Key note presentation: Methods to improve the reproductive of camel Julian A. Skidmore Camel Reproduction Centre, Dubai, United Arab Emirates

Transcription

1 Key note presentation: Methods to improve the reproductive of camel Julian A. Skidmore Camel Reproduction Centre, Dubai, United Arab Emirates ABSTRACT The reproductive performance of camels is generally regarded to be low due (40%). This could be due to the late age of reaching puberty (3-4 years), the long gestation period (13 months) and the relatively high incidence of abortions and non-conceptions possibly attributed to poor nutrition, poor management, limited breeding opportunities for the females due to seasonality of breeding and limited rutting potential of a single stud bull used to service too many females. Techniques such as artificial insemination and embryo transfer are therefore becoming increasingly important. Artificial insemination increases the productivity of male animals. It has been reported in camelids although insemination trials are rare, probably because of the difficulties involved in collecting and handling the semen due to the gelatinous nature of the seminal plasma. In addition, female camelids are induced ovulators, and therefore ovulation must be induced before insemination. This paper discusses different methods for collecting and handling camel semen and describes how to measure sperm concentration and morphology. It also examines the use of different buffers for liquid storage of fresh and chilled semen, the ideal number of live sperm to inseminate where to inseminate them and how to induce ovulation in the females. Embryo transfer offers the chance to greatly increase the productivity of female animals and thereby increase the rate of genetic improvement. This paper discusses the essential prerequisites of embryo transfer programmes, such as superovulation in donors and synchronising groups of recipient animals, as well as the recent advances in chilling and freezing of camel embryos. Key words: Camel, Improve, Reproduction INTRODUCTION Opportunities to improve reproductive performance in camels are limited by the long gestation period and the short breeding season, as well as the continuing use of traditional systems of reproductive management in most breeding herds. These age-old methods make it difficult to ensure an optimum number of females are pregnant at the end of the season and they can also lead to widespread venereal infections with consequent lowering of fertility. This low reproductive efficiency could be improved by better understanding of the reproductive cycle and increased use of assisted reproduction techniques such as artificial insemination and embryo transfer. This paper outlines methods that can be used to control the reproductive cycle and increase breeding efficiency. Mating at the correct time of the follicular wave cycle: Camels are induced ovulators and therefore normally only ovulate when mated. Thus follicles have a period of growth, maturity and regression, so it is more accurate to describe the changes in follicular development as a follicular wave pattern rather than 77

2 oestrous cycle. As oestrous behaviour in camels is highly variable (Musa and Abusineina, 1978), signs of oestrus are difficult to relate to follicular activity in the ovaries and cannot be used reliably to decide timing of breeding in camels. Ultrasonography of the ovaries is therefore regarded as the most reliable method to monitor follicular growth and determine the correct time for mating. It is very important that camels are mated at the correct stage of the follicular cycle to maximize the chances of ovulation and conception. Previous studies have shown that mating will induce ovulation in female camels when the dominant follicle measures between cm in diameter, but the ovulation rate is dramatically reduced to < 20% if the follicle diameter increases to cm and no follicle >3.0 cm ovulated (Skidmore et al., 1996). More recent studies indicate that the best time to mate or inseminate the camel to maximize the chances of conception is when the mature follicle measures between cm in diameter (Skidmore and Billah, 2006a). Artificial insemination Advantages Artificial insemination has several advantages. For