Rabbit artificial insemination : the current situation
Michèle Theau-Clément
INRA. Station d'Amélioration Génétique des Animaux BP 27-31326 CASTANET TOLOSAN Cedex, France
The European production of rabbit meat is approximately 1 million tonnes/year (Colin and Lebas, 1996). Because of the evolution of breeding systems, this production has undergone important changes during the last decade. In traditional systems, matings are done several times each week. So, the weekly organisation consists of a series of events (births, weanings, sales etc.). With cyclic production, the same breeding operation is repeated with a regular frequency : semen fractionation allows to inseminate numerous rabbit does (a "batch") on the same day. On a farm, the number of batches generally varies from 1 to 6. In an extreme case, very widespread in western France, breeders use a "single batch" : inseminations are only made every 35 or 42 days. Artificial insemination (A.I) allows better work organisation as well as decreased manpower costs. Often, breeders buy semen from selected bucks in insemination centres. So, on farms, bucks are replaced by does. Moreover, because of the suppression of physical contact, A.I offers a better sanitary guarantee and favours animal observations, although we should keep mind that semen can be a disease vector (Mercier and Rideau, 1992; Castellini et al., 1994; Cenci et al., 1996). These are essential points which lead to the increasing popularity of A.I. In France, for example, in 1987, its use was just beginning: 10 years later, several million inseminations are made each year. So, to assure good and regular production, it becomes more and more important to control factors affecting productivity.
In relation to does, we will first analyse some factors affecting reproductive performance. Secondly we will comment on some methods which induce sexual receptivity of does at the moment of insemination. Since several insemination centres have been created in Europe in the 4 last years (e.g. 14 in France, more than 10 in Italy...), we will briefly review published literature concerning some aspects of male animal husbandry and semen production.
FEMALE
Factors affecting reproductive performance of does
If rabbit does can be inseminated immediately after giving birth, their reproductive performance may vary with their parity, lactating status (lactating or non-lactating) and sexual receptivity at the moment of insemination.
Parity
Nulliparous does are typically very fertile (Szendrö et al., 1992) and moderately prolific. Primiparous does inseminated during their first lactation have low fertility (Chmitelin et al., 1990, Poujardieu et Theau-Clément, 1995), probably due to very marked energy losses (28%, Parigi Bini et Xiccato, 1993) and because of high and not fully satisfied requirements for lactation, pregnancy and growth. Lastly, lactating multiparous does have a fertility intermediate between nulliparous and primiparous does, while litter sizes are generally higher.
Physiological status
Lactation negatively influences sexual receptivity at the time of insemination, ovulation, and embryo development (Fortun and Lebas, 1994), and more generally the productivity of rabbit does (Theau-Clément and Roustan, 1992).
Sexual receptivity
Moret's studies on behaviour (1980), show that in pubescent nulliparous does, oestrus periods during which they will accept a male alternate with periods of dioestrus when mating is refused. The duration of oestrus and dioestrus periods respectively vary widely from one individual to another, and the hormonal and environmental factors determining these are not well understood. A doe is receptive if in the presence of a male, she displays mating acceptance behaviour (lordosis position). Vulva colour and turgescence can be a receptivity indicator but are quite subjective. So, it is difficult to detect receptivity in farm conditions. In comparison with non-receptive does (which present more frequent failures of ovulation and embryo losses), does which are receptive at the moment of insemination are more fertile (Theau-Clément and Roustan 1992 : + 37%; Alabiso et al. 1996 : + 31%) and more prolific.
At a physiological level, receptive does have more pre-ovulatory follicles (Kermabon et al., 1994) and a higher blood plasma oestrogen concentration than non-receptive does (Rebollar et al.,1992; Ubilla and Rebollar,1995). Also, Boiti et al. (1996) showed that 17% of does (21.1% of multiparous and only 3.3% of nulliparous, but results differ from farm to farm) have high progesterone levels (2.0 ng/ml); few are receptive (11.5%) and fertile (12.9%). Probably spontaneous ovulations occur after birth, probably due to stress factors like animal handling, controlled suckling, etc.. In order to avoid such stresses, we have to determine precisely the causes of these spontaneous ovulations.
