Monitoring and controlling follicular activity in camelids

Theriogenology xxx (2017) 1e9 Contents lists available at ScienceDirect Theriogenology journal homepage: www.theriojournal.com Monitoring and controlling follicular activity in camelids Ahmed Tibary Comparative Theriogenology, Department of Veterinary Clinical Science, College of Veterinary Medicine, Center for Reproductive Biology, Washington State University, USA a r t i c l e i n f o a b s t r a c t Article history: Received 1 December 2017 Accepted 1 December 2017 Available online xxx This paper reviews that state of our knowledge concerning follicular wave dynamics, monitoring and manipulation. All camelids have overlapping follicular waves in absence of ovulation which is induced by a seminal plasma factor (bNGF). The interval between follicular waves varies. The size of the ovulatory follicle varies between 11 and 25 mm in camels and between in 6 and 13 mm in South American Camelids. The interval between induction of ovulation and next ovulatory follicle is 15 ± 1 day for all camelids. Follicular activity is best monitored by transrectal ultrasonography. Progesterone therapy for 7 e15 days seems to suppress follicular dominance but does not completely inhibit follicular recruitment. Combination of estradiol and progesterone seems to provide better control of follicular activity. Both methods have provided variable results in the synchronization of follicular waves. Combination of induction of ovulation with GnRH and luteolysis at predetermined times shows some promise in synchronization of follicular dominance. These synchronization protocols require further investigation in order to provide practical approaches for fixed-time breeding. Ovarian superstimulation with FSH and eCG alone or in combination is somewhat successful. The best results are obtained when treatment is initiated at the emergence of a new follicular wave after induction of ovulation or following treatment with progesterone for 7e14 days. However, response remains extremely variable particularly in terms of ovulation rate and number of recovered embryos. Sources of this variability need to be studied including the effects of season, nutrition, doses and frequency of administration of gonadotropin. © 2017 Elsevier Inc. All rights reserved. Keywords: Camelidae Ovarian activity Nutrition Artificial breeding 1. Introduction The camelidae family includes 6 major species traditionally subdivided into Old-world camelids (OWC) or camels (Camelus dromedarius and Camelus bactrianus) and New-world camelids (NWC) (Lama glama, Lama guanicoe, Vicugna pacos and Vicugna vicugna). Domestic camelids (camels, llamas and alpacas) are important livestock in several parts of the word. It is predicted that these species will be increasingly important for animal production in harsh environments due to their adaptive characteristics to desert (camelids) or altiplano (llamas and alpacas). Wild camelids (vicugna, guanaco and some Bactrian camels) are important resources that are increasingly threatened due to habitat degradation. Reproductive management and multiplication of wild camelids through the use of interspecies embryo transfer has been investigated as a mean for the preservation of these species [1,2]. Efficient reproductive management and use of reproductive E-mail address: tibary@vetmed.wsu.edu. biotechnologies, such as artificial insemination (AI) and embryo transfer (ET), require a thorough understanding of follicular dynamics and factors governing ovarian activity. Prior to 1990's, most studies on ovarian activity in camelids relied on behavioral and hormonal observation [3]. Effort to characterize follicular wave dynamics was mostly driven by the desire to develop AI and ET technologies. The widespread use of ultrasonography allowed in situ observation of ovarian follicular activity and better characterization of follicular dynamics, ovulation and monitoring of responses to treatments [4]. The present paper discusses the state of our knowledge on ovarian follicular dynamics in camelids, factors governing it, and its monitoring and manipulation. 