Alpaca Female Reproduction | Alpaca Breeding Technologies

Alpaca Female Reproductive Physiology

Jorge Reyna

MSc (Hons), MscVetSC (Sydney Univ.)

Lecturer in Higher Education - Learning Design

Faculty of Science

University of Technology Sydney

< Back to Library

INTRODUCTION

Female reproductive physiology in alpacas has not been studied as extensively as in other domestic species. Lack of research in this area has been due to limited resources to conduct experiments in Peru, and also socio-political reasons like the terrorism of the 1980s which destroyed research stations, killed genetically improved herds and stopped further research on South American Camelids (SAC) in the Andes. Also, the priority for the government was to develop other breeding livestock like cattle and sheep, due to a poor understanding of the real importance of SAC breeding for more than 500,000 families who live in the Andes under conditions of extreme poverty.

Endocrinology in alpacas and llamas is poorly understand and there is a need to conduct basic experiments on hormonal profiles in prepubertal, pubertal and adult animals, to elucidate if the endocrine mechanism follows the patterns described in other domestic species.

Early studies in female reproductive physiology were conducted in the late 1960s and early 1970s in the areas of puberty, mating stimuli and ovulation, ovarian stimulation and corpus luteum function by Fernandez-Baca, Novoa and San Martin from San Marcos National University (UNMSM) in Peru. In the middle of the 1970s some research was conducted on ovarian dynamics using laparoscopy, and later on transrectal ultrasound as a non-invasive technique. Other studies were also conducted in the area of sperm collection, evaluation and artificial insemination. In the 1990s, some research was conducted on alpacas and llamas measuring hormones like oestradiol and progesterone during pregnancy, and luteinising hormone (LH) release during the copula stimuli. In the late 1990s, research was focused on superovulatory treatments and control of ovarian follicular waves (for some reason more in llamas than alpacas).

Recently a study on follicular waves in alpacas has been carried out in Australia by Vaughan et al. (2002) which confirms the occurrence of follicular waves in alpacas as postulated by Bravo et al. (1976). The later study points to the need for further research into efficiently controlling follicular waves in order to obtain better fertility rates upon natural mating, artificial insemination and also in embryo transfer protocols.

There are still important areas of research to be done on female reproductive physiology in alpacas. This paper reviews the knowledge up to now on alpaca female reproductive physiology in the areas of puberty, follicular waves, sexual receptivity and mating behaviour, ovulation, and pregnancy. It also describes briefly the current knowledge on the control of ovarian activity with hormonal treatments. Additionally, the paper discusses the need for research in the future to generate useful information that will improve fertility via the application of reproductive biotechnologies in alpacas.

PUBERTY

The age of onset of puberty in alpacas remains undetermined. It has been reported that puberty occurs between 10 and 24 months of age (Novoa M 1981; Sumar 1985; Bravo and Sumar 1989; Fernandez-Baca 1993; Pollard et al. 1995). There is evidence in alpacas, as in other domestic species, that puberty is associated with body weight. It has been established in the past that when the female reaches 40 kg she is ready for the first mating. (Novoa, Fernandez Baca et al. 1972). A relationship has been found between live weight, mating and subsequent birth rates. For each kilogram increase in live weight up to 33 kg there was a 5% increase in birth rates. In the case of animals weighing more than 33 kg this relationship was not found (Leyva et al., 1981). This is directly related to nutrition, with better nutritional status leading to faster growth rates (Elwishy 1988; Smith, Peter et al. 1994).

Under conditions prevailing in the Andes, alpacas are bred at 2 years of age when they reach 40 kg, although pregnant females have been observed at 6 months of age. This is indicative of ovarian functionality but is not recommended, as it will affect future growth of the female and could cause dystocia (Aba 1995). Regrettably there are no studies available on endocrinological changes during prepubertal to pubertal periods in alpacas. It has been reported in SAC that the time at which follicles acquire the ability to synthesise oestrogen may be as early as 5-6 months of age, by measuring the urine oestrone (Bravo et al. 1991). Using transrectal ultrasonography, no difference in terms of ovarian follicle populations has been found in females from 11 to 18 months of age in comparison with 3 year old adult females (Bravo and Sumar 1989).

OVARIAN FOLLICULAR WAVES CHARACTERISTICS

All camelids are considered induced ovulators which means that the copula stimuli are necessary to activate the GnRH release and then the LH surge occurs and ovulation occurs (Sumar, 1996). As in camels (Skidmore 2004) and llamas (Adams, Griffin et al. 1989), follicular growth occurs in waves in alpacas (Bravo and Sumar 1989; Vaughan, Macmillan et al. 2004). A new wave emergence in alpacas is characterised by the appearance of 8 to 10 follicles (Figure 1) (<3 mm) followed by continued growth of usually one follicle. This follicle will become dominant later on and the rest of the subordinate follicles will regress (Vaughan, Macmillan et al. 2004). This is a hormonal mechanism in which two hormones are responsible for the regression of the subordinate follicles: oestradiol and Inhibin, as described before in the cow and ewe (Reyna 2005). At present there are no studies which have been conducted on alpacas studying follicular dominance by measuring these hormones.

