36 CONTACT Ahmed Tibary tibary@wsu.edu © 2025 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Citation: Clinical Theriogenology 2025, 17, 11652, http://dx.doi.org/10.58292/CT.v17.11652 Review Report Manipulation of ovarian activity in camelids Muhammad-Salman Waqas,a Abdelhaq Anouassi,b Ahmed Tibarya aDepartment of Veterinary Clinical Sciences, College of Veterinary Medicine, and Center for Reproductive Biology, Washington State University, Pullman, WA, USA bVeterinary Research Center, Advanced Scientific Groups, Abu Dhabi, UAE Abstract Camelid production has increased in importance in the world in recent years. The high demand for genetically superior males and the implementation of reproductive technologies requires the synchronization of ovarian follicular waves. This paper describes the state of knowledge regarding seasonality and follicular wave patterns in camelids. Reproductive seasonality in camels can be manip- ulated using artificial photoperiod or melatonin treatment. As in other ruminants, protocols for follicular wave synchronization are based on the elimination of the dominant follicle (induction of ovulation) or progesterone treatment. These protocols are described for breeding management and ovarian superstimulation in embryo donors. Induction of ovulation and initiation of superstimula- tion 2-4 days later (emergence of a new follicular wave) results in higher response in terms of number of ovulations and collected embryos. Keywords: Camels, alpacas, llamas, superovulation, follicular wave Introduction Camelids are a vital production animal in several areas of the world. The renewed interest in these species in the last 3 decades has increased our understanding of their reproductive biology. In camels, the importance of the racing industry and the intensification of camel dairy production have increased the demand for assisted reproductive technology. A similar situation is observed in South American camelids (SAC), par- ticularly alpacas, where the show and fiber market increased the genetic selection pressure. Efficient reproductive management and the implementation of reproductive technologies such as artificial insemination (AI) and embryo transfer (ET) in camelids require manipula- tion of ovarian activity. This paper reviews the mechanisms controlling ovarian follicular dynamics and methods to manipulate follicular dynamics and ovulation in camelids. Follicular dynamics and ovulation in camelids Endocrine and ultrasonographic studies in the mid to late 1990s helped to define follicular dynamics in several domestic camelid species.1–8 All camelid species are induced ovulators. In the absence of an ovulatory stimulus (mating or hormonal induction), follicular waves occur in an overlapping manner (Figure 1).6,9 Seasonal variation of ovarian activity of female camelids has been described in South American camelids (SAC)10 but is more pronounced in camels.6,11,12 In camels, follicular activity (number and size) and oocyte quality are significantly lower during the nonbreeding season compared to the breeding season.12,13 However, a substantial proportion of females (20-60%) continue to have regular fol- licular activity outside the defined breeding season.6,14 Recent research has revealed that photoperiod is involved in the con- trol of ovarian follicular dynamics in the dromedary.15,16 Ovarian follicular activity is also affected by nutritional status (i.e. body condition score) in SAC17,18 and camels.19 Restricted nutrition decreases ovarian follicular growth and corpus luteum development, and lowers plasma progesterone and leptin concentrations. However, the interaction among photo- period, temperature, and nutrition in the control of seasonal- ity in camels remains poorly studied.12,20 Remarkable difference exists between camels and SAC regard- ing the effect of lactation on follicular dynamics. Resumption of ovarian activity occurs within 5-10 days after parturition in SAC,21,22 whereas in camels, lactational anestrus can last from 45 days up to several months, and has substantial impact on the productivity of this species.6,23–26 mailto:tibary@wsu.