Of course, the percentage of receptive does depends on other factors like the sanitary status of the herd, number of suckled young etc..
Finally, Theau-Clément and Roustan (1992) showed that the lowest reproductive performance is shown by lactating non receptive does. This antagonistic effect of lactation on the reproductive function represents a major problem since the intensive methods of production generally used require does to be inseminated at the start of the nursing period (from 0 to 11 days). This antagonism is particularly pronounced after 3-5 days of lactation. Effectively, the productivity of lactating non-receptive does inseminated 4 days post-partum is 3 times lower than ones inseminated 11 days post-partum (Table 1). It should also be emphasised that in the case of natural mating, the negative effect has often escaped notice until now, since this antagonism is hidden when non receptive lactating females refuse to mate.
At a physiological level, these effects could be related to a hormonal antagonism between lactation and reproductive functions and to an energy deficit. Prolactin, responsible for milk production, could participate in this negative effect (Rodriguez et al. 1989). It could act upon the pituitary (Rodriguez and Ubilla, 1988) and depress gonadotrophin secretion (FSH and LH, particularly responsible for follicular growth and ovulation respectively). At the level of the ovary, prolactin (the pituitary hormone responsible for milk production) could limit in vitro follicular growth (Hamada et al., 1980) and the positioning of LH receptors (Kermabon et al., 1994), thus decreasing the efficiency of endogeneous hormones. Furthermore, complex underlying physiological mechanisms could interfere with the hypothalamo-pituitary-ovarian axis.
So, to improve performance it is necessary to have reliable techniques to induce and synchronise oestrus of lactating does on a large scale.
Methods to induce sexual receptivity of does at the moment of insemination
On farms, the improvement and standardisation of reproductive performance are determined first by the choice of the reproduction rhythm and then by the use of methods able to improve and synchronise oestrus of lactating does. Many studies have been made on hormone treatments (Maertens et al., 1995) but only a few on bio-stimulations.
Choice of the reproduction rhythm
Optimising the productivity of a farm is a compromise between the parturition-insemination interval (which has to be as short as possible) and the percentage of receptive does (which has to be as high as possible).
Post-partum rhythm: does are very receptive (Stoufflet and Caillol, 1988; Beyer and Rivaud,1969; Maertens et al., 1988), probably because of inversion of the oestrogen/progesterone production rates. On the other hand, the number of corpora lutea and consequently litter size are lower (Lamb et al., 1990). 35 days rhythm : 3-4 days post-partum, does are generally less receptive: moreover, lactating does have poor productivity levels (Blocher and Franchet, 1990). 42 days rhythm : 11 days post-partum, does are more frequently receptive: their productivity justifies the common use of this rhythm. Post-weaning insemination: Females in this situation are appreciably more receptive and more fertile (Szendrö et al., 1992), and there is no longer any competition between gestation and lactation. But in view of production conditions today, an extensive post-weaning reproductive rhythm is unlikely to be cost-effective.
Hormonal treatments.
In lactating does, Pregnant Mare Serum Gonadotrophin (PMSG) is now largely used in rabbitries. Some authors have reported its efficiency in stimulating follicular growth (Bonnano et al., 1990), probably oestrogen production, and thus receptivity of does, consequently improving fertility. Nevertheless, Canali et al. (1991), Bourdillon et al. (1992) and Boiti et al. (1995) have demonstrated an immune response after successive injections due to its proteic and exogenous nature, and its high molecular weight. Consequently, fertility and injection efficiency decrease from the 4th injection. Production of PMSG antibodies is directly responsible for the number of injections and treatment interval. Nevertheless, after 9 injections, Lebas et al. (1996) did not find at the end of the experiment any clear relation between immune response (found in 30% of does), and fertility. Theau-Clément and Lebas (1996) noted that PMSG is effective only on lactating does (47% more weaned rabbits per A.I in comparison with control group). The ability of PMSG to increase prolificacy is observed by some authors (Maertens and Luzi, 1995), and is followed by higher birth mortality. Effectively, Stradaioli et al. (1994), Boiti et al. (1995) showed that in comparison with a control group, treated does have an abnormal ovarian response (a larger number of haemorrhagic follicles), and a perturbed in vitro embryo development.