As induced ovulators, female alpacas undergo repeated follicular waves with growth and regression of dominant and subordinate follicles and with lack of ovulation in both breeding and non-breeding seasons, in the absence of the mating stimuli. If the appropriate mating stimuli is present, the formation of a corpus luteum (CL) will occur (Bravo 1993). It has been

Figure 1: Ultrasound image of an alpaca ovary. Follicles appear as black circular structures surrounded by echogenic ovarian tissue, due to fluid absorbing rather than reflecting ultrasound waves.


Alpaca follicles


reported that 3-4 % of female alpacas are able to spontaneously ovulate, and this phenomenon has been associated with the presence of the male, as with the “ram effect” observed in the ewe.

The period of time between follicular waves is called the inter-wave interval. This has been reported in alpacas to be 11-12 days, measured by laparoscopic examinations (Bravo and Sumar 1989), and 12-16 days in 71% and 18-22 days in 29% of the females respectively, measured by transrectal ultrasonography (Vaughan et al. 2004). The growth period of the follicles in alpacas has been estimated to be 3-5 days, the static period to be 4 days and the regression period to be 4 days as well, in the case of follicular waves of 12 days length (Bravo and Sumar 1989). Emergence of the next successive dominant follicle occurred in the ipsilateral ovary in 60%, and in the contralateral ovary in 40%, of cases (Vaughan, Macmillan et al. 2004). The growth rate of ovarian follicles reported between days 0 and 10 was 0.43 ± 0.02 mm/day.

In the case of llamas, the same effect of the dominant follicle suppressing the growth of the subordinate follicles has been described. The inter-wave interval reported in this case was 11 days (8 to 14) corresponding to 5 days of growth, 5 days of stasis and 4 days of regression, respectively. The development of the subsequent dominant follicle usually began within 2-3 days after the onset of regression of the dominant follicle. This development tended to alternate between ovaries, as described in alpacas (Bravo, Fowler et al. 1990).


Taking into account follicular waves intervals, it is possible to postulate that there may be an optimum time for mating which can be defined as the time when the dominant follicle reaches a mature stage and the oocyte is capable of being fertilised. In this regard, it has been postulated that this time could be between days 6 to 8 after a wave emergence, based on follicle diameter (Vaughan, Macmillan et al. 2004). A field trial which considers oocyte quality along the follicular wave, to confirm if this optimum time can be predicted with more accuracy, would be interesting. This is a crucial area of research because many of the poor responses to AI may be related to different oocyte stages like the immature or regression stages which, even with the capacity to be fertilised, could have low viability upon fertilisation.

A protocol for inducing follicular waves in alpacas needs to be developed, perhaps testing progesterone/progestagens and thereby determining the possibility of synchronising a new follicular wave and predicting the stage of development of the ovulatory follicle, in order to induce ovulation. This may guarantee better fertility rates using AI, and may give a better yield of embryos in ET programs.

In summary, follicular waves occur in alpacas as in other domestic species like cows and ewes, the main difference being that ovulation takes place after the mating stimuli. In other words, if there is no mating there is no luteal phase in the cycle, with the exception of some spontaneous ovulations.

SEXUAL RECEPTIVITY AND MATING BEHAVIOR

In camelids, oestrus is not a cyclic, repeatable and predictable behaviour. In the case of alpacas there is a large individual variability in signs and length of oestrus (Sumar K 1993). The variability of oestrus length could presumably reflect the fact that in non-mated females the follicular phase does not end with ovulation and there is no luteal phase (Bravo, Fowler et al. 1990). It is possible to find females with different stages of development of the dominant follicle, and thus oestradiol concentrations may be variable, producing heterogenous oestrous behaviour in the herd. As was mentioned before, ovarian follicular waves in alpacas overlap, ensuring the adequate production of oestradiol from the dominant follicle at all times. Receptivity is usually maintained, but females exhibit brief periods of non-receptivity when no mature follicles are present (San Martin, Copaira et al. 1968; Bravo and Sumar 1989). It is important to mention that receptivity is not a guarantee of a well developed follicle able to ovulate and continue with fertilisation.

Few or none of the external signs of oestrus in the other domestic species are present in alpacas. Receptive females have a typical behaviour in the presence of the male. When a female is receptive and the male is in courtship mode she quickly sits down, assumes the posture known as sternal recumbency and exhibits passive behaviour. Some receptive females, when they are near a couple mating, sit down near them and frequently smell the male (Sumar K 1993). If the female is non-receptive (mated females or with the presence of a corpus luteum) she will reject the male, spitting or threatening to spit, orientating and elevating her head towards the male, with ears held back, kicking and vocalising or emitting a loud, high-pitched squeal and attempting to escape (Pollard, Littlejohn et al. 1995). It has been reported in Peru during the breeding season (December to March) that females sometimes mate other females, as observed in cows (Huanca 1993). This phenomenon coincides with apparently better follicular development and oestradiol production during this time of the year (Bravo and Sumar 1989).

When a male chases a receptive female, he will press his legs onto her back trying to force her to adopt the sternal recumbency position (Figure 2). Copula takes 20 minutes average (5 to 50 minutes). In some cases when copulation is prolonged, the female lies on her side while the male is mating. It has been observed as well that the female raises her neck and faces the male, and it has been postulated that this coincides with the passing of the penis through the cervix and the intra-uterine deposit of sperm.