example i) it allows more efficient use of genetically superior males by inseminating more than one female with a single ejaculate, ii) it eliminates the need to transport the male or female animal to the stud, as it is much easier to transport semen. This reduces the cost and risks of transporting valuable animals, iii) it reduces the risk of disease and infection, as all contact between male and female is prevented, and the semen can be treated with antibiotics before insemination and iv) it helps eliminate behavioural problems if, for example, a male camel refuses to mate a particular female. In addition, semen can be preserved for 24 h by chilling, which enables the semen to be shipped between farms or countries for insemination in other breeding herds, or it can be saved for many years by deep freezing, which extends the reproductive lifespan of the camel even beyond its death. Collection of semen Semen collection is camels presents many difficulties partly because they mate in sternal recumbancy, have a lengthy ejaculation throughout copulation, which lasts from 5 20 min, and the semen is highly viscous. Semen can be collected using a modified bull artificial vagina (AV: 30cm long, 5cm internal diameter) with a foam imitation cervix, of about 8cm in length, placed inside the AV (Bravo et al., 2000). The AV is prepared by filling with water at C to give an internal temperature of C, and air is pumped between the inner liner and outer rigid wall to create pressure inside the AV to stimulate ejaculation. A clear, glass waterjacketed (35-37 C) semen vessel is attached to the apex of the internal latex rubber liner to keep the semen warm during the lengthy ejaculation process and the entrance of the AV is lubricated with KY jelly before use. To collect the semen a sexually receptive female is used for the bull to mount but before he enters the female he is guided into the AV. The semen is usually ejaculated in fractions and this whole process can take between 5 and 10 min, although it may occasionally last for 20min or longer (Rai et al., 1988). As the collection procedure can be rather prolonged it can be advantageous to add about 1-2ml of extender to the collection vessel before the collection. 78

3 Semen Evaluation Once collected, the semen has to be evaluated and the following parameters noted: volume (usually between 2 10 ml), colour (creamy white), concentration (usual range between x 10 6 / ml, Anouassi et al., 1992) and motility. One of the main problems with camelid semen is its high viscosity which makes handling and estimation of the spermatozoa parameters difficult. This viscosity is usually attributed to the presence of mucopolysaccharides from secretions of the bulbourethral gland or the prostate, but the degree of viscosity depends on the individual male and on the quantity of the gel fraction in the ejaculate. According to some authors the semen will partially liquefy if stored at C for min (Abdel- Raouf and El Naggar, 1976; Musa et al., 1993) but other studies show it can take up to 8 h (Tibary and Anouassi, 1997a). Enzymatic methods using trypsin, collagenase and chymotrypsin as well as mechanical methods using centrifugation, density gradient centrifugation, vortexing and gentle pipetting have been investigated to try and reduce viscosity Gentle pipetting was the most successful as it did least damage to the spermatozoa. Short term semen preservation Several extenders have been used for dilution of freshly collected camel semen (Sieme et al., 1990) such as skimmed milk-glucose extender (Kenny et al., 1975), Dimitropolous 11, (Rasbech, 1984), Laiciphos (Cassou, 1959), Androhep (Waberski et al., 1989), glucose EDTA (Martin and Klug, 1979), sodium citrate-egg yolk (Kupper, 1954), lactose-egg yolk (Anouassi et al., 1992) and Green buffer- egg yolk (I.M.V. L Aigle, France; Skidmore et al., 2000). Most of these extenders contain an energy source (glucose or fructose), a protein for cold shock protection (lipoprotein from egg yolk or casein from milk), a buffering system and antibiotics. Once collected the semen is diluted with warmed (30-35 C) extender added slowly to the semen in a ratio of 1:1 3:1 (extender: semen) depending on the concentration of the ejaculate. It is