i th t h e a n i m a l s c o n c e r n e d .B e h a v i o u r d u r i n g i n s e m i n a t i o n is a u s e f u l g u i d e t o s e x u a l r e c e p ti v it y ( s e e b el o w ) .V u l v a l c o l o u r a l o n e is n o t a r e l i a b l e i n d i c a t o r o f r e c e p t i v it y s i n c e o c c a s i o n a l l y ad o e w i th a p a l e p i n k v u lv a m a y m a t e , w h i l e o n e w i th a p u r p l e v u lv a m a y r e f u s e t om a t e . F u r t h e r m o r e , p u r p l e v u lv a l c o l o u r a t i o n c a n b e o b s e r v e d in a p r o p o r t i o n o ff e m a l e s d u r i n g p r e g n a n c y : w h e n m o n i t o r i n g o f v ulv al c o lo u r a t i o n w a s c a r r ie d o u tt h r e e t im e s w e e k l y o v e r a 3 0 -d a y p e r i o d , p u r p l e c o l o u r a t i o n w a s o b s e r v e d a t l e a s to n c e i n 1 5 o u t o f 1 6 p r e g n a n t f e m a l e s a n d i n all o f 1 6 f e m a l e s d u r i n g p s e u d o p r e g -n a n c y ( u n p u b l i s h e d o b s e r v a t i o n ) . V u l v al c o l o u r is a s s es s e d u s i n g a s im i l a r te c h -n i q u e a s f o r s e x i n g r a b b i t s : t h e a n i m a l i s h e l d f i r m l y b y th e s c r u f f w i t h i ts b a c k t ot h e h o l d e r w h i le t h e l i ps o f t h e v u lv a a r e g e n t l y s p r e a d a p a r t t o re v e a l th e m u c o u sm e m b r a n e s .

F o r i n s e m i n a t i o n t h e f e m a l e is p l a c e d i n a s h a ll o w r e s t r a i n i n g b o x w i t h o u t n e c kr e s t r a i n t ( F ig . 4 ) . T h e b o t t o m o f th e b o x s h o u l d h a v e a no n - s li p s u rf a c e ; a r u b b e rb a t h m a t c a n b e u s e d t o l i n e a c o n v e n t i o n a l r e s t r a i n i n g b o x . A r e c e p t i v e f e m a l ew i l l e x h i b i t l o r d o s i s w h e n t h e t a i l i s l i f t e d g e n t l y , t h u s p e r m i t t i n g t h e i n s e r t i o n o ft h e l u b r i c a t e d i n s e m i n a t i o n p i p e t t e i n t o t h e v a g i n a . I t is n e c e s s a r y to i n s e r t t h ep i p e t t e a t a n a n g l e o f a p p r o x i m a t e l y 4 5 to n e g o t i a t e t h e r i m o f t h e p e lv is . O n c et h e d i l u t e d s e m e n is r e l e a s e d i n t o t h e u p p e r v a g i n a a n d t h e p i p e t t e i s r e m o v e d ,t h e d o e w i ll s it d o w n a g a i n . A n u n r e c e p t i v e d o e w ill n o t l if t h e r h i n d q u a r t e r s w h e n

~ i / / / / / / / / / / / / / / / ~ . , ~- , ~ . ~ . ~ , - , , u , , / / , , ~ _ :

~ W q / l l l , . - - - - - - ~ ~__ _~ .

Fig. 3. An ins em inat ion pipet te pre par ed from a stand ard 1 ml glass laboratory pipet te . Aben d i s p rod uced by hea t ing the p ipe t te i n a bunsen f l ame approximate ly 8 cm f rom on e end . W hen the glass i s soft, the pipet te c an be b en t to the req uired angle.

8/13/2019 1-s2.0-S0007193505800223-main

8/12

484 BRITISH VETERINARYJOURNAL, 151, 5the tail is raised: instead she clamps her tail firmly against the perineal region andremains sitting. Alternatively, the do e can be restrai ned in a supine position forinsemination (Adams, 1961; Hafez, 1970). Intracervical insemination is not poss-ible in sandy halflops using the type of insemination pipette shown in Fig. 3because of.the len gth of the vagina in this su'ain (average lengt h in 20 females was20.8 _+ 2.4cm). It is debatable whe ther intracervical insemi nation can beaccomplished with certainty in this species. Since there is no connection betweenthe two uterine horns in rabbits, it would be necessary to deposit the spermsuspension in both cervices if intracervical inseminati on was desired.