Time taken to adopt sternal recumbency is not a reliable indicator of either plasma oestradiol concentration or maximum ovarian follicle diameter. Females can adopt the copula position with follicles ranging from <3 to <13 mm and plasma oestradiol concentration from 0.32 to 9.96 pg/mL (Vaughan, Macmillan et al. 2003). In contrast, other authors reported a relationship between ovarian follicular growth and behavioural receptivity in alpacas and llamas (Bravo and Sumar 1989; Adams, Sumar et al. 1990). On the other hand, sitting has been considered to indicate that the female is receptive to

Figure 2: A male chasing a receptive female getting ready to adopt sternal recumbency.

Alpaca male chasing female


the male, but is also exhibited by alpacas under stressful conditions (Fowler, 1989). Sitting to allow mating could be confused with sitting in response to the stress of being chased by a male (Pollard, Littlejohn et al. 1994). The variability in sexual behaviour of female alpacas has been attributed to environmental differences, degree of domestication and social structure of the herd (Novoa M 1981).

The effect of season on sexual receptivity in alpacas has been reported to influence, but not limit, the sexual activity of the females. Studies conducted in Peru reported an increase in sexual receptivity from December to March, corresponding to the rainy season (Huanca 1993). Follicular activity examined by laparoscopy tended to be slightly lower during the non-breeding season (August to September) (Bravo and Sumar 1989). Under New Zealand conditions, it has been reported that females were less receptive in spring than in autumn (Pollard, Littlejohn et al. 1995).

In summary, the reproductive status of the female can be related to her behaviour in the presence of the male, particularly spitting and attempting to escape from the male. This behaviour may indicate that ovulation has taken place and the presence of a corpus luteum secreting progesterone. A sitting position might not necessarily indicate receptivity: as we mentioned before, it might be a sign of stress.

OVULATION

Induced ovulators

Alpacas and the other camelids belong to the group of animals called “induced ovulators” (Sumar K 1993). This means that the female does not ovulate until she has been mated by the male. The mating stimulates the release of the LH surge within 15 minutes after copulation, and ovulation will occur 30 h later in llamas (Bravo, Fowler et al. 1990). One of the first studies conducted in Peru showed that the type of stimulus modulates the response of the females. The percentage of females which ovulated upon mating with a male which had a device to prevent penis intromission, or upon mating by another female, was low. On the other hand, stimulation by a male without penile intromission and subsequent artificial insemination resulted in a significant increase in ovulation, in up to around 33% of the females tested. These results were explained by the stimulus of the vagina during artificial insemination. Copulation with a vasectomised male increased the percentage of females ovulating (77%). These results were explained by the stimulus of the vagina during artificial insemination. Interrupted copula had no effect on the percentage of females ovulating, which means there is no relationship between copulation length and ovulation. Multiple copulation also had no effect on ovulation (Fernandez Baca, Madden et al. 1970).

On the other hand, is has been reported that alpacas and llamas may ovulate spontaneously when they are isolated from males and are in contact with males for a few minutes for oestrus detection. These ovulations have been attributed to the presence of the male (‘the male effect') which involves pheromones (Leyva V and Sumar K 1987; Adams, Sumar et al. 1990).

In summary, alpacas are induced ovulators as they need the copula stimulus to release the LH surge and then to ovulate. On the other hand, they are capable of ovulating spontaneously as a response to the “male effect”. The nature of induced ovulation makes the application of artificial breeding techniques like artificial insemination difficult if it is not possible to predict the time of ovulation with accuracy, especially when frozen-thawed sperm is used, due to its short lifespan in the uterus.

Ovulation induction factor

The presence of an “ovulation induction factor” has been described in alpacas by injecting 0.8 to 1.0 ml of seminal plasma into female alpacas, where upon ovulation was achieved in 60% of cases (Sapana, Huanca et al. 2002). Studies conducted in vitro demonstrated that the seminal plasma of adult alpacas can induce rat hypophyseal gonadotrophic cells to secrete LH, but dilution (1:2 and 1:4) did not significantly increase LH secretion. Also, the addition of anti-GnRH antibodies did not modify the response of the cells to the stimulus of seminal plasma. This suggested that the stimulating effect could be mediated by factor(s) chemically different from GnRH (Paolicchi, Urquieta et al. 1999).

In Bactrian camels, a species phylogenetically related to SAC, the presence in the seminal plasma of a factor expressing GnRH-like biological activity which can stimulate the secretion of LH has been described (Xilong and Zhao 2002). Intramuscular injections with seminal plasma induce ovulation in camels via increase of LH and follicular stimulate hormone (FSH) in a similar manner to when ovulation is produced by natural mating (Paolicchi et al. 1999).

In summary, seminal plasma contains an ovulatory factor that improves the chances of the female ovulating when copula occurs, and could be an adaptive mechanism to the harsh conditions in the Andes to ensure ovulation, and thus fertilisation and pregnancy.

Follicle diameter and response to copulation

As was mentioned before, receptivity is not indicative of the presence of a mature follicle ready to ovulate, fertilise and continue with embryonic development. It has been described in both llamas and alpacas that the ovulatory release of LH depends on ovarian follicular diameter. Females with small follicles (4-5 mm) released less LH and did not ovulate, in comparison with animals which presented growing, mature or regressing follicles. This finding could be explained by the reduced gonadotrophin-related oestrogen priming of the hypothalamus and pituitary. On the other hand, copulation of females with regressing follicles provoked release of LH similar to that observed in animals with growing or mature follicles, but luteinisation took place instead of ovulation. This fact could indicate that regressing follicles have lost the capability to secrete factors like enzymes which are necessary for the rupture of the follicle (Bravo, Stabenfeldt et al. 1991).