2. Follicular dynamics in camelids 2.1. Follicular dynamics in absence of ovulation Ultrasonographic and hormonal studies in the mid to late 1990's helped define follicular dynamics in several domestic camelids species [5e8]. Field and experimental observations demonstrated https://doi.org/10.1016/j.theriogenology.2017.12.011 0093-691X/© 2017 Elsevier Inc. All rights reserved. Please cite this article in press as: Tibary A, Monitoring and controlling follicular activity in camelids, Theriogenology (2017), https://doi.org/ 10.1016/j.theriogenology.2017.12.011 2 A. Tibary / Theriogenology xxx (2017) 1e9 that ovarian activity in the female camelid is not seasonal under optimal nutritional condition [9]. However, under some conditions, female camels may display seasonal variation in follicular activity that may be partly regulated by photoperiod. All camelid species are induced ovulators, thus in absence of ovulatory stimulus (mating or hormonal induction), follicular waves occur in an overlapping manner [6,10]. Follicular waves present the typical phases of recruitment, growth, maturation and regression. The duration of each of these stages of the follicular waves is variable (Table 1). Follicles that surpass a certain diameter (25 mm in camels, 12 mm in alpacas, 13 mm llama) have decreased ovulatory response. Some of these follicles may continue to grow and develop into large anovulatory follicles that may become hemorrhagic or luteinized (Fig. 1). Anovulatory hemorrhagic follicles (AHF) seems to be more frequent in camels [4,6] and llamas [11] than in other camelids species. The tendency for development of AHF seems to be dependent on individual female. The pathophysiology of AHF is poorly understood [6]. Clinical observations suggest that some of these AHF may be triggered by metabolic disorders in the female as well as adverse climatic conditions. Presence of AHF does not seem to disturb follicular wave patterns. Follicular dynamic is dependent on FSH and LH stimulation [12e14]. Presence of 2 or more co-dominant follicles is not uncommon in camelids and may occur in up to 40% of follicular wave (Camels [4,15,16], NWC [17]). Behavioral changes during the follicular wave are not strongly correlated to size of the follicle and readiness for ovulation (Camels [4,18]; NWC [19e22]). Therefore the best method for monitoring follicular dynamics is transrectal ultrasonography. At peak follicular development (pre-ovulatory stage), the uterus is toned and present characteristic edema pattern on ultrasonography (Fig. 2). Color-Doppler ultrasonography can be used to monitor blood flow to the follicle during various stages of development. Blood flow to the dominant follicle increased with follicular growth [23]. Fig. 1. Ultrasonographic appearance of anovulatory follicles in the dromedary. A) 48.6 mm thin-walled anovulatory follicle with anechoic fluid, b) 80 mm thick-walled anovulatory follicle with echogenic fluid, c) 42 mm anovulatory follicle showing some hemorrhage and intralumunal fibrin, d) 83.3 mm anovulatory hemorrhagic follicle (AHF) with characteristic multiloculated appearance, e) and f) AHF with varying degrees of luteinization. 2.2. Ovulation The induced nature of ovulation in camelids has long been suspected based on clinical and hormonal studies [30]. However, the major breakthroughs in defining the mechanisms of induction of ovulation came in two main groups of studies. The first demonstrated the hypothalamo-pituitary response to mating represented by a sharp increase in luteinizing hormone within minutes following mating in presence of a mature follicle in OWC [31,32] and NWC [12,13]. The second group of studies led to the hypothesis of the presence of an ovulation-inducing factor (OIF) within the seminal plasma [33]. Recent studies identified the OIF as b nerve growth factor (bNGF) in llamas and alpacas [34,35] and in camels [36]. Both bNGF and endometrial inflammation are required to Table 1 Characteristics of follicular dynamics and corpus luteum development in camelids Dromedary [4,5,24,25]; Alpaca and llama [7,26]; Bactrian camel [27,28]; Vicuna [17]; Guanoco [29]. Parameter C. dromedarius C. bactrianus V. pacos L. glama V. vicugna L. guanacoe 10.5 ± 0.5 7.6 ± 0.8 11.9 ± 0.8 10.9 ± 3 7 ± 4.2 11.9 ± 4.2 3e9 2e8 3e8 3e9 2e8 3e8 3.0 ± 0.2 1.4 ± 0.1 2.0 ± 0.3 7.0 ± 2.4 3.0 ± 1.2 5.2 ± 2.1 9 1.8 10e18 25 40e50 8e45 9 0.7e1.8 10e18 25 ? ? 6 0.43 8e10 12 5 ? 7 0.5e0.9 9e12 13 10e40 4e22 6.2 1.8 ± 0.1 8.4 ± 0.9 11.2 ? ? 7.2 1.0 ± 0.3 10.2 ± 2.1 16.1 ? ? 