INDUCTION OF OVULATIONThr ee met hod s of indu cing ovulation are available for this species: (1) matin gwith a vasectomized buck; (2) administration of hu man chorionic gona dotro pin(hCG); or (3) administration of a gonadot ropin releasing ho rmo ne (GnRH) ana-logue, e.g. buserelin (Receptal; Hoechst, UK) (Battista et al. 1987; Morrell, 1990).For preference the author uses 0.8 ptg buserelin, administered by subcutaneousinjection.

Although the surgical prepa ration of vasectomized males is no t complicated,their use can negate some of the benefits of AI, for example in disease control a ndin reducing the n umb er of animals kept. In addition, females exhibit preferencesconc erni ng which buck they will allow to mat e with them. Thus several vasectom-ized bucks would have to be kep t to provide an alternative if the first one does no tmeet with 'approval', presenting considerations for husbandry and management.Secondly, vasectomized males can develop anti-sperm antibodies in their seminalplasma because of the alteration of the blood-testis barrier after vasectomy(Singh & Yang, 1984). Th eref ore matin g with such a male immedi ately after

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

8/13/2019 1-s2.0-S0007193505800223-main

9/12

ARTIFICIAL INSEMIN ATION IN RABBITS 85

i n s e na i n a fi o n c o u l d r e d u c e t h e n u m b e r o f v i a b le s p e r m a v a il a bl e a n d t h e r e f o r ea d v e r s e l y a f f e c t f e rt il it y . H o w e v e r , a l l o w i n g t h e m a t i n g t o o c c u r 1 - 4 h a f t e r i n s e m i -n a t i o n c a n c i r c u m v e n t t h e p r o b l e m b e c a u s e o f s p e r m m i g r a t io n a w ay f r o m t h es it e o f s e m e n d e p o s i t i o n . T h i r d ly , i t is p o s s i b le f o r a v a s e c t o m i z e d b u c k t o m o u n t ad o e a n d e j a c u l a t e w i t h o u t i n d u c i n g o v u l a t i o n ( H a f e z , 1 9 7 0 ) . A l l o w i n g t h e p a i r t om a t e m o r e t h a n o n c e d o e s n o t a p p e a r t o a f f e c t t h e s iz e o f t h e s u b s e q u e n t l it te r.T h e f e m a l e s h o u l d a lw a y s b e t a k e n t o t h e m a l e s c a g e f o r m a t i n g , n o t v i c e v e rs a , toa v o i d f i g h t in g a n d i n h i b i t io n o f t h e m a l e ( N a p i e r , 1 9 6 3 ) .

T h e a d m i n i s t r a t io n o f h C G i n t r a v e n o u s l y to i n d u c e o v u l a t i o n a ls o h a s s ev e r a ld i s a d v a n t a g e s . S i n c e h C G is h i g h l y a n t i g e n i c , r e p e a t e d a d m i n i s t r a t i o n r e s u l t s i na n t i b o d y f o r m a t i o n a n d c o n s e q u e n t l y t h e r e i s n o o v u l a t o ry re s p o n s e w h e n h C G isa d m i n i s t e r e d o n s u b s e q u e n t o c c a s i o n s ( A d a m s , 1 9 6 1 ) . M o r e o v e r , t r a i n i n g i sr e q u i r e d t o p e r f o r m t h e i n t r a v e n o u s i n j e c t i o n c o r r e c t l y .

T h e r e c e n t d e v e l o p m e n t o f t h e G n R H a n a l o g u e , b u s e r e li n , a p p e a r s t o h a v es o l v e d t h e p r o b l e m o f i n d u c t i o n o f o v u l a t i o n in r a b b it s , s i n c e t h e d e c a p e p t i d em o l e c u l e i s n o n - a n t i g e n i c . T h e p r e p a r a t i o n is i n j e c t e d s u b c u t a n e o u s l y a t th e t i m eo f i n s e m i n a t i o n a n d c a u s e s o v u l a t i o n a p p r o x i m a t e l y 1 0 - 1 2 h la te r . T h i s m e t h o d iss i m p l e a n d r e l ia b l e a n d c a n b e u s e d i n s u b s e q u e n t A I a t t e m p t s i n t h e s a m ef e m a l e s w i t h o u t l o ss o f e ff ic a c y .