The application of this knowledge is very important when reproductive technologies like artificial insemination and embryo transfer are involved. If the ovulation-induced female has small or regressing follicles, she may not be able to ovulate and to form an appropriate corpus luteum. This may cause low fertility rates upon application of reproductive techniques. Synchronising follicular waves to establish follicular status is necessary to ensure better fertility rates.

Time of ovulation

The female alpaca ovulates 24 h after copulation (San Martin, Copaira et al. 1968; Fernandez Baca, Madden et al. 1970), although different intervals have been reported in the past - from 24 to 72 h (Sumar 1985; Sumar K, Bravo et al. 1993). The size of the ovulatory follicle has been reported to be between 6-8 mm (Figure 3 and Figure 4). In the case of llamas, an interval from 24 to 96 h has been reported (Adams, Griffin et al. 1989; Adams, Sumar et al. 1990; Bravo, Fowler et al. 1990; Sumar K 1993; Aba, Forsberg et al. 1995; Bourke, Kyle et al. 1995; Ratto, Huanca et al. 2005).

There are many factors which have been described in other species like sheep and cattle which may affect the time of ovulation. The factors which have been found to modify this interval are: season, age, breed, nutrition, lactation, ram

Figure 3: Ovulatory follicle in alpaca around 6 mm, captured by transrectal ultrasound.


Fig 3

Figure 4: Ovulatory follicle in an adult 23 year old alpaca around 9 mm. the follicle contains fluid reach in oestradiol.

alpaca ovary

exposure, the kind of progestagen and the dose of exogenous gonadotrophin used in oestrus synchronisation protocols (Colas, 1979; Robinson, 1979; Romano, 1996), and differences between flocks (Walker et al., 1988). These factors affect the efficacy of both fixed time artificial insemination and embryo transfer programs (Walker et al., 1989; Romano et al., 1998). Regrettably, in the case of SAC there are no studies that evaluated the effect of these variables on the time of ovulation. Research has been limited to GnRH and hCG analogue applications which we will discuss later on in this review. It will be extremely important to conduct a study on alpacas to determine whether these factors are significant, because they could provide alternatives which improve fertility upon application of artificial breeding techniques.

Ova transport

Transport of ova from the oviducts to the uterus in alpacas is similar to that reported in other species (6-7 days) like ewes (Rowson and Moor, 1966); cows (Rowson et al., 1969); pigs (Hunter, 1974); and mares (Oguri and Tsutsumi, 1972). Development of embryos in alpacas after fertilisation also appears to be similar to that observed in other livestock species. Four days after copulation it is possible to collect embryos from the oviducts which are at the morulae stage with 4-8 blastomeres. By day 7, embryos collected from the left oviduct were compact morulae and it was not possible to count the number of cells (Figure 5). By day 10, embryos were collected from the left uterine horn as blastocysts (Bravo, Moscoso et al. 1996). This knowledge is very important in ET programs in order to flush the females at the right time.

Figure 5: Alpaca hatched blastocyst flushed from a donor at day 7 under stereoscopy. Embryos are visible to the eye and look like white-milky spheric structures.

alpaca embryos




Corpus Luteum function

The corpus luteum originated by natural mating or HCG application in the alpaca attains a maximum diameter at day 8-9 in the absence of pregnancy (Figure 6). Morphological regression changes are measurable as early as day 12, but secretory changes are more dramatic than morphological changes. A sharp decline in progesterone secretion by the corpus luteum is observed at day 13 and complete regression at day 15. This indicates a short lifespan of the corpus luteum in alpacas in comparison with other domestic species. Factors involved in this early regression are unknown. A possible explanation for it could be that the mechanism controlling luteal maintenance and regression in alpacas differs from those in other species.

In the case of pregnant females, after day 8 post copula no significant differences in corpus luteum size and progesterone levels have been found, with the exception of a transient decline in weight and progesterone production on day 13. This may suggest that once the maximum development is reached, the embryo prolongs the functional life of the corpus luteum without marked increase in size, total mass or secretory profile. The transient decline may suggest that this stage represents the critical period of zygote survival (Fernandez Baca, Hansel et al. 1970).

Additionally, it was reported that corpus luteum located in the right ovary regress more rapidly than those in the left ovary. This suggests that this differential regression may influence embryo survival and may explain the

Figure 6: Ultrasonographic image of corpus luteum. The structures had a hyperechogenic shape, similar to the ovarian parenchyma but slightly less even. On the right, a 5 and 3 mm follicles.

alpaca CL

high rate of migration of embryos from the right to the left uterine horns in alpacas (Fernandez Baca, Sumar et al. 1973). This regression is under the influence of the uterus, as partial hysterectomy extends the lifespan and secretory activity of the corpus luteum ipsilateral to the missing horn (Fernandez-Baca, Hansel et al. 1979).