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/CT.v17.11652 Citation: Clinical Theriogenology 2025, 17, 11652, http://dx.doi.org/10.58292/CT.v17.11652 37 Ovarian follicular dynamics are regulated by follicle stimulat- ing hormone (FSH) and luteinizing hormone (LH).1,27–29 Duration of follicular development phases, ovulation, corpus luteum development, and luteolysis are presented (Table). The presence of 2 or more codominant follicles is common in camelids and may occur in up to 40% of follicular waves.30–33 Follicles ovulate in response to mating or hormonal treat- ment (GnRH or hCG) when they reach an appropriate size (camels: 12 mm; SAC: 7-8 mm). Receptivity to the male is not strongly correlated with the size of the follicle and readiness for ovulation (camels;33,34 SAC35–38). Therefore, the best method for monitoring follicular dynamics is transrectal ultrasonography. At the peak of follicular development, the uterus is toned and has a characteristic edema pattern on ultrasonography (Figure 2). A sharp rise in serum LH concentration occurs minutes after mating (camels;2,3,39 SAC28,29,40). Ovulation occurs on average 30 hours (range: 26-72 hours) after mating. Ovulation is induced by the β nerve growth factor (βNGF) present in camelid seminal plasma (SAC;41,42 camels43). The presence of βNGF and endometrial inflammation are required to maxi- mize the ovulatory response.41 The action of the ßNGF involves kisspeptin neuron activation44,45 and a local mechanism on the ovary;46 ßNGF treatment by various routes (intravenous [IV], intramuscular [IM], or intrauterine induces LH surge. However, the dose is higher with intrauterine route.47 βNGF appears to have a luteotrophic effect on the CL.48–50 However, this effect was not observed after intrauterine treatment.51 Spontaneous ovulation has been described in up to 5% of the follicular waves in llamas and camels.52 This phenomenon appears to be more common in lactating animals.53 Follicular recruitment starts 2-4 days after ovulation, and a mature follicle develops 4-6 days after completion of luteoly- sis, which occurs 9-10 days after a sterile mating. This short luteal phase results in a single follicular wave per cycle in most females. Preliminary data in our laboratory revealed that 90% of females (camels and alpacas) have only 1 follicular wave per ovulatory cycle. Thus, after a sterile mating or hormonal induction of ovulation, the average interval between 2 consec- utive ovulatory follicles is 14 days (Figure 3).6 In the absence of mating or other ovulatory stimuli (i.e. GnRH or hCG treatment), follicles continue to grow and reach a maximum diameter (25 mm in camels, 12 mm in alpacas, 13 mm in llamas) then undergo atresia. Some follicles con- tinue to grow and develop into large anovulatory follicles that may become hemorrhagic or luteinized (Figure 2). The inci- dence of anovulatory HAF is higher in camels,6,33 and llamas55 than other camelid species. There is an individual predisposi- tion for HAF in the absence of ovulation, but the exact patho- physiology of AHF is poorly understood.6 Manipulation of ovarian follicular activity is an important aspect of reproductive management and implementation of synchronization, timed-AI, and multiple ovulation and embryo transfer technologies. Several techniques have been used to control ovarian follicular dynamics, including inhibition of fol- licular activity, manipulation of seasonality, synchronization of follicular waves, and ovarian superstimulation. Inhibition of follicular activity Inhibition of follicular activity may be desired for manage- ment reasons (contraception) or to control the emergence of a new follicular wave. There are limited observations on hor- monal inhibition of follicular activity in camelids. In alpacas, daily subcutaneous (SC) buserelin (50 µg/female) treatment for 10 days suppressed follicular activity, starting on day 6 after treatment.56 Immunization against GnRH has not been stud- ied thoroughly in camelids, but preliminary experiments in our laboratory in males and females produced variable