Davoust et al. (1994) demonstrated that the combination of 8 I.U PMSG with 4 I.U hCG significantly improves fertility of 3-4 day lactating does. Recently, Theau-Clément et al. (1998b-1998c) concluded that as little as 8 IU of PMSG can improve the productivity (+62%) of multiparous and lactating does inseminated 3-5 days post partum (single batch). They studied the kinetics of anti-PMSG antibodies in relation to the number of injections and injection dose (8 or 25 IU). None of the does developed detectable anti-PMSG antibodies before the 6th injection. After 11 consecutive injections, 15 and 39 % of them treated with 8 or 25 IU respectively, developed immunity to PMSG. Nevertheless, the number of weaned young per insemination is not related to PMSG dose or immune response.
Different conclusions could be the result of different experimental conditions (dose, treatment interval, doe renewal rate, genotype etc.). So new studies are necessary to define more clearly the optimal conditions for the use of PMSG. Nevertheless, it seems that PMSG efficiency is greater when does are in an unfavourable condition for reproduction (lactating or with a low initial fertility level). In order to reduce PMSG use, Bonnano et al. (1995) propose a different protocol. On insemination day, only receptive does (observation of vulva colour) are inseminated (without any treatment). Non-receptive does are then treated and inseminated 3 days later. This method improves reproductive performance, but unfortunately is not very suitable for cyclic production.
So, it is not possible to draw definite conclusions about conditions of use for PMSG. Only a few recommendations can be suggested : • Only treat lactating does, • Don't inject more than 20 I.U. (possibly less, especially in association with hCG), • Avoid short intervals between treatments.
Biostimulations
During the 6th World Rabbit Congress, Castellini (1996) mentioned that, in the near future, the European Community will impose a restriction on the use of hormones (gonadotrophins) in relation to their residues in meat, animal welfare and the desire to preserve a "natural" image of meat. So the International Rabbit Reproduction Group (I.R.R.G.) decided to study some "Bio-stimulation" methods designed to contrast with gonadotrophin treatment, in order to increase sexual receptivity and consequently the productivity of the rabbit does (Boiti, 1998; Theau-Clément et al. 1998a). We will comment on these recent results (Theau-Clément and Boiti, 1998).
It has long been recognised that the environment has an important role in the regulation of reproductive function and it now appears obvious that such environmental stimuli must act through the nervous system and the hypothalamo-pituitary axis. Environmental stimuli, such as varying daylength or temperature and feeding, affecting animals by stress, auditory and/or olfactory stimuli can positively or negatively modify reproductive performance.
Photoperiod.
Within the European latitudes, Hammond and Marshall (1925) and Boyd (1986) reported that wild rabbits (Oryctolagus cuniculus), have a well defined seasonal cycle of reproduction: most pregnancies occur between February and early August with a peak in May. It means that fertility is maximal in increasing daylength. Walter et al. (1968) showed that 16h/24h of constant lighting all the year round reduces the reproduction problems normally associated with decreasing daylength periods. Moreover, Lefevre and Moret (1978) showed the importance of an increasing daylength and cage changing on the onset of oestrus in nulliparous does.
Theau-Clément et al. (1990) found that a modification of the daylength 8 days before AI (from 8h to 16h/day) significantly improved the sexual receptivity of the does : 71.4 % vs 54.3 % for the control (16h light/day). However, the effect on fertility was not significant (61.4 % vs 48.9 %), as confirmed by Maertens and Luzi (1995). Mirabito et al. (1994a), using a similar experimental lighting programme (except that in the week after AI there was a progressive return to 16 h/ day) but with a longer reproductive rhythm (6 vs 5 weeks minimum between 2 fertile inseminations) obtained a significantly higher fertility in the experimental group (+9%). However, in these studies, the weight of the litter at weaning was significantly lower in the treated group, suggesting that the lighting programme can adversely affect the suckling ability of females and/or the feeding behaviour of the young rabbits.