There seems to be a different luteolytic effect of the right and left uterine horn in alpacas. In females with a corpus luteum on the left ovary, absence of the ipsilateral uterine horn extends the luteal function, evidenced by high progesterone levels, large corpus luteum size and lack of sexual receptivity for a period of up to 70 days. Contrarily, removal of the right uterine horn in females with a functional corpus luteum on the right ovary causes a slight delay in luteal regression. In females with a corpus luteum on each ovary, removal of the left uterine horn results in regression of the corpus luteum on the right ovary and persistence of the corpus luteum on the left ovary. Removal of the right uterine horn results in regression of the corpus luteum in both ovaries. This finding indicates that the luteolytic effect of the left uterine horn is local and systemic while the luteolytic effect of the right uterine horn is local. This may explain why corpus luteum located on the right ovary regress more rapidly than those located on the left ovary (Fernandez-Baca et al. 1979).

In summary, corpus luteum lifespan in alpacas is shorter than in other domestic species, and the regression is under the influence of the uterus. Additionally, there is a differential luteolytic effect of the right and left uterine horns, being local for the right and local and systemic for the left. The reason for this difference is unknown.

Pregnancy

Length of gestation in alpacas of the Suri and Huacaya breeds has been reported to be 345 and 341 days, respectively. In almost all cases, alpacas foetuses occupy the left uterine horn, even when ovulation takes place from both ovaries without statistical differences. This indicates that embryos originating from the right side migrate to the left side. The reason for this is unknown, but it could possibly be explained by the differential luteolytic effect of the right and left uterine horn discussed above. The corpus luteum is necessary to maintain pregnancy in alpacas during the whole gestational period (Sumar 1985).

Parturition

Labour under conditions prevailing in the Andes in Peru higher than 4,000 m above sea level lasted 200 and 190 minutes for primiparous and multiparous females, respectively. More than 90% of births occur between 07:00 and 13:00 hours and this has been considered an adaptive mechanism that gives the newborn a chance to get warm and dry before the cold of the night.

Puerperium

Up to the fourth day after parturition the female alpaca is submissive and will be receptive to being mounted by the male. However, luteal regression, follicular growth and uterine involution are not complete and the female will not become pregnant from such early mating. Mating of alpaca females is recommended within 15 to 20 days after giving birth to obtain good fertility rates and one “cria” per year.

Control of the ovarian activity with hormonal treatments

For some reason, most of the research conducted in this area has been limited to llamas. This information needs to be used carefully as, although llamas and alpacas belong to the same family (Camelidae), it has been found that the llama descends from the guanaco and the alpaca from the vicuña. We may expect to find similarities in reproductive endocrinology, but further studies need to be conducted to confirm if both species present the same endocrine patterns.

Induction of ovulation

The importance of a reliable technique to induce ovulation in alpacas is critical for successful development of fixed-time insemination protocols and for recipient synchronisation in embryo transfer protocols. Moreover, considering the peculiar characteristics of alpaca sperm, which is low in concentration and motility and difficult to freeze without considerable damage, induction of ovulation is a key part of the process to synchronise the deposit of the sperm in the female reproductive tract with ovulation. This provides the opportunity to use low sperm concentration per female inseminated and the possibility of maximising the use of an ejaculate from a particularly valuable male.

One of the first successful experiments inducing ovulation in alpacas was with the use of 750 IU of hCG. The time of ovulation reported after the application was 24 h, however the method of detection was necropsy and this limited characterisation of the mean interval and distribution of ovulations (San Martin, Copaira et al. 1968). Later the same dose of hCG was used successfully to induce ovulation in females for artificial insemination (Bravo, Flores et al. 1996). In llamas, a study using transrectal ultrasound to follow up ovulation reported an interval of 27 and 28 h after 750 IU of hCG and 8 ug of GnRH analogue treatment, respectively (Adam, Bourke et al. 1992).

Gonadotrophin hormones (Sumar 1985; Ratto, Singh et al. 2003) and GnRh analogues (Bourke, Kyle et al. 1995; Correa, Ratto et al. 1997; Bourke, Kyle et al. 2000; Aller, Rebuffi et al. 2002) have been used to induce ovulation in receptor llamas for embryo transfer programs. The time of ovulation varied from 24 to 36 h, according to dose and probably different environmental conditions like photoperiod, body condition, presence of mature follicles etc.

A comparative study on the time of ovulation has been conducted recently in llamas upon natural mating and upon the application of 5 mg of LH or 50ug of GnRH. Time of ovulation was 30, 29 and 29 h after natural mating, LH and GnRH application, respectively (Ratto, Huanca et al. 2005). A similar study on alpacas is necessary under Australian conditions to determine whether there are significant differences, also taking into account season, body condition, age and presence of the male.

In summary, treatments with both hCG and GnRH have been shown to be effective in inducing ovulation in alpacas, although further studies to elucidate the influence of other variables on the time of ovulation like season, nutrition and body condition, presence of the male etc; would be desirable.

Ovarian follicular wave synchronisation

Establishing a protocol to control follicular waves in alpacas will dramatically improve breeding management, reducing the time taken for detecting sexual receptivity, and will possibly increase fertility rates upon artificial insemination and increase the yield of embryos in superovulation programs. This protocol needs to ensure the presence of a healthy mature ovulatory follicle able to ovulate and be fertilised for AI, and probably the absence of a dominant follicle for superovulatory treatments.