results. Manipulation of seasonality The seasonal reproductive pattern of camels is driven in large part by photoperiod. Peak reproductive activity in this species is observed during the short photoperiod.57 The photoperiod effect on reproduction is confirmed by changes in the noctur- nal melatonin secretion. Melatonin secretion peak is shorter under an artificially long photoperiod.15,16,58 Similar findings were reported in guanacos in the wild, indicating a circadian pattern of melatonin secretion.59 Melatonin treatment advanced the breeding season in female dromedaries.60 Dromedary females exposed to an artificial long photoperiod (16 light:8 dark) for 41 days and then treated with a subcutaneous melatonin implant (1 implant of 18 mg melatonin per 28 kg of body weight), displayed ovarian cyclicity 3.5 months earlier compared to females that did not receive melatonin.16 Figure 1. Schematic representation of follicular dynamics in camelids showing overlapping follicular waves in absence of ovulation http://dx.doi.org/10.58292/CT.v17.11652 38 Citation: Clinical Theriogenology 2025, 17, 11652, http://dx.doi.org/10.58292/CT.v17.11652 Induction of ovulation Ovulation can be reliably induced by GnRH (SAC buserelin 8 µg, camels 20 µg camels; SAC GnRH 20-50 µg, camels 100 µg) or hCG (SAC 500-750 IU IV; camels 1,500-3,000 IU IV) in the presence of a growing or mature follicle. The optimal ovula- tory response after treatment is observed when the dominant follicle is 7-10 mm in alpaca, 8-12 mm in llamas, and 11-18 mm in camels.8,54,61–63 If given outside the optimal time, ovula- tion rate decreases. In llamas, there was no difference in ovu- lation rate, interval to ovulation, or luteal development when ovulation was induced by copulation or hormonal (LH or GnRH) treatment.64 Synchronization of follicular waves Synchronization of follicular waves is essential for timed-AI, superovulation, and embryo transfer.27,65 Methods used in other ruminants have been adapted to camelids with varying degrees of success. Several approaches have been used to con- trol ovarian follicular dynamics and eliminate dominant folli- cles prior to gonadotropin treatment. These include manual ablation (not recommended), ultrasound-guided aspiration of the dominant follicles, and hormonal treatments.54,63,66,67 Follicular ablation In the dromedary camel, a new follicular wave emerges 2.3 ± 0.5 days after ablation of the dominant follicle.68 In SAC, a new follicular wave emerges 2-3 days after induction of ovula- tion of the dominant follicle. In camels, follicular wave emer- gence occurs 70.6 ± 1.4 h (range: 60-84 hours) after GnRH injection to induce ovulation, and follicular deviation occurs 58.6 ± 2.7 hours (range: 36-84 hours) after emergence.31 In dromedary camels, the percentages of females that ovulated within 14 days were: no treatment (47%), ultrasound-guided follicular ablation (40%), GnRH treatment (80%), or GnRH followed by a luteolytic dose of cloprostenol 1 week later (87%).68 In llamas, elimination of the dominant follicle by ultrasound-guided aspiration or LH treatment is effective in inducing follicular wave synchronization.69 Eliminating dominant follicles by aspiration or induction of ovulation allows initiation of gonadotropin treatment for Table. Characteristics of the follicular wave, ovulation, and corpus luteum in camelids (adapted from54) Dromedary camel (Camelus dromedarius) Bactrian camel (Camelus bactrianus) Llama (Llama glama) Alpaca (Vicugna pacos) Vicuna (Vicugna vicuga) Guanaco (Llama guanaco) Number of follicles at emergence 8-30 - 8-12 8-12 - - Growth phase (days) 9-12 9-12 3-9 3-9 3-6 5-9 Growth rate (mm/day) 1.6 1.6 0.7 1.4 1.8 1.0 Plateau (days) 5-9 5-11 3-8 3-8 2-4 2-4 Minimum ovulatory follicular size (mm) 8 9 8 7 7 8 Average ovulatory follicular size (mm) 15 15 10 9 8 10 Maximum ovulatory follicular diameter (mm) 25 22 14 12 10 13 Regression phase (days) 10-12 7-15 3-8 3-8 3-5 3-7 Hemorrhagic anovulatory follicle (HAF) incidence (%) 40-55 - 4-22 - - - HAF size (mm) 30-90 30-50 14-35 13-35 - - HAF regression (days) 8-45 - 4-22 - - - Codominance (%) 45 - 10-30 25 12 Inter-wave interval (days) 18.2 ± 3.8 19.1 ± 0.6 15.8 ± 0.5 12-16 7.2 ± 0.5 12.6 ± 5.6 Ovarian alternance (%) - - - - 77 93 Interval mating to ovula- tion (hours) 32.