32 to 40 15e25 7.2 ± 1.7 10 ± 1.2 30 to 48 15e25 7.3 10.5 28 to 30 11e15 7e8 10e12 27e36 11e18 8 10e12 e e e e e e e e a Follicular wave phases duration Growth (days) Maturation (days) Regression (days) Ovulatory follicle characteristica Minimum size (mm) Growth rate (mm/day) Average size (mm) Maximum size (mm) Incidence of anovulatory follicles (%) Anovulatory follicle regression (days) Corpus luteum characteristics Interval from mating to ovulation (hours) Size (mm) Day at CL maximum size Days post-ovulation to complete luteolysis a Extreme variation in onset of postpartum ovarian follicular activity is primarily due to nutritional condition and effect on lactation anestrus and seasonality. Please cite this article in press as: Tibary A, Monitoring and controlling follicular activity in camelids, Theriogenology (2017), https://doi.org/ 10.1016/j.theriogenology.2017.12.011 A. Tibary / Theriogenology xxx (2017) 1e9 3 frequently but not systematically [6]. Double ovulations are common in most domestic camelids in good health and nutritional status [15]. Triple and quadruple ovulations have also been documented in the dromedary camel [4,16]. Spontaneous ovulation has been described in up to 5% of the follicular wave in llamas and camels [45]. This phenomenon seems to be more common in lactating animals [25]. Follicular recruitment in mated females start 2e4 days following ovulation and results in an available mature follicle within 2e4 days of completion of luteolysis which occurs around day 10 post-ovulation. This short luteal phase results in a single follicular wave per cycle in the majority of the females. Preliminary data in our laboratory show that 90% of the female (camels and alpacas) have only one follicular wave per ovulatory cycle. Thus after a sterile mating or hormonal induction of ovulation, the average interval between two consecutive ovulatory follicles is 14 days in all camelids [6,46]. Fig. 2. Maximum uterine edema and ontrcation at the peak follicular growth (arrow indicated cranial aspect of the uterus). maximize ovulation rate [34]. Mating in absence of seminal plasma (urethrotomized males) does not induce ovulation [37]. Administration of bNGF by various routes (IV, IM or intrauterine) induced LH surge however the dose required is higher with the intrauterine route [38]. The bNGF seems to have a luteotrophic effect on the corpus luteum in llamas [39e41]. However, this effect was not observed after intrauterine administration [42]. In absence of copulation, ovulation can be reliably induced by GnRH (Buserelin 8 mg NWC, 20 mg camels; GnRH 20e50 mg NWC, 100 mg camels) or hCG (NWC 500e750 IU IV, Camels 1500e3000 IU IV) as long as a growing or mature follicle is present (Fig. 3). In llamas, there is no difference in ovulation rate, interval to ovulation and luteal development whether ovulation is induced by copulation, im administration of LH or GnRH [43]. In camels, optimal ovulation rate is achieved when the dominant follicle is at least 11 mm in diameter [44]. Ovulation occurs on average 30 h after mating. Both ovaries are equally active and alternation of ovulation between ovaries occurs 2.3. Factors affecting follicular dynamics The major factors influencing follicular dynamics are puberty, season, postpartum, lactation and nutrition. Studies on age at puberty in the female camelids are very scarce and limited to field observation. In well fed animals, follicular wave start as early as 4 months in alpacas, 6 months in llamas and 18 months in camels. However, under traditional management, puberty may be delayed until 3 or 4 years in camels [6,47]. Seasonal variation of ovarian activity of female camelids has been described in both OWC [6] and NWC [48]. However, the control of this apparent seasonality of reproduction in camelids remains poorly studied. In camels, slaughterhouse studies show a significantly lower follicular activity (number and size) and oocyte quality during the non-breeding season [49]. However, some females continue to have normal follicular activity outside of the defined breeding season [50]. Although some studies in camels have shown some degree of control of ovarian follicular activity by photoperiod [51], several aspects of the interaction between photoperiod, temperature and nutrition remain to be elucidated [52,53]. In the dromedary, seasonal variation in follicular activity may be exacerbated by lactation. In one study, lactating dromedary Fig. 3. Effect of follicular size on ovulatory response after GnRH intramuscular GnRH injection in camels (100 mg) and alpacas (50 mg) (12e25 females per group) (Tibary A, unpublished). Please cite this article in press as: Tibary A, Monitoring and controlling follicular activity in camelids, Theriogenology (2017), https://doi.org/ 10.1016/j.theriogenology.2017.12.011 4 A. Tibary / Theriogenology xxx (2017) 1e9 females had smaller dominant follicles at the beginning of the breeding season than non-lactating females [54]. Postpartum resumption of follicular activity occurs in NWC within 5 days of delivery and good conception rates are obtained by 2e3 weeks postpartum [55,56]. There seem to be an