A follicular wave in alpacas lasts 11-12 days and can last up to 22 days (Vaughan, Macmillan et al. 2004). This extended lifespan of the follicle causes atresia, which can be defined as a combination of biochemical, physiological and histological processes in the follicle leading to degenerative changes and loss of integrity/viability. These changes are associated with altered passage of nutritive substances from the plasma in the follicle and loss of receptors for various hormones (Scaramuzzi et al., 1993). Ovulatory follicles with a degree of atresia could be a cause of low fertility at natural mating or upon application of reproductive technologies like artificial insemination. There is no information in alpacas regarding how atresia affects the competence of the oocyte to be fertilised, and studies in this area could provide the key to this matter. Interesting techniques to study in this area could be ovum pick-up at different days during the follicular wave, as well as histological studies and in vitro fertilisation experiments.

In cattle, ovarian follicular wave synchronisation has been reported using hormonal treatments like GnRH, LH and oestradiol in combination with progestagens. The principle is removing the suppressive effects of the dominant follicle, either inducing its ovulation (GnRH, LH) or atresia (Oestradiol). After that a new follicular wave starts, followed by the development of a dominant follicle capable of ovulating at a predictable time.

In the case of llamas, progestagens have been used extensively to synchronise follicular development, with variable results (Aller and Alberio 1996; Aller, Ferre et al. 1997; Aba, Quiroga et al. 1999; Ferrer, Aguero et al. 2002; Ratto, Singh et al. 2003).The rational use of progestagens alone to synchronise follicular waves is unclear, as luteal phases are not characteristic of the reproductive cycle of induced ovulators (Adams et al. 1990). Treatments in llamas with Oestradiol + progesterone, LH, or Oestradiol + progesterone + LH, achieved the same ovulation rates as animals naturally mated, but the pregnancy rate was higher in treated animals (Ratto, Singh et al. 2003). Another study, using 0.33 g of progesterone (CIRD®) inserted for 16 days in llamas, reported an inhibition of the ovarian follicular activity regardless of the stage of follicular development at the time of insertion. In addition this study reported the lowest ovarian activity between days 5 and 7. This time could be appropriate to start a superovulation protocol, as there is then no dominant follicle and the new cohort of follicles that will emerge will have more chance of being viable (Chavez, Aba et al. 2002).

Using sponges containing 120 mg of medroxyprogesterone acetate (MAP) for a period of 9 days, and the application of GnRH at day 6 after removal, has been shown to be effective as a method of synchronising ovarian follicular waves in llamas (Aba, Quiroga et al. 1999). Nevertheless, this study monitored corpus luteum lifespan and progesterone concentrations only, and not fertility after the treatment.

Norgestomet implants (6 mg) for 14 days (winter) and 9 days (summer) + an injection (3 mg of Norgestomet + 5 mg of oestradiol valerate) at implant insertion + a dose of GnRH at implant removal, produced ovulations in 78% of llamas in both seasons (Aller, Ferre et al. 1997). Contrarily, another experiment using the same protocol produced the same percentage of females ovulating in comparison with natural mating (Aller, Ferre et al. 1997). The reason may be that the first report did not use a control group to make a comparison.

Injection of 50 mg/day of progesterone for 13 days efficiently synchronised a new follicular wave in llamas. Seven days after the last injection, all the females had follicles (>6 mm) susceptible to ovulation. After natural mating 85% of the females ovulated and presented a corpus luteum (Aller and Alberio 1996). No fertility rates were reported in this experiment.

In alpacas, a single injection of 1 mg of oestradiol alone, or combined with 100 mg of progesterone or 2-5 mg of oestradiol, did not induce a new follicular wave. Contrarily, treatments with 10 or 100 mg of progesterone at 12 h intervals over a period of 4 days, 25 mg of progesterone twice daily for 21 days or 25 mg of progesterone twice daily for 9 days + 2 mg of oestradiol, were effective in inducing a new follicular wave. The most practical protocol was the injection of 200 mg of progesterone on days 0, 2 and 4 of the cycle. Oocytes from females treated with progesterone were retrieved using ultrasound-guided transvaginal aspiration and it was concluded that morphology and capacity to respond to LH application was not affected by progesterone treatment. Field trials confirmed that the quality of the oocytes was not affected by progesterone treatment, but pregnancy rates were no different than in placebo-treated animals (Vaughan 2002).

In summary, several hormones have been tested with a view to synchronising follicular waves, mostly in llamas and one test in alpacas. These hormones include the use of progesterone, progestagen, oestradiol, and combinations of progestagens and oestradiol, and have been shown to be effective in inducing a new follicular wave, but no significant differences have been reported in comparison with untreated females. A comparative study that considers all these different hormone applications needs to be done using a large group of alpacas, to draw a conclusion.

An interesting approach to the use of synchronisation protocols could be in trying to control the presence of a dominant follicle prior to a superovulatory treatment. An improvement of the yield of embryos in cows and ewes when there is no dominant follicle present at the ovary during the superovulatory treatment has been reported. This finding has been explained by the fact that the dominant follicle produces oestradiol and inhibin, which affect the growth of the subordinate follicle. In the absence of a dominant follicle, follicles at the ovary have a better chance of responding to the superovulatory treatment.