-40 30-48 28-30 27-36 30 30 Corpus luteum size (mm) 15-25 15-25 11-18 11-15 11-15 11-15 Day at maximum size of corpus luteum 7.2±1.7 7.3 8 7-8 - - Day of luteolysis 10 ± 1.2 10.5 10-12 10-12 - - Return to receptivity (cycle-days) 12-16 12-16 12-15 12-15 - - http://dx.doi.org/10.58292/CT.v17.11652 Citation: Clinical Theriogenology 2025, 17, 11652, http://dx.doi.org/10.58292/CT.v17.11652 39 superovulation that coincides with the emergence of a new follicular wave.70 Embryo recovery rates are improved when gonadotropin treatment is started 2-4 days after induction of ovulation (SAC;71,72 camels73–75) Synchronization with a combination of GnRH and PGF2α Ovulation synchronization using a combination of GnRH and PGF2α was investigated in camelids (Figure 4). In camels, 2 GnRH injections given 14 days apart or 2 GnRH injections 14 days apart with PGF2α 7 days after the first GnRH was effec- tive in synchronizing ovulation.68 Timed-breeding on day 22 after a hormonal treatment protocol consisting of GnRH on day 0, PGF2α on day 7, GnRH on day 10, and PGF2α on day 17 resulted in 46-60% pregnancy rates.76–78 However, several studies lacked a proper control group. Two injections of GnRH given 14 days apart followed by timed-mating (14 days after the second injection of GnRH) resulted in a 57.7% pregnancy rate.79 In Bactrian camels, 2 injections of GnRH given 14 days apart resulted in better synchronization of follicular waves and response to ovarian superstimualtion with equine chorionic gonadotropin (eCG) or FSH.80 In llamas, GnRH treatment on day 0 and PGF2α after 7 days, then a second GnRH on day 10 resulted in a good synchroni- zation of ovulation in females that ovulated after the first GnRH.81 There was no advantage in synchronization of the follicular wave, ovulation rate, or pregnancy rate using a treat- ment consisting of 2 injections of GnRH 1 week apart, fol- lowed by PGF2α on day 14.54,82 Progesterone treatment Several progestogens have been used to attempt to control fol- licular dynamics in camelids, including daily progesterone injection (50-100 mg in SAC and 100-150 mg in camels), intravaginal devices (PRIDs or CIDRs with 1.38 g or 1.9 g pro- gesterone in camels, CIDRs 0.3 g progesterone and medroxy- progesterone acetate (MAP) sponges in llamas and alpacas) or SC implants of norgestomet (3 mg) in llamas and alpacas. In several studies, the length of treatment varied from 7 to 14 days.54,67,83,84 Figure 2. Ultrasonographic images of follicular dynamics in an alpaca: a. ovary quiescent stage; b. ovary with follicular recruit- ment and growth (follicles 3-4 mm in diameter); c. ovary with dominant follicle (9 mm in diameter); d. uterine horn during peak follicular growth (edema and tone); e. ovary with corpus luteum 7 days after ovulation; f. ovary with anovulatory follicle (19 mm) and g. ovary with hemorrhagic anovulatory follicle http://dx.doi.org/10.58292/CT.v17.11652 40 Citation: Clinical Theriogenology 2025, 17, 11652, http://dx.doi.org/10.58292/CT.v17.11652 In camels, there are conflicting reports on the efficacy of PRIDs85 and CIDRs19,86 for synchronization of follicular waves. In addition, some studies have reported an increase in sponta- neous ovulation when these devices are used.85 Treatment with PRIDs containing 1.55 g of progesterone for 7 days did not synchronize follicular waves.85,87 However, camels treated for 17 days with PRIDs containing 1.9 g progesterone and receiv- ing a large dose of eCG (3,000 IU) had a better synchrony of follicular growth than those not treated with PRIDs.88 Treatment with CIDRs containing 1.38 g of progesterone for 10 days did not synchronize follicular waves in the nonbreeding season.19 In dromedary camels, 70 and 75% had a preovula- tory follicle on days 16 and 18, respectively, after treatment with CIDRs (1.9 g of progesterone) for 14 days.89 However, this study has no control (untreated) group. Norgestomet implants were not