P R E G N N C Y D I G N O S I SU n t i l r e c e n t l y t h e r e w a s n o s i m p l e r e l i a b l e m e t h o d f o r p r e g n a n c y d i a g n o s i s i nr a b b it s . S e x u a l r e c e p t iv i t y a t a t e st m a t i n g d o e s n o t i n d i c a t e n o n - p r e g n a n c yb e c a u s e p r e g n a n t f e m a l e s m a y p e r m i t m a t i n g ( A d am s , 1 9 83 ) a n d b u c k s a r e n o td e t e r r e d f r o m m a t i n g a fe m a l e b y e i t h e r p r e g n a n c y o r p s e u d o p r e g n a n c y . L ik e-w i se , t h e a b s e n c e o f h a ir p l u c k i n g a t a r o u n d d a y 1 8 a f t e r in s e m i n a t i o n is n o t ar e li a b le i n d i c a t o r t h a t th e f e m a l e is p r e g n a n t a n d n o t p s e u d o p r e g n a n t .D e v e l o p i n g f o e t u s e s c a n b e p a l p a t e d i n t h e u t e r u s 1 0 d a y s ( S u it o r, 1 9 4 6 ) o r 1 3 - 1 4d a y s ( H a f ez , 1 9 7 0 ) a f t e r m a t in g , b u t t h e t e c h n i q u e c a n b e r is k y b e c a u s e t h e f o e -t us e s m a y b e d a m a g e d , c a u s in g a b o r t i o n o r t o x ae m i a . F u r t h e r m o r e , t h e t e c h n i q u em a y n o t b e e f f e c ti v e w h e r e o n l y o n e o r t w o fo e t u s e s a r e p r e s e n t . R a d i o g r a p h y i sp o s s i b l e a f te r t h e 11 t h d a y o f p r e g n a n c y b u t is n o t a p ra c t ic a l m e a n s o f p r e g n a n c yd i a g n o s i s fo r m o s t b r e e d i n g u n i t s . H o w e v e r , i t h a s b e e n s h o w n t h a t e n z y m e - l i n k e di m m u n o a s s a y ( E L IS A ) k i ts f o r t h e a ss ay o f p l a s m a p r o g e s t e r o n e i n o t h e r s p e c i e sc a n b e u s e d w i t h r a b b i t p l a s m a a n d s e r u m ( M o r r e l l, 1 9 9 0, 1 9 9 3) . T h e O v u c h e c kE L I S A k i t ( C a m b r i d g e L i f e S c i e n c e s ) f o r b o v i n e p r o g e s t e r o n e h a s b e e n u s e d t om e a s u r e r a b b i t p l a s m a p r o g e s t e r o n e . A c o m p a r i s o n b e t w e e n m e a s u r e m e n t s o f t h es a m e s a m p l e s b y E L I S A a n d r a d i o i m m u n o a s s a y ( R IA ) s h o w e d t h a t t h e r e w a s ag o o d r e l a t i o n s h i p b e t w e e n E L I S A a n d R I A v a l u e s (F ig . 5 ) , a l t h o u g h t h e a b s o l u t ec o n c e n t r a t i o n s a s a s s ay e d b y E L I S A w e r e h i g h e r t h a n R I A , a s is a l so t h e c a s e f o rb i tc h p la s m a p r o g e s t e r o n e ( E n g l a n d e t a l . 1989) .U s i n g t h e O v u c h e c k E L I S A ki t i t is p o s s i b l e to d i f f e r e n t ia t e b e t w e e n p r e g n a n ta n d p s e u d o p r e g n a n t a n im a l s r e l ia b ly at a r o u n d d a y 1 7 -1 8 a f t e r in s e m i n a ti o n .S i n c e t h e o p t i m a l t i m e f o r r e - m a t in g p s e u d o p r e g n a n t r a b b i t s is a t d a y 1 8 (F i s c h e re t a l . 1 9 8 6) m e a s u r e m e n t o f t h e p r o g e s t e r o n e l ev e l a t th is t im e p r o v i d e s a u s e f u l

8/13/2019 1-s2.0-S0007193505800223-main

10/12

486 BRITISH VETERINARY OU RN AL, 151. 5

7

r

6

5

4

3

2

I I I I I t I I I0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0RIA progesterone ng m1-1F i g . 5 . T h e r e l a t i o n s h i p b e t w een p l a s m a p r o g es t e r o n e co n cen t r a t i o n s m eas u r ed b yrad io imm unoassay (R IA ) and enz}qne-l inked imm unoassay (ELISA) . Th e c oef f i c ien t o fregre ssion is significant.

t oo l f o r c o l o n y m a n a g e m e n t . T h u s p r e g n a n t f e m a le s c a n b e h o u s e d a n d f e da p p r o p r i a t e l y d u r i n g l a te p r e g n a n c y w h il e n o n - p r e g n a n t f e m a l e s ca n b e in s e m i -n a t e d a g a in . 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 i n p l a sm a o r s e ru mf r o m s a m p l e s t a k e n 2 h a f t e r t h e a d m i n i s t r a t i o n o f t h e o v u l a t o r y s t i m u l u s c a n b eu s e d t o p r e d i c t o ~ d a t i o n s in c e 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 r e a c h e s a p e a ka p p r o x i m a t e l y 1 0 h b e f o r e o v u l a t i o n i n r a b b i t s ( M il ls G e r a d o t , 1 98 4 ) a n d t h e nd e c l i n e s s h a r p l y a g a i n .