interaction between lactation and ovarian activity. In llamas, lactation was associated with decreased diameter of the dominant follicle [57] and CL size and pregnancy rate following embryo transfer were lower in lactating female alpacas [58]. OWC have a slightly delayed postpartum resumption of ovarian activity and adequate conception rate which occurs between 30 and 45 days postpartum [3,59,60]. However, prolonged lactational anestrus is frequent in camels reared under desert conditions and may last several months. This long lactational anestrus is one of the reason why MOET has a great impact on the generation interval and genetic improvement in camels [6,47]. Early weaning in camels hastens postpartum follicular activity and shorten the inter-calving intervals in intensive camel production systems [47,61]. Clinical and experimental observations show a pronounced effect of nutrition on ovarian activity. In llamas, females under nutritional restriction (BCS ¼ 2.5) experience follicular suppression, lower CL development and lower concentration of progesterone after ovulation than females averaging a BCS of 3.9 [62]. In alpacas, administration of leptin prior to induction of ovulation improved CL size and progesterone levels [63]. These studies as well as the effects of lactation on ovarian follicular dynamics confirm the effect of negative energy balance on follicular dynamics and CL quality. 3. Synchronization of follicular waves Synchronization of follicular waves is important for development of fixed time artificial insemination and embryo transfer programs [14,64]. Methods used in ruminant species have been adapted to camelids with varying degrees of success. Several approaches have been used to control ovarian follicular dynamics and eliminate dominant follicles prior to gonadotropin treatment. These include manual ablation, ultrasound guided aspiration of the dominant follicles or initiation of treatment following a period progestogen treatment [65e69]. There are limited observations on hormonal inhibition of follicular activity. In one study on alpacas, daily administration of Buserelin (50 mg/female SQ) for 10 days results in suppression of follicular activity starting on the 6th day of treatment [70]. aspiration or LH treatment are effective in inducing follicular wave synchronization [57]. Ovulation synchronization using a combination of GnRH and PGF2a was investigated in camels. Timed breeding on day 22 following a series of treatment (GnRH on Day 0, PGF2a on day 7, GnRH on Day 10, PGF2a on day 17) resulted in pregnancy rates of 46e60%. However, this study lacked a control group [76]. In Bactrian camels, two injections of GnRH at 14 days interval provided a better synchronization of follicular waves and response to superstimualtion with eCG and FSH [77]. A study in our laboratory did not show any advantage any advantage in terms of synchronization of follicular wave, ovulation rate and pregnancy rate with a treatment consisting of 2 injection of GnRH at one week interval followed by PGF2a at 14 days (Fig. 4) [78]. 3.2. Progesterone treatment Progestogen treatments that have been tested in camelids include daily progesterone injection (50e100 mg in SAC and 100e150 mg in camels), intravaginal devices PRIDs or CIDRs with 1.38 g or 1.9 g progesterone in camels, CIDRs 0.3 g progesterone and Medroxyprogesterone acetate (MAP) sponges in llamas and alpacas or subcutaneous implants of norgestomet (3 mg) in llamas and alpacas. The length of treatment varies generally from 7 to 14 days (OWC [30,64,68]; camels [14,30,79]). A 9 day treatment with MAP vaginal sponges (60 mg) was shown to synchronize follicular activity in llamas and produce a preovulatory follicle 6 days after treatment [80]. In llamas, CIDRs (0.33 mg progesterone) treatment for 16 days reduced follicular diameter from day 5 [81]. Similar results were obtained in our laboratory in llamas and alpacas with a 14 day treatment (Figs. 5e7). In vicunas, treatment with CIDRs for 5 days exerted a negative effect on follicular development and allowed a better superstimulation response to eCG [82]. In llamas, intravaginal devices containing 0.5 mg of progesterone seem to provide better control of follicular activity and provide better response to superovulation with eCG. The shape and area of contact of the vaginal device for progesterone delivery may affect absorption of progesterone [83]. Vaginal devices containing 0.78 g progesterone (Cue-mate®) inserted for 7 days reduced follicular development. A new dominant follicle was available in all females 6 days after removal of the device [84]. In camels, there are conflicting reports on the efficacy of PRIDs 3.1. Follicular ablation Dominant follicles can be eliminated by follicular aspiration or induction of ovulation. Removal of the dominant follicle by ultrasound-guided aspiration provides consistent results for ovarian superstimulation 48 h after [64]. Studies in llamas and alpacas, show improved embryo recovery rates when gonadotropin treatment is started after administration of LH to synchronize follicular wave emergence [71,72]. Initiation of gonadotropin treatment at a specific time (2e4 days) following induction of ovulation has been shown to be more reliable in Bactrian [1] and dromedary camels [73,74]. In camels, follicular wave emergence occur about 70.6 ± 1.4 h (range 60e84 h) after GnRH injection to induce ovulation and follicular deviation occurs 58.6 ± 2.7 h (range 36e84 h) from emergence [16]. In one study on dromedary camels, there was not difference in the percentage of females that ovulated 14 days (47% vs 40%) after follicular ablation by ultrasound-guided transvaginal aspiration or control (saline injection), whereas 80 and 87% of females that received receptively GnRH or GnRH followed by a luteolytic dose of cloprostenol a week later ovulated after GnRH treatment 14 days later [75]. In llamas, follicular ablation by Fig. 4. Proportion of female llamas that were mated, ovulated and their pregnancy rate at 45 days. Treatment: Females (n ¼ 31) received a synchronization treatment protocol consisting of 2 GnRH injections at 7 days interval followed by an injection of cloprostenol 7 days after the second injection and mated on day 25 (Day 0 ¼ First GnRH injection). Control: females (n ¼ 10) mated based on receptivity on the same day [78]. Please cite this article in press as: Tibary A, Monitoring and controlling follicular activity in camelids, Theriogenology (2017), https://doi.org/ 10.1016/j.theriogenology.2017.12.011 A. Tibary / Theriogenology xxx (2017) 1e9 5 Fig. 5. Serum progesterone level in alpacas (n ¼ 7) and llamas (n ¼ 7) during and following a 14 day treatment with vaginal CIDR (0.33 mg progesterone) [78]. [85] and CIDRs [9] in synchronization of follicular waves. In addition, these devices have been associated with increased spontaneous ovulation in some studies. Treatment with PRIDs containing 1.55 g of progesterone for 7 days did not synchronize follicular waves [85,86]. However camels treated for 17 days with PRIDs containing 1.9 g progesterone and receiving a large dose of eCG (3000 IU) had a better synchrony of follicular growth [87]. Treatment with CIDRs containing 1.38 g of progesterone for 10 days did not synchronize follicular waves in the non-breeding season [9]. In a recent study, 70 and 75% of camels had a preovulatory follicle on days 16 and 18 respectively after treatment with CIDRs containing 1.9 g of progesterone for 14 days [88]. However in there was no control (untreated) group in this study. Nogestomet implant were not efficacious in synchronizing follicular wave in Bactrian camels [77]. Daily injection of progesterone (50 mg, IM) for 12 days induced reduction follicular diameter on day 7 in llamas [89]. In camels, daily injections of progesterone (100 mg/day) for 10e16 days provided promising results in MOET programs [66]. A recent study in our laboratory showed that long-acting progesterone injection can be used in camels and may be more advantageous then daily injections (Fig. 8) [90]. In summary, progesterone therapy in camelid suppresses the growth of large follicles but does not completely suppress follicular activity and therefore is not efficient in synchronizing follicular wave emergence and fixed time breeding [6,69,79,86]. The combination of estradiol and progesterone has been shown to be more effective in the control of follicular wave in OWC in some studies [91] but not others [68]. In llamas, daily pretreatment with 100 or 150 mg progesterone for 5 days after a single injection of estradiol benzoate (1 mg) resulted in a higher embryo recovery rate following superstimulation in some trials [92]. In our laboratory, daily administration of estradiol and progesterone for 7e10 days to alpacas produced a more uniform response however the ovulatory response was poor [93]. In llamas, a single injection of combined estradiol-17b (1 mg) and progesterone (25 mg) provided some synchronization of follicular wave but not as good as induction of ovulation or follicle aspiration [57]. In camels, a single injection of estradiol benzoate (5 mg) and progesterone (100 mg) was not effective in synchronization of follicular wave [75]. 