efficacious in synchronizing follicular waves in Bactrian camels.80 In dromedary camels, daily IM progesterone injections (100 mg/day) for 10-16 days were used with rela- tively good results to synchronize recipients in an embryo transfer program.90 Long-acting progesterone injection can be used in camels and may be more advantageous than daily injections or intravaginal devices, but this compound still needs to be thoroughly investigated for synchronization of fol- licular waves.54,91 Daily IM injection of progesterone (50 mg) for 12 days inhibited follicular growth by day 7.92 In llamas, 9 days of MAP treatment vaginal sponges (60 mg) syn- chronized follicular activity, resulting in the emergence of a pre- ovulatory follicle 6 days after treatment.93 However, in another study, sponges containing 120, 240, or 480 mg MAP had no inhibitory effect on follicular development.94 Treatment with CIDRs (0.33 mg progesterone) for 16 days reduced follicular diameter starting on day 5 of the treatment.95 Similar results were obtained in our laboratory in llamas and alpacas with a 14-day treatment.54 Intravaginal devices containing 0.5 mg of progester- one appears to provide better control of follicular activity and bet- ter response to superovulation with eCG. The shape and area of contact of the vaginal device for progesterone delivery may affect the absorption of progesterone.54,96 Vaginal devices containing 0.78 g progesterone (Cue-mate®) inserted for 7 days reduced fol- licular development. A new dominant follicle was available 6 days after the removal of the device.97 Daily IM injections of progester- one (50 mg) for 12 days inhibited follicular growth by day 7.92 In vicuñas, treatment with CIDRs for 5 days exerted a negative effect on follicular development and allowed a better super- stimulation in response to eCG.98 In summary, progesterone therapy in camelids reduces the growth of large follicles and inhibits LH release,3 but does not Figure 3. Camelid cycle in presence of an ovulation Figure 4. Examples of camelid follicular wave synchronization using GnRH and PGF2α http://dx.doi.org/10.58292/CT.v17.11652 Citation: Clinical Theriogenology 2025, 17, 11652, http://dx.doi.org/10.58292/CT.v17.11652 41 completely suppress follicular activity; therefore, its use for synchronization of follicular wave emergence and timed-breed- ing remains questionable.6,54,63,83,87 Combination of progesterone and estrogens The combination of estradiol and progesterone is more effec- tive in controlling follicular waves in some studies99 but not in others.84 In llamas, superstimulation at the end of a 5-day daily treatment with 100 or 150 mg progesterone after injection of estradiol benzoate (1 mg) resulted in a higher embryo recovery rate.100 In another study, 1 injection of estradiol-17ß (1 mg) and progesterone (25 mg) provided some synchronization of follicular waves, but not as good as that accomplished by the induction of ovulation or by follicle aspiration.69 In alpacas, daily estradiol benzoate (5 mg) and progesterone (50 mg) treatment for 7-10 days produced a more uniform follicular wave response; however, the ovulatory response was poor.101 In camels, 1 injection of estradiol benzoate (5 mg) and pro- gesterone (100 mg) was ineffective for synchronizing follicu- lar waves.68 Ovarian superstimulation In vivo and in vitro embryo production are important tech- nologies to multiply genetically superior females and have become common place in camels.27,102 Additionally, these technologies are critical for the reproductive management and multiplication of endangered wild camelids using interspecies embryo transfer.73,103 Ovarian superstimulation is a crucial step for in vivo production of embryos and oocyte collection. Protocols for ovarian superstimulation used in camelids have been largely adapted from those used in ruminants with vari- able success (camels;27,54,85,87,90,104 SAC65,84,105). The 2 main hormones used are FSH and eCG, either alone or in combination. As for other species, response to these hor- mones depends on the timing of treatment in relationship to follicular