The future

Female reproductive physiology in alpacas is still not well understood and further research needs to be conducted in several areas. Time of ovulation under different hormonal protocols in different seasons and locations needs to be studied to elucidate if these variables affect ovulation and to find the best way to synchronise the deposit of the sperm in the uterus with ovulation. Another important area of study is how nutrition and body condition may affect follicular waves, and to find if there is a relationship with embryo loss.

There is a need to develop a technique to predict when a mature follicle able to ovulate and facilitate fertilisation is present on the ovary. Follicular wave synchronisation with the use of progesterone/progestagens needs to be reviewed carefully, and probably a field trial with a large group of animals should be performed to validate its use. Finally, it is necessary to study the viability of oocytes at different developmental stages, perhaps with technologies like ovum pick-up. All this information generated will be useful for the application in these species of new reproductive techniques like artificial insemination, embryo transfer and in vitro production of embryos.

ACKNOWLEDGMENTS

The author would like to thank to Janie Hicks from Coolaroo Alpaca Stud and Dr. Jane Vaughan from Criagenesis for the opportunity to take photographs during a recent embryo transfer trial. Special thanks to Helen and Nestor Ellinopoullos from Orrapoora Alpacas to provide the alpaca reproductive tract. Special thanks to Mr. Peter Krockerberger for editorial assistance.
 

REFERENCES

Aba, M. A. (1995). Studies on the reproductive endocrinology of llamas and alpacas. faculty of Veterinary medicine. Uppsala -Sweden, Swedish University of Agricultural Sciences.

Aba, M. A., M. Forsberg, et al. (1995). "Endocrine changes after mating in pregnant and non-pregnant llamas and alpacas." Acta Veterinaria Scandinavica 36(4): 489-498.

Aba, M. A., M. Quiroga, et al. (1999). "Control of ovarian activity in llamas (Lama glama) with medroxiprogesterone acetate." Reproduction in Domestic Animals 34: 471-476.

Adam, C. L., D. Bourke, et al. (1992). Ovulation and embryonic recovery in the llama. 1st International camel conference.

Adams, G. P., P. G. Griffin, et al. (1989). "In situ morphologic dynamics of ovaries, uterus and cervix in llamas." Biology of Reproduction 41: 551-558.

Adams, G. P., J. Sumar, et al. (1990). "Effects of lactational and reproductive status on ovarian follicular waves in llamas (Lama glama)." Journal of Reproduction and Fertility 90(90): 535-545.

Aller, J. F. and R. Alberio (1996). "Dinamica folicular ovarica en llamas y su control con progesterona exogena." Revista Argentina de Produccion Animal 16(Sup 1): 69-70.

Aller, J. F., L. B. Ferre, et al. (1997). "Synchronisation of follicular wave in llama (Lama glama) in two seasons (winter and summer)." Revista Argentina de Produccion Animal 17(1): 243.

Aller, J. F., L. B. Ferre, et al. (1997). Ciclo folicular natural vs ciclo sincronizado en llamas (Lama glama). Abra Pampa, INTA: 246.

Aller, J. F., G. E. Rebuffi, et al. (2002). "Successful transfer of vitrified llama (Lama glama) embryos." Animal Reproduction Science 73: 121-127.

Bourke, D., C. E. Kyle, et al. (2000). "Recipient synchronisation and embryo transfer i south american camelids." Theriogenology.

Bourke, D. A., C. E. Kyle, et al. (1995). "Superovulatory responses to eCG in llamas (Lama glama)." Theriogenology 44: 255-268.

Bravo, O. F. (2001). Estudio comparativo de tres metodos de colleccion de semen en alpacas. Departamento de produccion animal. Lima, Universidad Nacional Agraria La Molina - UNALM.

Bravo, P. W. (1993). Basic reproductive physiology in female South American camelids, Zoo and wild animal medicine.. W.B. Saunders Company Philadelphia USA: 1993. 532-536. 28 ref.

Bravo, P. W., U. Flores, et al. (1996). "Collection of semen and artificial insemination of alpacas." Theriogenology 47: 619-626.

Bravo, P. W., M. Fowler, et al. (1990). "Endocrine responses in the llama to copulation." Theriogenology 33(4): 891-899.

Bravo, P. W., M. Fowler, et al. (1990). "Ovarian follicular dynamics in the llama." Biology of Reproduction 43: 579-585.

Bravo, P. W., J. Moscoso, et al. (1996). "Transport of spermatozoa and ova in female alpaca." Animal Reproduction Science 43(2/3): 173-179.

Bravo, P. W., G. H. Stabenfeldt, et al. (1991). "The effect of ovarian follicle size on pituitary and ovarian responses to copulation in domesticated South American camelids." Biology of Reproduction 45(4): 553-559.

Bravo, P. W. and J. Sumar (1989). "Laparoscopic examination of the ovarian activity in alpacas." Animal Reproduction Science 21(3-4): 271-281.

Chavez, M. G., M. A. Aba, et al. (2002). "Ovarian follicular wave pattern and the effect of exogenous progesterone on follicular activity in non-mated llamas." Animal Reproduction Science 69: 37-46.

Chipana, O. (1997). Colleccion de semen y evaluacion de dilutores sobre la viabilidad espermatica de alpacas. Escuela de Post Gardo - Produccion Animal. Lima - Peru, Universidad Nacional Agraria La Molina - UNALM.