better to let the semen liquefy before insemination to aid better mixing of the semen with the extender and to allow more accurate assessments of concentration and motility. Seime et al. (1990) assessed sperm motility to be better in Laiciphos (60%) and Androhep (68%) compared with Glucose EDTA (42%) but no pregnancy results were reported, whereas Anouassi et al. (1992) achieved pregnancy rates of 50% (5/10) with an extender containing 11% lactose and 20% egg yolk. In a subsequent study the best pregnancy rates were achieved with Laciphos + 20% egg yolk (53%; 7/13), although two subsequently aborted, and Green Buffer + 20% egg yolk (47%; 10/21) compared with other extenders such as skimmed milk extender (0%; 0/6; Skidmore et al., 2000). More recently, further studies have compared the extender INRA 96 (I.M.V.) with Green Buffer. Results showed that whereas motility was higher after dilution in Green Buffer (67%) compared with INRA-96 (59%) pregnancy rates were unaffected by diluent, Green Buffer (34%) and INRA-96 (34%; Morton et al., 2010). Optimum number of spermatozoa to inseminate and site of insemination Fibroscopic evaluation of the camel s cervix before and after mating has shown that semen is deposited partly intra-uterine and partly intra-cervical (Tibary and Anouassi, 1997b). Therefore in AI, semen is generally inseminated directly into the uterus using a bovine insemination catheter passed through the camel s cervix. Pregnancy rates of 50% have been achieved after insemination of 300 x 10 6 live spermatozoa (Bravo et al., 2000) or as few as 100 x 10 6 (Anouassi et al., 1992) directly into the uterine body. However, 79

4 when it is deposited just into the body of the uterus there can be a considerable loss of spermatozoa, due to backflow of semen through the relatively short cervix. A further study was therefore carried out to investigate whether better results could be achieved if the semen was deposited at the tip of the horn, rather than the body of the uterus (Skidmore and Billah, 2006a). The advantage of deep uterine insemination is that the semen is deposited nearer the UTJ and therefore should reduce the number of spermatozoa needed for successful fertilization (Lopez Gatius, 2000). In this study by Skidmore and Billah (2006a) a total of 40, 80 or 150 x 10 6 motile spermatozoa were deposited either just through the cervix into the uterine body or at the tip of the uterine horn ipsilateral with the ovary containing the dominant follicle and the results are shown in Table 1. These results indicate that although a pregnancy rate of 53% or 43% could be achieved after insemination of 150 x 10 6 motile spermatozoa into the body of the uterus or at the tip of the horn respectively, a pregnancy rate of 40% could also be achieved when a reduced number of only 80 x 10 6 spermatozoa were deposited at the tip of the uterine horn. However, as only one camel inseminated with 40 x 10 6 spermatozoa at the tip of the horn conceived it would suggest that perhaps 80 x 10 6 is the minimum number of sperm needed to establish a pregnancy by deep intrauterine insemination. Table 1: Pregnancy rates following uterine body or deep insemination of 150, 80 or 40 million spermatozoa. Number of spermatozoa inseminated x 10 6 Method of insemination Pregnancy rate %; (no. pregnant / no. inseminated) Deep Uterine 43 a (6/14) 40 (6/15) 7 (1/14) Uterine body 53 a (8/15) 7 b (1/14) 0 c (0/14) Columns with same superscript are not significantly different ( aa p = 0.8); whereas rows with different superscripts are significantly different ( ab p = 0.014; ac p = 0.002). (From Skidmore and Billah 2006a) Timing of insemination in relation to ovulation As mentioned previously, mating induces ovulation in all camelids; however, when preparing animals for AI the camel cannot be mated so alternative hormonal methods have to be used to induce ovulation. A single injection of either 20µg of the GnRH analogue (Buserelin), or 3000 IU of hcg are most commonly used and will induce ovulation if injected when the dominant follicle measures between cm in diameter in >80% of cases (Skidmore and Billah, 2006a; Tibary and Anouassi, 1997c). One of the most important aspects of insemination in camelidae is its timing in relation to