dynamics, dose, frequency of treatment, and indi- vidual animal variation. Although FSH and eCG treatments have been initiated during the receptive or luteal phase of the cycle with some success, the best results in camels are obtained when the treatment is initiated in the absence of any follicles > 3 mm in diameter (camel).27 Follicle stimulating hormone Ovine (oFSH) and porcine (pFSH) FSH have been used for ovarian superstimulation with variable success.6,27 Induction with camel FSH (cFSH), purified from camel pituitary extract, or equine pituitary extract, was not successful in the authors’ experience. The manner of FSH treatment (dose, frequency, and timing during the cycle) has been investigated to some degree; however, detailed descriptions of the treatment proto- cols are not always provided in publications. In the dromedary, a total dose of 20-30 mg of oFSH was given in 2 daily injections of decreasing doses over 6 days. The treatment starts 2 days before and continues up to 1 day after the comple- tion of 7 days course of progesterone treatment by intravaginal device (PRID).85 FSH was also given in a single dose (3.3 units) followed the next day by 1 injection of 3,500 IU of eCG, resulting in an average of 7 embryos recovered per treated female.87 In another study, oFSH was given twice daily (1-3 mg per injection) for 3-5 days after 10-15 days course of progesterone treatment (100 mg per day).90 Single SC dose of oFSH has been tested with variable (5.7 ± 2.32 embryos recovered) results.106 In the dromedary, ovarian superstimulation has been obtained by twice daily eFSH treatment in decreasing doses over 3, 5, or 7 days after 10-15 days progesterone treatment. (dromedary27,90,106–109; Bactrian73,80). In practice, superovulation protocols are modified on an individual basis, based on ultrasonographic monitoring of the follicular response. The interval from pFSH treatment to the development of mature follicles (10-16 mm in diameter) varies between 6 and 8 days (Figure 5).6,27,54,63 The number of FSH injec- tions can be reduced using a slow-release FSH preparation (hyal- uronan solution) resulting in the same superovulatory response and embryo collection rate.109 A single epidural dose of FSH resulted in multiple ovulations in camels; however, the response was lower than for traditional protocols using multiple injections.110 A recent study reported an adequate embryo recovery response in dromedaries superovulated with a single dose of recombi- nant bovine FSH given IM (5.1 ± 3.6 embryos flushed per donor for 120 µg FSH and 5.0 ± 2.9 for 100 µg FSH) given 4 days after ovulation.111 In llamas and alpacas, ovarian superstimulation with pFSH alone or in combination with eCG has been used after 12 days of pro- gesterone treatment.84,112,113 The best results were obtained with pFSH treatment (IM) given twice daily for 5 days in decreasing doses (32, 27, 22, 17, and 12 mg).84,113 Superovulation was obtained in 95% of the alpaca cycles after treatment with FSH (decreasing doses) starting 2 days after induction of ovulation.114 The number of embryos (4.2 ± 3.3) obtained after pFSH stimula- tion is generally lower compared to the number of ovulations (10 ± 4.4).114,115 Generally, alpacas produce a more variable response to superstimulation protocols than llamas.84 Equine chorionic gonadotropin Ovarian superstimulation with eCG has been extensively used in camelids. In general, a single dose is given IM a day before or on the day of completion of progesterone treatment for 5-15 days. The dose of eCG used varies from 1,000 to 6,000 IU in camels,27,75,80,85,90,104,106,107,110,116,117 from 500 to 2,000 IU in lla- mas,72,84,85,93,96,99,100,113,118,119 and from 500 to 750 IU in alpacas84,113 and vicuñas.93 In the dromedary, eCG is given as a single injection (2,000 IU, 2,500 IU, 3,000 IU, or 4,000 IU), 1 day before or 1 day after PRID removal, resulted in superovulation in 40% of treated animals. However, only 42% of ovulating females yielded > 1 embryo. The interval from PRID removal to mating was 5 and 4.5 days for females receiving 2,500 IU and 4,000 IU of