Correa, J., M. Ratto, et al. (1997). "Superovulation in llamas (lama glama) with pFSH and equine chorionic gonadotropin used individually or in combination." Animal Reproduction Science 46: 289-296.

Elwishy, A. B. (1988). "Reproduction in the male dromedary (Camelus dromedarius): a review." Animal Reproduction Science 17: 217-241.

Fernandez-Baca, S. (1993). "Manipulation of reproductive functions in male and female New World camelids." Animal Reproduction Science 33(1-4): 307-323.

Fernandez-Baca, S., W. Hansel, et al. (1979). "Differential luteolytic effects of right and left uterine horns in the alpaca." Biology of Reproduction 20(3): 586-595.

Fernandez Baca, S., W. Hansel, et al. (1970). "Corpus luteum function in the alpaca." Biology of Reproduction 3(252-261).

Fernandez Baca, S., D. H. L. Madden, et al. (1970). "Effects of different mating stimuli on induction of ovulation in the alpacas." Journal of Reproduction and Fertility 22(261-267).

Fernandez Baca, S., J. Sumar, et al. (1973). "Relationship between the location of the corpus luteum and that of the embryo in the alpaca. [Spanish]." Revista de Investigaciones Pecuarias, Peru 2(2): 131-135.

Ferrer, M. S., A. Aguero, et al. (2002). "Sincrnizacion de la ond afolicular mediante el uso de buserelina en la llama (Lama glama)." Investigaciones Veterinarias 4(1): 7-11.

Huanca, T. (1993). Manual del alpaquero. Lima - Peru, INIA.

Leyva V, V. and J. Sumar K (1987). Indicence of oestrus and spontaneous ovulation in Huacaya type alpacas. Utah.

Lichtenwalner, A. B., G. L. Woods, et al. (1995). "Seminal collection, seminal characteristics and pattern of ejaculation in llamas." Theriogenology 46: 293-305.

Novoa, C., S. Fernandez Baca, et al. (1972). "Pubertad en la alpaca." Revista de Investigaciones Pecuarias 1: 29-35.

Novoa M, C. (1981). South American Camelidae. [Spanish], Recursos geneticos animales en America Latina. Ganado Criollo y especies de altura.. FAO. Rome Italy: 1981. 118-128.

Paolicchi, F., B. Urquieta, et al. (1999). "Biological activity of the seminal plasma of alapcas: stimulus for the production of LH by pituitary cells." Animal Reproduction Science 54: 203-210.

Pollard, J. C., R. P. Littlejohn, et al. (1995). "Seasonal and other factors affecting the sexual behaviour of alpacas." Animal Reproduction Science 37: 349-356.

Pollard, J. C., R. P. Littlejohn, et al. (1994). "The effects of mating on the sexual receptivity of female alpacas." Animal Reproduction Science 34(3-4): 289-297.

Ratto, M., W. Huanca, et al. (2005). "Comparison of the effect of natural amting, LH and GnRH on interval to ovulation and luteal function in llamas." Animal Reproduction Science In press.

Ratto, M., J. Singh, et al. (2003). "Ovarian follicular wave synchronization and pregnancy rate after fixed-time natural mating in llamas." Theriogenology 60: 1645-1656.

Reyna, J. (2005). Studies in time of ovulation in Merino ewes by transrectal ultrasound. Veterinary science. Sydney, University of Sydney: 250.

San Martin, M., M. Copaira, et al. (1968). "Aspects of reproduction in the alpaca." Journal of Reproduction and Fertility 16: 395-399.

Sapana, R., T. Huanca, et al. (2002). Estudio del plasma seminal para inducir la ovulacion en alpacas. Puno - Peru, INIA: 191-192.

Skidmore, L. (2004). Reproductive physiology in the female camel, www.ivis.org, Ithaca, New York, USA.

Smith, C. L., A. T. Peter, et al. (1994). "Reproduction in llamas and alpacas: a review." Theriogenology 41(3): 573-592.

Sumar, J. (1985). Reproductive physiology in South American camelids, Genetics of reproduction in sheep.. Butterworths London UK: 1985. 81-95. 44 ref.

Sumar K, J. (1993). "Efectos de los estimulos de induccion en la ovulacion de alpacas y llamas." Investigaciones pecuarias 6(1): 1-6.

Sumar K, J., P. W. Bravo, et al. (1993). "Sexual receptivity and time of ovulation in alpacas." Small Ruminant Research 11(2): 143-150.

Vaughan, J. L. (2002). Improving the efficiency of reproduction and breeding in alpacas, RIRDC: 20.

Vaughan, J. L., K. L. Macmillan, et al. (2003). "Effects of mating behaviour and the ovarian follicular state of female alpacas on conception." Australian Veterinary Journal 81(1/2): 86-90.

Vaughan, J. L., K. L. Macmillan, et al. (2004). "Ovarian follicular wave characteristics in alpacas." Animal Reproduction Science 80: 353-361.

Xilong, L. and X. X. Zhao (2002). "Separation, purification and identification of ovulation-inducing factors in the seminal plasma of bactrin camel (Camelus bactrianus)." Unpublished.

Please cite as:

Reyna, J (2006). Alpaca Female Reproductive Physiology.The Camelid Quarterly Magazine, Canada. Vol 1, March. 1-7 p.

Copyright © 2016 Artminds Digital Media www.artminds.com.au