4. Ovarian superstimulation 4.1. Induction of follicular activity in anestrous females Induction of follicular activity with eCG or FSH in prepubertal animals and during lactational and seasonal anestrus is possible and was demonstrated in dromedaries [94]. However, this is not a viable management option for natural mating and may only be useful for in vitro or in vitro (follicular aspiration) embryo production. 4.2. Superstimulation for MOET programs Fig. 6. Serum estradiol 17b levels in alpacas and llamas with and without a 14 day treatment with CIDRs (Group 1 ¼ 7 treated llamas, Group 2 ¼ 7 treated alpacas, Group 3 ¼ 7 untreated llamas, Group 4 ¼ 9 untreated alpacas) [78]. Approaches to ovarian superstimulation in camelids have been largely adapted from protocols used in ruminants. The primary hormones used are FSH and eCG alone or in combination. As for other species, response to these hormones depends on timing of initiation of treatment in relationship to follicular dynamics, dose and schedule on administration, and individual variation. Response to gonadotropin treatment is largely affected by the stage of follicular wave recruitment and the presence of a dominant follicles. Although FSH and eCG treatments have been initiated during Please cite this article in press as: Tibary A, Monitoring and controlling follicular activity in camelids, Theriogenology (2017), https://doi.org/ 10.1016/j.theriogenology.2017.12.011 6 A. Tibary / Theriogenology xxx (2017) 1e9 Fig. 7. Proportion of females with ovulatory follicles (9e12 mm in llamas; 8e11 mm in alpacas) following a 14 days CIDR (0.33 mg progesterone). Group 1 ¼ 7 treated llamas, Group 2 ¼ 7 treated alpacas, Group 3: 7 untreated llamas, Group 4: 9 untreated alpacas [78]. the receptive or luteal phase of the cycle with some success, better results are obtained when the treatment is initiated in absence of any follicles greater than 2 mm [14]. 4.2.1. Follicle stimulating hormone (FSH) Both ovine (oFSH) and porcine (pFSH) FSH, have been for ovarian superstimulation with variable success [6]. The manner of administration of FSH (dose, frequency and timing during the cycle) has been investigated to some degree. Unfortunately detailed description of the treatment protocol is often not clearly presented in publications. In the dromedary, a total dose of 20e30 mg units of oFSH is given over 6 days (two injections daily) starting 2 days before and up to 1 day after completion of a 7-day course of progesterone treatment by intravaginal device (PRID) [85]. FSH was also given in a single small dose (3.3 units) followed by an injection of 3500 IU of eCG, resulting in an average of 7 embryos recovered per treated female [86]. In another study, oFSH was given twice a day (1e3 mg per injection) during 3e5 days following a 10e15 day course of progesterone treatment (100 mg per day during 10e15 days) [66]. Single subcutaneous dose of oFSH has been tested with variable results. Porcine FSH given twice daily in decreasing doses over 3, 5, or 7 days after a 10e15 day progesterone treatment resulted also in superstimulation of dromedary female [14,66]. The interval from pFSH treatment to development of a mature follicle (10e16 mm in diameter) varies between 6 and 8 days [6,69]. Similar protocols have been used for superstimulation of Bactrian camels [1,77]. In llamas and alpacas, FSH alone or in combination with eCG, has been used following a 12 day progesterone treatment [95,96]. The best superovulation and embryo collection results were obtained following administration of pFSH twice a day for 5 days in decreasing doses (32, 27, 22, 17 and 12 mg, im) [68,96]. The number of embryos obtained after pFSH stimulation is generally low. Alpacas reportedly produce a more variable response to superstimulation protocols than llamas [68]. 4.2.2. Equine chorionic gonadotropin (eCG) Superstimulation with eCG has been extensively used in camelids. In general, a single dose is administered intramuscularly one Fig. 8. Mean (±SEM) serum progesterone concentration in female camels (n ¼ 12) following intramuscular injection of 5 mL of BioRelease P4 LA 300 containing 300 mg or progesterone/ml on day 0 [90]. Please cite this article in press as: Tibary A, Monitoring and controlling follicular activity in camelids, Theriogenology (2017), https://doi.org/ 10.1016/j.theriogenology.2017.12.011 A. Tibary / Theriogenology xxx (2017) 1e9 day before or on the day of completion of a 5e15 days progesterone regime. The dose of eCG used vary from 1500 to 6000 in camels, ~ as 500e2000 IU in the llama and 500 to 750 in alpaca