eCG, respectively. This interval was 1 day shorter in females treated with eCG 1 day before the removal of PRID.85,87 When eCG (2,000-3,000 IU) is given to females at the beginning of a fol- licular wave (no follicle > 3 mm in diameter), the interval from treatment to mating (follicular diameter of 12 mm) is relatively constant (8 days).27,120 Follicular response is variable (0-19 follicles) and 20% of the females did not respond to eCG treatment.27 Treatment of 2,500 IU eCG after a CIDR http://dx.doi.org/10.58292/CT.v17.11652 42 Citation: Clinical Theriogenology 2025, 17, 11652, http://dx.doi.org/10.58292/CT.v17.11652 (1.38 g progesterone for 3 days) produced superstimulation during the nonbreeding and transitional seasons, with folli- cles reaching ovulatory size 12-13 days after treatment.116,117 Treatment with eCG is on 2-4 days after induction of ovula- tion. The ovulation and embryo recovery rates are improved with doses ranging 3,000-4,000 IU. Higher doses (5,000- 6,000 IU) of eCG produced a larger number of follicles; how- ever, the ovulation rate and the number of transferrable embryos collected were lower.109 A small number of animals had better response and fewer anovulatory follicles when 2,500 IU eCG was given at the end of a 13-day progesterone treatment in decreasing doses over 3 days compared to 1 dose.116 For the decreasing dose regimen, 2,500 IU eCG was splitted in 6 decreasing doses at 12-hour interval over 3 days as follows: 1,250, 600, 300, 200, 100, and 50 IU, respectively. In llamas, eCG (1,000 IU) is given after progesterone priming (luteal phase after ovulation induction with hCG or GnRH, CIDR, or SC implants).72,96,119,121 Follicular response is variable (0-13 follicles), and the number of ovulations ranged from 0-7 with a mean of 1.3, and an average of 2.3 embryos col- lected per donor (range: 0-6). Follicles reached the mature size (9-13 mm) 5-11 days after eCG treatment. Several treated females had premature luteinization 7-9 days after eCG treat- ment. Increasing the dose of eCG to 2,000 IU increased the incidence of anovulatory follicles.54 Supplementation with exogenous progesterone during the eCG treatment in the luteal phase appears to inhibit excessive follicular growth and improve ovulation rate and embryo quality.121 Superstimulation with eCG and timed mating can be performed after synchro- nization with GnRH and PGF2α (Figure 6).122 As eCG is not available in the USA, the use of PG600 can result in ovarian superstimulation in alpacas but the ovulation rate was low.123 In alpacas, the average number of recovered embryos per ovu- lating female was 3.7 after superovulation with 750 IU of eCG daily for 3 days.84,112 The main disadvantage of eCG is the high incidence of follic- ular luteinization and failure of ovulation, most likely due to its long half-life. Additionally, females tend to become refrac- tory to eCG after multiple use. This suggests that there is a risk of inducing anti-eCG antibodies.27,63 Combination of FSH and eCG Ovarian superstimulation protocols combining FSH and eCG have been published in alpacas, llamas,84,105,118 vicunas,32 and camels.27,80,110,124 These protocols consist of giving a Figure 5. Examples of FSH ovarian superstimulation protocols Figure 6. Example of eCG ovarian superstimulation protocol after induction of ovulation http://dx.doi.org/10.58292/CT.v17.11652 Citation: Clinical Theriogenology 2025, 17, 11652, http://dx.doi.org/10.58292/CT.v17.11652 43 single dose of eCG followed by FSH injection twice daily in decreasing doses.124,125 Generally, these protocols do not pro- vide much superiority compared to protocols using FSH alone.123 Human menopausal gonadotropin Human menopausal gonadotropin (hMG) is a hormone with equal FSH and LH activity. Its use for superovulation in came- lids has been limited. Although hMG produced superovula- tion in camels, ovulation rate (FSH: 22.4 ± 2.25; eCG + FSH: 11.6 ± 2.58; hMG: 7 ± 3.19) and embryo yield (FSH: 16.2 ± 2.72; eCG + FSH: 7.2 ± 3.1; hMG: 1.6 ± 1.17) were less predict- able and lower than those obtained with superovulation pro- tocols based