Interrupted lighting programs have also been studied with rabbits. Uzcategui and Johnston (1992), concluded that Rex rabbits need at least 14 h of continuous light to reach their reproductive potential and that intermittent lighting schedules of 10, 12 and 14 h are equally as effective as 14 h of continuous light in promoting doe reproduction. Feed consumption appears to be inversely related to total hours of light. Arveux and Troislouches (1994), submitting commercial hybrid does to different lighting programs (continuous : 16h light/day or discontinuous : 8h light, followed by 4h dark and so on), increased fertility (82.6 vs 67.6 % for natural mating) without any reduction of litter weight at weaning.
In conclusion, these results illustrate the need to study photoperiodism more intensively in the rabbit. Moreover, lighting programs are easy to apply and do not need large manpower costs. They will be all the more efficient as rabbit does will be in the same physiological condition. So, lighting programs are perfectly suited to cyclic production.
Controlled lactation
In some species of livestock, a hormonal mechanism is responsible for the post partum anoestrus: the persistence of prolactin, allowing continued lactation, delays or blocks the gonadotropin secretions and consequently the onset of oestrous. For rabbits, zootechnical results show that immediately after birth there is a short period (1-2 days) during which nearly all does exhibit good sexual receptivity. On the other hand, 3 to 5 days after giving birth, does show very little sexual activity.
Pavois et al. (1994), by preventing access to the nest box by the doe (10-12 days lactating) for 24 or 36 hours before the AI, obtained an increased fertility of 13 and 11% respectively in comparison with a freely suckling control group. The litter weight at weaning was adversely influenced only for a 36-hour separation. Verifying the effect of this technique (with some modifications) in 33 commercial rabbitries, Duperray (1995) concluded that a temporary doe-litter separation results in an 8.5 % increase in fertility (in 70% of rabbitries) without any harmful consequences to the doe or the litter.
Within the IRRG program, Castellini (1998) submitted does to 24 hours of mother-litter separation (closing the nestbox), 3 days before A.I. Mother-litter separation did not significantly affect reproductive results of lactating does. On the other hand, nulliparous and multiparous non-lactating does showed a significant positive reaction to biostimulation (gain of fertility rate: 12.9% and 14.0%, respectively). Maertens (1998) and Alvariño et al. (1998) clearly demonstrate that a 36-40h mother-litter separation, immediately before insemination on day 11 post partum, leads to at least a 10% gain in fertility rate. Sometimes the performance was better than for PMSG treated does. But the problem of the decreased weaning weight (-5 to -10 %) still remains. Finally, further experiments are needed to find out whether mother-litter separation has an effect on post weaning performance, and if compensatory growth can minimise the growth retardation. It would also be of interest to study the effect of separation on milk production in the phase following this separation. For the sake of animal welfare, new experiments are still necessary to define the optimal moment for, and duration of, a brief mother-litter separation.
Animal manipulation
Mirabito et al. (1994b), by putting three females together immediately before insemination, did not obtain any improvement of performance even in nulliparous does. Rebollar et al. (1994) showed that a change of cage in nulliparous does, 48 hours before insemination affected the fertility to the same degree as 25 IU PMSG (81.8 vs 79.6 %, respectively). Luzi and Crimella (1998) transferred does (and their litter when lactating) to another cage 2 days before insemination. The fertility rate in non-nulliparous does (whatever their lactating status) was increased compared to the control group (+ 14 %). But in contrast with earlier published results, this method had no effect on nulliparous does. Moreover, this biostimulation method is time consuming and difficult to manage in large rabbit farms
Feeding
Flushing aims to increase the does' feeding (energy) level before insemination. In nulliparous does, Van Den Broeck and Lampo (1977) demonstrated that flushing following a restricted feeding period improves reproductive performance. With lactating does, Maertens (1998), performed a 4 day flushing with a high-energy diet before insemination. However this failed to improve sexual receptivity and fertility, in comparison with the control group (-1.2%, -12.2 %, respectively). The author attributed these results to the low palatability of the experimental diet. The daily energy intake during the flushing period was lower than in the control group (-0.15 MJ ME/day). Although these first results are not encouraging, new experiments should be done to counterbalance the energy deficit of does during lactation and to improve their reproductive performance. With lactating does, some rabbit breeders also claim to use flushing before insemination, but no scientific study has really defined a nutritional programme suitable for artificial insemination and for various production systems (depending on reproductive rhythm) and for various physiological statuses of the doe. This program should be really well suited to cyclic production.