C K N O W L E D G E M E N T S

I a m g r a t e f u l t o P r o f e s s o r D a v i d N o a k e s , R o y a l V e t e r i n a r y C o l l e g e , f o r a r r a n g i n gt h e R I A o f r a b b i t p l a s m a s a m p l e s .

R E F E R E N C E S

An,~xls, C. E. (196 1) . Art i f icial ins em ina t io n in th e ra bbi t . Jou~zal o f Reproduct ion an d Fer t i li ty2, 521-2 .ADA,XlS, C . E . (1970) . Age ing an d rep ro du ct i on in the f em ale m am m al w i th par t i c u la r r e fe r -e n c e t o t h e r abb i t Jou~ z a l o f Repr oduc tion and F er ti li ty Supp lemen t 12, 1-16.

8/13/2019 1-s2.0-S0007193505800223-main

11/12

ARTIFICIAL INSEMINATION IN RABBITS 487ADAMSC . E . ( 1 9 8 3 ) . S o m e r e c e n t s tu d i e s o n r e p r o d u c t i o n i n t h e r a b b it . Anim al T echno lo~3 4 1 3 7 - 9 .AD..x~ls, C . E. (19 87 ) . T h e labo, -a to l 3 , rab bi t , C h ap te r 26 . In T he U FAWH andbook on the Car ean d Ma nageme nt of Laboratory, Animals, 6 t h E d n , e d . T . P o o l e , p p . 4 1 5 - 3 5 . A v o n , U K :

L o n g l n a n S c i e n ti f ic a n d T e c l~ n i ca l.A M..\N N, R . P . ( 1 9 6 6 ) . E l t e c t o f e j a c u l a t i o n f i ' e q u e n c y a n d b r e e d o n s e m e n c h a r a c t e r i s t i c sa n d s p e r m o u t p u t o f r a b b i ts. Jou rnal o f Reproduct ion and Fer t il i ty 1 1 , 2 9 1 - 3 .B ..v rr ,s -rA , M. G . , P opi ., , W . F. F oor~ :, R . H . (1987 ) . P l a sm a a nd u t e r i n e p r og e s t e r on e a n de m b , ' y o su r vi va l i n r a b h i t s f o l l o w i n g a s y n c h r o n o u s t r a n s f e r t o u n i l at e r a ll y o v a , -i e c to m i z e dr e c i p i e n t s . Theriogenolo~, 2 7 , 8 9 7 - 9 0 5 .BI~t.:E),)I.:R~,..\XX, P .J ., Fo or E, R. H. Y.~ssi:.N, A. M. (1 96 4) . A n i m p r o v e d ar tifi cia l va g in a fo rc o l l e c t i n g r a b b i t s e m e n . Jo urn al of Reproduction an d Fertili ty, 7 , 4 0 1 - 3 .C,n-Y, Y., LI, J . , S,,~IKXN, M . E . , Y.XN{;, X. FOOTI.., R. H . (1 98 9) . Fe rt i l i ty o f fi 'esh a n d fr o ze nr a b b i t s e m e n i n s e m i n a t e d a t d i f f e r e n t t i m e s is i n d ic a ti v e o f m a l e d i f f e r e n c e s i n c a p a c it -a t i o n t i m e . Biolo

8/13/2019 1-s2.0-S0007193505800223-main

12/12

4 8 8 B R I T I S H V E T E R I N A R Y .J O U R N A L , 1 5 1 , 5SA~AqNP. B., DENENBERGV. H ., Ro ss, S. , HAFTER, E. & ZARROWX . ( 1 9 6 0 ) . M a t e r n a l b e h a v i o ri n t h e r a b b i t : h a i r l o o s e n i n g d u r i n g g e s t a t i o n . Am e) q ca n Jo u rn a l o f Ph ys io log y 198 ,1 0 9 9 - 1 0 2 .SI-L~NNON, P . ( 1 9 6 5 ) . C o n t r i b u t i o n o f s e m i n a l p l a s m a , s p e r m n n n l b e r s a n d g a s p h a s e t o