ovulation. The interval from mating to ovulation is not precisely known in camels although in a recent study where the ovaries of dromedary camels were scanned at regular 2 h intervals from h after induction of ovulation with 20 µg GnRH (i.v), the majority of camels ovulated between h after injection (Skidmore and Adams, unpublished data). To investigate the most appropriate time to inseminate the camels a study was carried out where females were inseminated with 150 x 10 6 live spermatozoa either at the same time or 24 h after the GnRH injection. The results indicated that whereas 53% of camels conceived if they were inseminated 24 h after GnRH injection, only 36% conceived if they were inseminated at the same time as the GnRH injection (Skidmore and Billah, 80

5 2006b). This would suggest that to maximize the chances of conception after AI the camel should be inseminated 24 h after GnRH injection. Embryo Transfer The technique of embryo transfer can also be used to increase the reproductive efficiency of camels as it can be used to produce multiple progeny from desirable genetic combinations of sire and dam. There are also other advantages as the female can continue in competitions without taking a year out carrying a pregnancy and conformational problems that may prevent her from carrying a calf to term or giving birth can be overcome. However, there are two essential prerequisites of successful embryo transfer in large domestic species; firstly induction of superovulation in donor animals by exogenous gonadotrophin therapy, and secondly simple methods for preparing groups of synchronised recipients. Superovulation Superovulation treatments, to stimulate the growth of multiple follicles, include the use of exogenous gonadotrophins such as equine chorionic gonadotrophin (ecg) or Follicle Stimulating Hormone (FSH) which may or may not, be given after a period of progesterone priming. This progesterone priming can be given either as a Progesterone Releasing Intravaginal Device (PRID) inserted into the vagina for a period of seven days, or as daily injections of mg progesterone - in - oil for up to 15 days (Cooper et al., 1990; Skidmore et al., 1992; McKinnon et al., 1992, 1994). i) Follicle stimulating hormone (FSH). Porcine or ovine FSH is generally used. In the dromedary a total dose of 18mg of ovine FSH (ofsh) or 400mg porcine FSH (pfsh) in 20ml is given over a period of 4 days in twice daily injections in gradually decreasing doses for example: Day 1: 2 x 4ml, Day 2: 2 x 3ml, Day 3: 2 x 2ml, Day 4: 2 x 1ml (Cooper et al., 1990; Skidmore et al., 1992). ii) Equine Chorionic Gonadotrophin (ecg). The dosage of ecg used varies from iu. and is injected in a single dose one day before, or on the day of completion of a 5-15 day progesterone regime (Skidmore et al., 1992; McKinnon et al., 1992) iii) Combined ecg and FSH. Previous studies by Skidmore et al., (2002) has shown that the best response is seen when a combination of both FSH and ecg are given. The ecg (2500iu) is given as a single injection on Day 1 of treatment together with pfsh injected over a period of 4 days, as described above, also starting on Day 1 of treatment (Skidmore et al., 2002). Following superovulation treatment, follicular growth is monitored by ultrasonography and the camel mated when the majority of follicles measure between cm in diameter. This is approximately 10 days after the start of treatment. Non-Surgical collection of embryos It is now well established that in camels the embryo does not reach the uterus until day 5.5 or 6 after ovulation (Day 0 = one day after mating). Therefore any attempt to collect embryos before day 6 post ovulation, results in low recovery rates. In practice the best recovery rates from dromedaries are achieved when the uterus is flushed on day 7 or 8 after ovulation. The most widely used method for the collection of embryos from camelidae remains the non-surgical technique. A flexible, two-way Gibbon balloon catheter (20 Gauge) is passed through the cervix, then the balloon inflated with 30-40mL of air to seal the internal os. The uterus is filled to moderate capacity with embryo flushing medium and 81