on FSH or FSH + eCG.106,107 Immunization against inhibin Immunization against inhibin, inhibin subunits or recombi- nant DNA vaccines result in high concentrations of circulating FSH, and consequently, an increase in the number of recruited and mature follicles. The vaccination was followed by a booster 28-231 days later, depending on the species and study. Immunization against inhibin has been used successfully to improve ovarian superovulation in several species (cattle,126,127 horses,128 goats,129 sheep130). In the dromedary, a trial on immunization against inhibin gave encouraging results. An increase in ovulation number (4-10) was observed in 60% of the immunized females.63 Further studies reported a high rate  of triple ovulations up to 5 months after the initial immunization.131,132 Problems with superovulation in camelidae Major problems encountered in superovulation of camelids are the high incidence of non-responsive females (20-30%), high incidence of follicular luteinization, overstimulation in some females, and loss of efficacy after multiple treatments. Additionally, ovulation response and embryo yield remain highly variable.6,27,65,84 The recovery rate (number of embryos recovered/number of corpora lutea) is also variable, ranging from 30-90%. Initiation of gonadotropin treatment 2 days after induction of ovulation or after a progesterone treatment results in the best superovulation and embryo recovery out- comes (Figure 6). Failure of ovulation may be due to premature regression of follicles and may be associated with inappropriate FSH dose or method of delivery.74 Luteinization of follicles before ovulation may be due to increased concentrations of LH in response to high serum estradiol concentrations. Preliminary trials in our laboratory using recombinant  highly purified FSH are encouraging. Most females with overstimulated ovaries do not produce embryos, possibly because of alter- ation of gamete transport. Finally, some females become refractory to superovulation with either FSH or eCG, per- haps because of the development of antibodies against these hormones. Sources of variations in the response to superovulation in camelids that need to be investigated include species, breed, and individual animal. In alpacas, ovarian superstimulation response is positively correlated to serum AntiMüllerian hor- mone concentrations.133 A similar trend was reported recently in dromedary camels.134 Conclusion Research on camelid reproductive endocrinology and clinical monitoring of the reproductive function in female camelids led to the adaptation of several hormonal strategies to alter this function positively or negatively. Knowledge gained on factors governing seasonality in camels, in particular photope- riod, allowed the use of artificial photoperiod or melatonin treatments to extend the breeding season in camels. Multiple approaches to synchronize follicular waves have been adapted from other species, with variable results. Progesterone therapy alone or in combination with estradiol has some efficacy for the control of follicular waves; however, it is still not optimal for timed-AI. Synchronization based on induction of ovula- tion followed by natural or induced luteolysis provides better synchronization of follicular dynamics and allows optimiza- tion of ovarian superstimulation protocols. Ovarian super- stimulation treatments were adapted mainly from ruminant protocols. However, these treatments are still far from being optimized and need to be adjusted on an individual animal basis. Factors affecting ovarian response to superstimulation treatment remain poorly studied; however, recent observa- tions indicate that serum AntiMüllerian hormone concentra- tions could be used to predict this response. Conflict of interest Authors declare no conflict of interest. References 1. Anouassi A: Hormones antéhypophysaires du dromadaire (Camelus dromedarius): purificataion, caracterisation, dosages et utilisation pour l’idnuction de la superovulation. PhD Thesis, Université Paris VI, France, 1999. 2. 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