d i l u t i o n e f f ec t s o f b o v i n e s p e r m a t o z o a . Jou rna l o f Dai)y Science4 8 , 1 3 5 7 - 6 1 .SHANNON, P . ( 1 9 7 2 ) . T o x i c e f f ec t a n d a c t io n o f d e a d s p e r m o n d i l u te d b o v i n e s e m e n .J o u r n a l o f D a i D, Science 5 5 , 6 1 4 - 2 0 .SINC,H, G. & MINC,Y ~'~ c,, W . ( 1 9 8 4 ) . T h e b l o o d - t e s t i s b a r r i e r i n t i le r a b b i t a f t e r v a s e c t o m y : al i g h t m i c r o s c o p i c s t u d y u s i n g h o r s e r a d i s h p e r o x i d a s e . ICI~ Med ical Science 12, 549.STAVt.ES, R . E . ( 1 9 6 7 ) . B e h a v i o u r a l i n d u c t i o n o f o v u l a t i o q i n t h e o e s t r o u s r a b b i t . J o u r n a l o fRep)od,l ction a nd Fertil i ty 1 3 , 4 2 9 - 3 5 .S'rEVHENS, D . T ., HICKMA N,R . & H O Sg JN S, D . D . ( 1 9 8 8 ) . D e s c r i p t i o n , v a l i d a t i o n a n d p e r f o r m -a n c e c h a r a c t e ri s t ic s o f a n e w c o n a p u t e r - a n t o m a t e d s p e r m m o t i l it y an a ly s is sy s te m . Biolo~,o f Reproduct ion 3 8 , 5 7 7 - 8 6 .STRANZINGERG. F. , M AU:~R, R. R . & PAUFLERS . K . ( 1 9 7 1 ) . F e r t i l it y o f f r o z e n r a b b i t s e m e n .Jou rna l o f Reproduct ion an d Fe~i li tr 24 , 111-13 .SLTN'OR, A . E . ( 1 9 4 6 ) . P a l p a t i n g d o m e s t i c r a b b i t s t o d e t e r m i n e p r e g n a n c y . L e a f l e t 2 4 5 ,U n i t e d S t at es D e p a r t m e n t o f A g r i c u lt u r e.TH~TrOR O, C . R . , MORR ZLL, . M. & C OCKLE, S . M. (199 2) . T R H -re l a t e d pe p t i d e s i n r a b b i tp r o s t a t e d u r i n g s e x u a l m a t u r a t i o n . Biochemica et Biophysica Aa a 1 1 1 5 , 2 5 2 - 5 8 .WA LES, R . G ., IVL.~RTIN,L . & O 'S H EA , T . ( 1 9 6 5 ) . E f f e c t o f d i l u t io n r a t e a n d o f t h e n u m b e r o fs p e r m a t o z o a i n s e m i n a t e d o n t h e fe r ti li ty o f r a b b i ts o v u l a t e d w i th c h o r i o n i c g o n a d o -t r o p h i n . Jou rna l o f Reproduction a nd Fert i li tr 1 0 , 6 9 - 7 8 .WICI-IER,V., WITH ER, K. & GRUI-IN,R . F . (1 98 7 ) . I n c r e a s e d p r o d u c t i o n o f a n t i b o d i e s t o s p e r m -a t o z o a a n d s e m i n a l f l u id i n r a b b i ts u s e d a s s e m e n d o n o r s . I n t e ~ a t i o n a l A r ch iv es o f A l l e ~ ,an d Appl ied hnmunology 8 3 , 1 0 4 - 1 0 8 .W n.LETT, E . L . & S ALtSBURV, G. W . (194 2) . T he e f fe c t o f va r i ou s d i l u t o r s , c o o l i ng r a t e ,t e m p e r a t u r e o f s to r a g e, a n d s o m e o t h e r f a c to r s o n t h e l iv ab ili ty o f s p e r m a t o z o a i n s t o r e ds a m p l e s o f b u l l s e m e n . Mem oirs of the Cornell University Agricultum.l E xpel~m ental Station N o .249 .YOt,NC,, R. J . , BOOT, B. A ., I 'rURV, t.DE, T. G. & ST.am~t. W . C. (1 99 2) . A u to m a te d ana lys is o fr a b b i t s p e r m m o t il it y a n d t h e e f fe c t o f c h e m i c a l s o n s p e r m m o t i o n p a r a m e t e r s . MolecularReproduct ion and Development 3 3 , 3 4 7 - 5 6 .

(Accepted 3 Februal 199 5)