In conclusion, the results of these biostimulation methods are very promising and offer an alternative to hormonal induced oestrus.
MALE
At the 6th World Rabbit Congress, Castellini (1996) pointed out that few studies have analysed the effect of management and environmental conditions on semen production. It is true that it was not very important with natural mating, but now because of the spread of A.I, insemination centres specialised in semen diffusion. So we will first briefly review methods of buck management for good libido and sperm production (qualitative and quantitative aspects). Secondly, we will discuss some semen evaluation methods before ending with the little scientific knowledge which exists of the optimal insemination dose.
Buck management, libido and sperm production
With A.I the possible space/time separation between sperm collection (at its place of production) and insemination means that males can be kept in special units. Then, it becomes possible and important to study environmental conditions favouring maximal and less variable sperm production. Numerous authors have found a seasonal effect on libido and semen biological quality, but the calendar season effect is the resultant of numerous parameters like temperature, photoperiod, humidity etc.. Some factors could be easily controlled if their optimal values were known.
In experimental situations, sometimes of long duration, exposure of bucks to high temperature (30°C) both decreases libido and biological semen characteristics (such as concentration, motility, percentage of live or abnormal cells). But in our latitudes it is not so easy to control summer temperature. Theau-Clément et al. (1995) showed that during the whole experimental period (6 months), a 16L : 8D photoperiod improved the number of live spermatozoa per ejaculate (+31 %) compared with a 8L :16D one, as well as sperm motility.
In the bibliography, few authors studied relations between feeding and sperm output. Only Luzi et al. (1996) showed that the dietary protein level (19.7 % vs 14.5%) did not influence either libido or semen biological characteristics. However the feeding level (ad libitum vs restricted at maintenance energy requirement) allows a higher sperm output without any effect on semen quality.
During the sixties, the relationship between collection frequency and rabbit semen characteristics was studied. These early studies, whose duration was often short (less than a spermatogenetic cycle) with an insufficient number of males and sometimes different genotypes, suggest however that intensive rhythms induce a volume, concentration and motility decrease. Bencheikh (1993, 1995), making 4000 collections over a year, studied semen production of 26 males (2 collections 15 min. apart) one day each week (extensive rhythm) or 3 days each week (intensive rhythm). Compared with the intensive group, bucks submitted to an extensive rhythm had a greater percentage of useful collections (79 vs 70%), a greater number of live spermatozoa per ejaculate and a greater percentage of ejaculate with good motility (presence of waves: + 66%). The gain observed in semen biological characteristics of the bucks on the extensive rhythm had repercussions on fertility (69.5 vs 57.2 %) and litter size (9.3 vs 6.7). To summarise, the extensive rhythm increased the number of spermatozoa collected on each collection day by 2.3-fold. But because there was only one collection day per week, the weekly production of the extensive rhythm resulted in a loss of 22% of semen. In conclusion, in these experimental conditions, one collection day (2 ejaculates collected 15 min. apart) provides the best compromise between the number of spermatozoa per ejaculate, their quality, the production per unit time, and the rational time management of sperm collections. Bodnar et al. (1996) also showed that all semen quantitative characters are improved when an extensive collection rhythm is used. Moreover, these works confirm the high variability between and within bucks found by Battaglini et al. (1992) and Panella and Castellini (1990).
Bencheikh (1995) showed (as was also suggested by Lopez et al. 1996), the superiority of the second ejaculate compared with the first one. To improve sperm output, sexual preparation can be practised: McMillan and Hafs (1967) demonstrated that a false mount followed 3 minutes later by the re-introduction of the doe results in a 2.8-fold increase in the number of cells collected in the ejaculate and a smaller improvement in the number of cells in the second one.
All these environmental factors; feeding, collection rhythm, stimulation, can influence the bucks' libido, semen quantity and quality. A knowledge of optimal conditions in which bucks have to be housed should allow us to obtain a greater number of better quality semen doses.