and vicun [30]. In the dromedary, eCG given as a single injection of 2000 IU, 2500 IU or 4000 IU, one day before or one day after PRID removal, resulted in ovulation in 40% of treated animals. Only 42% of ovulating females yielded one or more embryos. The interval from PRID removal to mating was 5 and 4.5 days respectively for females receiving 2500 IU and 4000 IU of eCG. This interval was one day shorter in females treated with eCG one day before removal of PRID [85,86]. When eCG (2000e3000 IU) is administered to females with no dominant follicle, the interval from treatment to mating (follicular diameter of 12 mm) is relatively constant (8 days) [30]. Follicular response is variable (0e19) with about 20% of the females not responding [14]. In the llama, eCG (1000 IU) was used following progesterone priming either in the form of natural corpus luteum (ovulation induced by hCG or GnRH injection), CIDR's or subcutaneous implants [97]. Follicular response was variable (0e13 follicles) and the number of ovulations ranged from 0 to 7 with a mean number of embryos collected of 1.3e2.3 per donor (range 0e6). Follicles reached the mature size (9e13 mm) 5e11 days following eCG treatment. Several treated females show premature luteinization 7e9 days following eCG treatment. Increasing the dose of eCG to 2000 IU results in an increase in incidence of anovulatory follicles. In the alpaca, injection of 750 IU of eCG resulted in average 3.7 embryos per ovulating female [95,96]. The main disadvantage of eCG is the high incidence of follicular luteinization and disturbance of ovulation probably due to its long half-life. Dromedary females tend to become refractory to eCG following multiple use. This suggests that at least in the camel, the risk of inducing anti-eCG antibodies is real [69]. Superstimulation protocols combining both FSH and eCG have been published in alpacas, llamas [68], vicunas [98] and camels [69]. In general, these protocols do not provide much superiority to protocols using FSH alone. Superstimulation treatments of the female camelidae are far from being perfect. Ovulation response and embryo yield remain highly variable [6,14,64,68]. The major problems that need to be addressed are: the high incidence of non-responsive females (20e30%), incidence of follicular luteinization, hyperstimulation in some females and loss of efficacy after multiple treatments. In addition, the recovery rate number of embryos recovery/number of corpora lutea is low (40%). Sources of variations that need to be investigate in response to superstimulation include species, breed, and individual animal variation. 4.2.3. Immunization against inhibin Immunization again inhibin results in very high levels of circulating FSH and consequently an increase in the number or recruited follicles which will continue to grow until maturation. In the dromedary, immunization against inhibin resulted in encouraging ovulatory response. An increase in ovulation number (4e10) was observed in 60% of the immunized females [69]. Recent studies showed a high rate of triple of ovulations up to 5 months from the initial immunization [99,100]. 5. Conclusion In the last 3 decades, research on camelids reproduction has helped further our knowledge on follicular dynamics in these species. Transrectal ultrasonographic monitoring of follicular waves is now current practice in all species of camelids. This has been an effective tool in the management of breeding and to monitoring 7 effects of various factors on ovarian activity. This technique has replaced the need for laborious endocrine assays. Factors affecting follicular dynamics and in particular season and nutrition need more investigation. Multiple approaches to synchronize follicular wave have been adapted from other species but met with variable results. Progesterone therapy alone or in combination with estradiol shows some efficacy for superstimulation in MOET programs but is not sufficient for fixed time mating or artificial insemination. Ovulation synchronization protocol may be more suitable for timed breeding and artificial insemination. References [1] Niasari-Naslaji A, Nikjou D, Skidmore JA, Moghiseh A, Mostafaey M, Razavi K, et al. Interspecies embryo transfer in camelids: the birth of the first Bactrian camel calves (Camelus bactrianus) from dromedary camels (Camelus dromedarius). Reprod Fert Dev 2009;21:333e7. [2] Sumar JB. Embryo transfer in domestic South American camelids. Anim. Reprod. Sci 2013;136:170e7. [3] Tibary A, Anouassi A. Theriogenology in camelidae. 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