6 then recovered by gravity flow into sterile beakers. This procedure is repeated a total of three times before the recovered medium is passed through a sterile embryo filter and the residual filtrate searched for embryos using a stereoscopic binocular dissecting microscope. When embryos are located they are assessed morphologically and graded 1 to 5 (Grade 1 = excellent, Grade 2 = good/fair, Grade 3 = poor, yet expanded, Grade 4 = collapsed and degenerate, Grade 5 = fragmented and degenerate) before being individually washed 4 times by passing them through 4 drops of fresh flushing medium. As many as 20 or more embryos have been recovered in a single flush but because not all the follicles will ovulate at the same time these embryos can vary greatly in size and development (Skidmore et al., 2002). Non-surgical embryo transfer technique For transfer the embryos are individually aspirated into 0.25 ml straws which are placed in the embryo transfer gun. The embryo transfer gun is passed through the cervix and then manually guided into the left horn per rectum before the embryo is deposited. Pregnancy in the recipients is diagnosed by ultrasound scanning of the uterus initially between days 18 and 20, after ovulation followed by confirmation of a viable conceptus with a heart beat between days 25 and 30, as described by Skidmore et al. (1992). Synchronization with donors Synchronization of the reproductive cycle between the donor and the recipient is critical, and embryo transfer results in the dromedary suggest that the best recipients should ovulate hours after the donor. Transfer of embryos into recipients that have ovulated one day before the donor, or three of more days afterwards, result in very low pregnancy rates of <10% (McKinnon et al., 1994; Skidmore et al., 2002) To achieve this synchronization one of two methods can be used; either recipients can be selected from a random group. If using this technique a group of recipients at known stages of their reproductive cycles are examined 24 and 48 hours after the donor is bred and all females that have a mature follicle ( cm in diameter) are treated with GnRH or hcg (Anouassi and Ali, 1990; Skidmore et al., 2002). Alternatively better results can be obtained when recipients are induced to ovulate with hcg or GnRH following a treatment combining progesterone and ecg. The recipients are treated daily with progesterone-in-oil (100mg/day) for 10 to 15 days, to try and dampen the development of more follicles, and on the last day of progesterone treatment, i.u. ecg is injected to induce follicular development. Progesterone treatment is scheduled to end on the day of injection of gonadotrophin in the donor in an attempt to synchronize the recipient and donor. The ecg treatment guarantees the presence of mature follicles in the recipient at the same time or hours after the donor (McKinnon et al., 1994). CONCLUSIONS The reproductive performance of camels can be improved by mating at the correct stage of the cycle, using embryo transfer and transferring day 7 embryos in synchronized recipients on day 5 or 6 after ovulation or using artificial insemination and inseminating 150 x 10 6 live sperm in the uterine body or horn 24 h after GnRH injection. REFERENCES Abdel-Raouf, M. El-Naggar A., (1976). Studies on reproduction in camels (Camelus dromedarius). VI. Properties and constituents of ejaculated semen. Proc. VIIIth Int. Congr. Anim. Reprod. and A.I. (Cracow),

7 Anouassi, A. and Ali, A. (1990). Embryo transfer in the camel (Camelus dromedarius). In Proceedings: Is it Possible to Improve the Reproductive Performance of the Camel? Paris. pp Anouassi, A., Adnani, M., Raed, E.L., (1992). Artificial insemination in the camel requires induction of ovulation to achieve pregnancy. Proc. 1 st Int. Camel Conf., W.R. Allen, A.J. Higgins, I.G. Mayhew, D.H. Bravo, P.W., Skidmore, J.A., and Zhao, X.X., (2000). Reproductive aspects and storage of semen in Camelidae. in: Salamon, S., Maxwell, W.M.C. (Eds). Storage of semen in domestic animals. Anim. Reprod. Sci. 62: Cassou, M.R., cited by Krause D., (1961). Derzeitiger stand der verfahren zur konservierung von bullensperma. Dtsch. Tierarztl. Wschr. 