Semen evaluation
Semen assessment requires trained eyes and a microscope. Whereas motility and percentage of live cells are subjective measurements (visual estimation), concentration can be estimated objectively (haemocytometer counts). Nevertheless, this technique is time consuming and can be used only in experimental stations. In contrast to other species, concentration cannot be precisely estimated by optical density measurements because particles from the prostate gland interact with spermatozoa light absorption. Different systems of Computer Assisted Semen Analyses (C.A.S.A) exist (Pizzi et al., 1996, Castellini et al., 1997). Theau-Clément et al. (1996a, 1996b) demonstrated the accuracy and reliability of the Hamilton Thorne system for motility analyses, but point out the importance of analysis conditions on the result of C.A.S.A. Nevertheless, this technique allows the measurement of new parameters like cell velocity, linearity, percentage of progressive cells (rapid with a linear displacement). Of course, other less expensive techniques might prove useful: as Micu et al. (1996) for example suggested that the electric current intensity of the seminal cell suspensions should depend upon the cellular viability and could be related to biological quality of rabbit semen.
Few works quantify the relationship between quantitative and qualitative biological semen characteristics and fertility. Panella and Castellini (1990) obtained a significant correlation (r=0.49) between the percentage of live cells in the inseminated semen and doe fertility. More O'Ferral (1990) found that the pH is significantly negatively related to fertility (r= - 0.51). Bencheikh (1995) demonstrated that in contrast to fertility, semen characteristics are positively related to prolificacy. But correlations are quite low (0.2 to 0.4) for qualitative parameters (percentage of live cells and motility).
So, in order to evaluate biological semen characteristics more efficiently, we need instruments able to make rapid, objective and reliable measurements in order to find parameters or combinations of parameters which are good fertility predictors.
Optimal semen dose
Few studies try to define the minimum number of spermatozoa per insemination giving maximum fertility. Some of the very old ones are more applicable to experimental than to production conditions. In the bibliography between 1948 and 1980 this quantity varies between 90 000 and 1 million. More recently, Farrel et al. (1993) obtained normal fertility when only 50 000 spermatozoa were inseminated. Since then, Pizzi et al. (1996) obtained 75% fertility with 7.5 million spermatozoa inseminated (does at the end of lactation, treated with PMSG). Alvariño et al. (1996) showed that 26 to 30 million cells are necessary (24h semen preservation, 4 day lactating does previously treated with PMSG). Probably this number depends on semen collection-insemination interval, diluent, preservation temperature but especially on the doe environment in which semen is deposited. Of course, a favourable vaginal and uterine environment of a receptive doe helps spermatozoa to reach the fertilisation site.
So it is now a matter of urgency define the optimal number of spermatozoa per insemination to allow insemination centres to fully estimate semen potentiality and to give breeders better guaranties. This number probably depends upon the physiological status of the does at the moment of insemination.
In conclusion, to summarize :
Does : • Efficient and lasting improvements can be achieved only in a healthy herd. • A better understanding of physiological mechanisms could help us to devise some "bio-stimulation" methods able to improve the sexual receptivity of rabbit does. • Before applying methods in rabbitries to induce receptivity of does, we have to test whether they improve not only reproductive performance, but also the general productivity (which depends on milk production, welfare, health and young growth etc.) during the whole lifetime's production. • These methods have to be: easy to apply, inexpensive (low manpower costs), consistent with animal welfare and suited to cyclic production. Even if they are not, they can offer alternative systems which can interest breeders. Lighting programs, temporary doe-litter separation and feeding programs seem to be worthwhile avenues to explore.
Male, semen :
We need better analytical tools to evaluate rabbit semen. They are essential : • to define optimal insemination dose, management and environmental conditions allowing bucks to express a better sperm output, good fertility predictors, but also • to predict semen freezing ability, • and to progress more rapidly in semen cryopreservation techniques.
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Table I. Influence of physiological status on productivity components of inseminated does
Fresh semen Frozen semen
Reproduction rhythm Experiment 1 (1) 35 days Experiment 2 (2) 42 days Experiment 3 (3) 42 days