68: Cooper, M.J., Skidmore, J.A., Ali, M., Billah, M., Wensvoort, S., Billah, A.M. and Allen, W.R. (1990). An attempt to induce and synchronise ovulation and superovulation in dromedary camels for embryo transfer. In Proceedings: Is it Possible to Improve the Reproductive Performance of the Camel? Paris.pp Kenney, R.M., Bergmann, R.V., Cooper, W.L., Morse, G.W., (1975). Minimal contamination techniques for breeding mares: Technique and preliminary findings. Proc. of the Ann. Conf. Am. Assoc. Equine Pract. 21: Kupper, H., (1954). Versuche zur Werdunnung des Hengstspermas. Hannover Tierarztl. Hochsch. Thesis. Lopez-Gatius F., (2000). Site of semen deposition in cattle: A review. Theriogenology 53: Martin, J.C., Klug, E., (1979). Zur Samenubertragung beim Pferd-Samenkonservierung in Kunststoffrohrchen. Prakt. Tierarzt 3: McKinnon, A.O. and Tinson, A.H. (1992). Embryo transfer in dromedary camels. In Proceedings: 1 st Int. Camel Conf., Dubai, U.A.E McKinnon, A.O., Tinson, A.H. and Nation, G. (1994). Embryo transfer in dromedary camels. Theriogenology. 41: Morton, K.M., Billah, M., Skidmore, J.A., Artificial insemination of dromedary camels with fresh and chilled semen: Effect of diluent and sperm dose, preliminary results. Reprod. Dom. Rum. VII. Abs Musa, B.E. and Abusineina, M.E. (1978). The oestrous cycle of the camel (Camelus dromedarius.) Vet. Rec. 102, Musa, B.H., Sieme, H., Merkt, B., Hago, M.J., Cooper, M.J., Allen, W.R., Jochle, W., (1993). Manipulation of reproductive functions in male and female camels. Anim. Reprod. Sci. 33: Rai A.K., Tandon, S.N., Khanna, N.D., (1988). Copulation time of Bikaneri male camels. Ind. J. Anim. Sci. 58: Rasbech, N.O., (1984). Instrumental inserinering i hesteavlen. Dansk. Vet. Tidsskr. 67: Sieme, H., Merkt, H., Musa, B., Badreldin, H., Willmen, T., (1990). Liquid and deep freeze preservation of camel semen using different extenders and methods. Proc. Unite de Coordination pour l'elevage Camelin. Workshop: Is it Possible to Improve the Reproductive Performance of the Camel? Paris, pp

8 Skidmore J.A., Billah M., Allen W.R., (1996). The ovarian follicular wave pattern and induction of ovulation in the mated and non-mated one - humped camel (Camelus dromedarius). J. Reprod. Fertil. 106: Skidmore, J.A., Allen, W.R., Cooper, M.J., Chaudhry, M.A., Billah, M. and Billah, A.M. (1992). The recovery and transfer of embryos in the dromedary camel: results of preliminary experiments. In Proceedings: 1 st Int. Camel Conf., Dubai, U.A.E. pp Skidmore, J.A., Billah, M. (2006b). Investigation of the most appropriate time for insemination of female camels (Camelus dromedarius) after GnRH injection and comparison of pregnancy rates after deep intra-uterine versus cervical insemination. Proceedings of First Conference of International Society of Camelids Research and Development (ISOCARD). pp. 54. Skidmore, J.A., Billah, M. and Allen, W.R. (2002). Investigation of factors affecting pregnancy rate after embryo transfer in the dromedary camel. Reproduction, Fertility and Development, 14: Skidmore, J.A., Billah, M., (2006a). Comparison of pregnancy rates in dromedary camels (Camelus dromedarius) after deep intra-uterine versus cervical insemination. Theriogenology 66: Skidmore, J.A., Billah, M., Allen, W.R., (2000). Using modern reproductive technologies such as embryo transfer and artificial insemination to improve the reproductive potential of dromedary camels. Revue Elev. Med. Vet. Pays. Trop. 53 (2): Snow, J.F. Wade, (Eds.). R & W Publications (Newmarket) Ltd. UK, pp Tibary, A., Anouassi, A., (1997a). Male breeding soundness examination, in: Institut Agronomique et Veterinarie Hassan II, Rabat, Maroc. (Eds.), Theriogenology in camelidae. Abu Dhabi Printing and Publishing Company. U.A.E. pp Tibary, A., Anouassi, A., (1997b). Artificial breeding and manipulation of reproduction in camelidae, in: Institut Agronomique et Veterinarie Hassan II, Rabat, Maroc. (Eds.), Theriogenology in camelidae. Abu Dhabi Printing and Publishing Company. U.A.E. pp Tibary, A., Anouassi, A., (1997c). Reproductive phusiology of the male, in: Institut Agronomique et Veterinarie Hassan II, Rabat, Maroc. (Eds.), Theriogenology in camelidae. Abu Dhabi Printing and Publishing Company. U.A.E. pp Waberski, D., Weitze, K.F., Rath, D., Sallmann, H.P., (1989). Wirkung von bovinem Serumalbumin und Zwitterionenpuffer auf flussigkonservierten Ebersamen. Zuchthygiene 24: