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The sperm physiology of South American camelids from samples collected by vaginal aspiration of the ejaculate reveals that it is perfectly sound for semen analysis. There are no significant differences between llama and alpaca semen. Volume (4 mL), motility (70%), viability (85%), concentration (32 million), normal spermatozoa (76%), abnormal heads (8%), cytoplasmic droplets (5%), abnormal tails (8.5%) and morphology is comparable to semen collected with an artificial vagina. The most salient feature is the epididymal spermatozoa’s concentration (327 million/mL). Glucose and fructose are present in uterine and oviductal fluid with less glucose concentration by 24 h after ejaculation which is concomitant with decreased spermatozoa motility. Supplemental nutrition of the male effectively improved spermatozoa concentration to 192 million spermatozoa/mL rather than 60 million in males not receiving any supplement. Altogether, practitioners and scientists could use the information in this chapter, especially spermatozoa features within the uterus and the oviduct.
Escuela de Medicina Veterinaria, Filial Sicuani, Universidad Nacional de San Antonio Abad del Cusco, Cusco, Peru
*Address all correspondence to: pwbravo@gmail.com
1. Introduction
The reproductive process of South American camelids is partially known and in the process of being examined. The knowledge on the male side differs from what is known on the female side. Fifteen years ago, for each report on the semen of camelids, there were 50 reports on females. This difference is due to how semen was collected and the length of copulation. Historically, semen collection has undergone different approaches and tries to mimic what is used in bulls and rams. The other aspect is the length of copulation dictated by the male. Llamas and alpacas copulate for more than 15 min, completely different from rams and bulls, with the penis reaching the uterine horns and changing between the two uterine horns. Since the use of semen collected by an artificial vagina, the understanding of semen characteristics has been steady and progressing. Thus, the knowledge of the male side, especially on semen, has been moving gradually. This aspect of having semen readily available and in the laboratory has opened the doors to work on different world latitudes.
Males are also important, like females, in the reproductive process, and when large numbers of females are ready to reproduce, about 10% of the best males are maintained, with the rest being destined to be a source of meat. In North America, since alpaca meat is unacceptable, a surplus of males has been maintained and is offered as part of sales accompanying other animals. Thus, males are kept only as sire quality in America, North, and South, which is still a tiny proportion of the female population. In this sense, raising llamas and alpacas in South America constitutes an integral part of the people inhabiting the Andean places over 3000 m from sea level. Many people still rely on fiber harvested from their animals as a source of meat for themselves and for sale, especially as dried meat.
The physiology of semen from domesticated South American camelids, llamas, and alpacas is still developing. Reports from wild camelids, vicuña, and guanaco are scanty due to their difficulty to work with and strict restrictions from the government entities. Semen collection has undergone several steps. Reports from the last century using vaginal sponges, cannulation of the urethra, condoms, electroejaculation, artificial vagina, and deviation of the vas deferens have developed into a more practical and clinical method of vaginal aspiration. Using this last method, samples of the ejaculate are perfectly fitted to determine physical characteristics and their biochemical components. In addition, the physiology of camelid spermatozoa collected from the uterus and oviduct of the female following copulation has been used to unravel the physical characteristics and the use of some chemical compounds, especially two carbohydrates inside the female’s oviducts. These last results could be used to fine-tune the outcome of in-vitro fertilization and use specific information for camelids, which is now extrapolated from other livestock species. Finally, the use of spermatozoa in the process of in-vitro fertilization is taking place with some good promises. This chapter includes the semen characteristics of spermatozoa collected by vaginal aspiration, a comparison of spermatozoa collected from the epididymis with the ejaculate, physical characteristics, and the use of two carbohydrates, the features of spermatozoa used for in-vitro fertilization.
2. Semen characteristics collected from vaginal aspiration
Semen and its components are the exocrine products of the male reproductive system. The ejaculate is part of the spermatogenic function of the testicles and is subject to evaluation. In South American camelids, due to the length of copulation (20 min) it was challenging to come up with a system to collect semen efficiently. Since the last century, diverse methods have been applied, each with advantages and disadvantages. The first report [1] indicated that alpaca ejaculate was absent from 70% of males, and it was challenging to collect it. Later, intravaginal sponges were placed into the vagina, a fistulation of the urethra was also applied, and even a condom from human use and electroejaculation were used. An artificial vagina inside a dummy with a stricture simulating the female’s cervix was used [2]. This latter method with an electric pad, like those used to warm up different parts of the human body and wrap the artificial vagina [3], has sparked many reports on semen. Another method used was a surgical deviation of the vas deferens.
In this section, the method of vaginal aspiration is emphasized as a clinical method, easy to use, applicable in any part of the world, without sophisticated equipment, with no need to train males to an artificial vagina, and as natural as possible for females and males. Briefly, this method considers the behavior of the male and copulation time. After a copulatory period, a prewarmed vaginal speculum is inserted into the vagina of the female with a stylet. The stylet is withdrawn, and at the caudal end of the speculum, a 15-mL collecting tube is attached. A gentle movement up and down, or introducing and partially removing the speculum to catch semen deposited near the cervix, is applied until a reddish semen sample appears in the collecting tube. The speculum is inclined towards the collecting tube, allowing the semen sample to flow into the collection tube. Then the speculum is withdrawn. The semen sample is kept warm [4]. In the case of semen collection in the field, where llamas and alpacas are copulating and away from the laboratory, they are kept close to the body and within a flannel or polar material with divisions large enough to accommodate the diameter of the collecting tube. Then, once in the lab, collecting tubes are placed in a water bath at 36°C until the examination happens. Then, semen samples are ready to be evaluated, and the same protocols to define motility, concentration, morphology, and endosmosis test, or HOST test, as in samples collected by an artificial vagina are used. It is necessary to state that the speculum does not enter the uterus corpus through the cervix. The sample collected using this method results from semen deposited during penis withdrawal and change of uterine horns during, taking advantage of continuous ejaculation. A concern is also expressed when some people believe that this method damages the vaginal wall. This is unsubstantiated because the speculum has a smooth opening and the external surface is entirely polished. In addition, the speculum does not penetrate the cervix. The reddish appearance of the ejaculate results from mixing with blood coming from the uterine horns and due to the rubbing of an engorged penis process of the male and threading movement of the penis (Figure 1). This coloration is not artificial; it is natural and has been happening for thousands of years. Following are the physical and biochemical characteristics of the ejaculate from llamas and alpacas using this method. Physical features are presented in Table 1.
Figure 1.
Damage provoked by the penis of the male into the uterus of the female alpaca. (A) Uterine horns and body of the uterus are hemorrhagic immediately after copulation, and (B) the appearance of the uterine horns 48 h following copulation is still damaged and hemorrhagic.
Main semen physical characteristics of spermatozoa from llamas and alpaca ejaculate collected by vaginal aspiration and alpaca epididymis.
13% with progressive motility and 26% active. 61% nonmotile.
Volume: It is the first physical characteristic to evaluate that anybody evaluating semen is in charge. Ejaculate volume collected by vaginal aspiration varies from 2 to 15 mL, averaging 4 mL (Figure 2).
Figure 2.
Semen samples were collected from the vaginal fundus by aspiration after copulation to an intact male for at least 15 min.
Color: The semen collected by vaginal aspiration varies from white to intense red (Figure 2). White indicates that the penis has not penetrated the cervix and that there is no friction between the penis and the uterine mucosae. On the contrary, a reddish color indicates rubbing between the penial processes and an intense red suggests that the penis frictions the uterus mucosae vigorously. More than 40% of the samples collected are reddish or red.
Viscosity: Spermatozoa in the ejaculate are trapped within a gelatinous and viscous seminal plasma and do not show progressive motility. The origin of this viscid seminal plasma is bulbourethral glands. Viscosity is challenging to measure, and a practical way is to pipette a drop of semen, let it encounter a glass slide, and try to pull it apart. In this process, a thread will be formed, and the length of it will be compared to a ruler maintained next to it. Thus, it is measured in cm, and Argentinian scientists [5] started to use the term “filancia,” which has been adopted in South America and does not have an equivalent word in the English dictionary. Thus, llamas and alpaca’s semen viscosity [6] are present and vary from 1 to 7 cm (Figure 3).
Figure 3.
Variety of color semen collected from vaginal aspiration from the female alpaca and llama.
Motility: The average motility is 70%, and the most striking feature is that spermatozoa appear agglutinated and non-motile due to a thick seminal plasma. Most scientific literature reports that motility is only around 30%; however, a patient evaluation of at least three microscopic fields indicates that motility is greater than 30%. Another situation to consider is that the poor motility reported earlier has to do with the presence of a thick seminal plasma. The presence of this plasma limits the expression of the total motility of spermatozoa. As reported from samples collected by an artificial vagina, motility is nonprogressive, i.e., in place and with a slight tail movement.
Viability: It is impressive that more than 90% of spermatozoa are alive, and this high value contrasts with reports that viability only reaches 60%. In addition, this high value reiterates that nonmotile spermatozoa are alive. Even if the endosmosis test is applied for 2 h, only up to 20% show membrane disruption.
The hypo-osmotic swelling test uses a hypoosmotic fructose solution at 200 mOsm/L. Since spermatozoa are ejaculated in an osmotic liquid and subjected to a hypoosmotic solution, they will try to compensate for getting bigger, as with red blood cells in the same solution. However, the plasma membrane of spermatozoa does not swell at its head; instead, its tail swells and presents a loop. The incubation time in the hypoosmotic solution is essential, with 2 h at 37°C being the most favorable. Using this protocol, up to 15% of spermatozoa present the swelling of their tail. This value is comparable to the viability of 90% of spermatozoa not becoming black to the stain used to determine viability.
Concentration: The range of sperm concentration varies from 6 to 100 million spermatozoa/mL. It is one of the semen characteristics that changes the most, even in ejaculates from the same male. In addition, it represents only a million compared to a billion spermatozoa in the ram and the bull. However, it should be considered that alpaca testicles weigh 16–20 g in contrast to 500 g in the ram. See Table 1 for the mean concentrations in the alpaca and the llama.
Morphology: The classification of normal spermatozoa, abnormal heads, cytoplasmic droplets, and abnormal tails is also applied to South American camelids. The different categories of spermatozoa are generally similar to other livestock species. Table 1 summarizes the percentage of spermatozoa morphology for the llama and the alpaca.
Semen characteristics collected from the epididymis tail.
Spermatozoa from the epididymis are widely used for in-vitro fertilization. The first report of this advanced reproductive technology was used in llamas to fertilize oocytes from slaughterhouse material [7]. Since that time, more than 30 publications have used sperm from the epididymis. Table 1 depicts some of the characteristics of spermatozoa collected from the epididymis tail, even though it is not ejaculate is presented in Table 1 for comparative purposes.
Among the salient characteristics is motility, which is higher (76.7%) than semen collected by vaginal aspiration. As observed in semen from vaginal aspiration, 61% of immotile spermatozoa are less than 30% collected by vaginal aspiration. In addition, 13% of spermatozoa show progressive motility, and 26% are active spermatozoa. This is due to the absence of seminal plasma in semen collected from the epididymis. Concerning sperm concentration, it is eight times more concentrated than ejaculated semen. This is due to a high sperm concentration and the place of spermatozoa storage in the cauda epididymis. Regarding spermatozoa morphology, it is noteworthy that 29% of them present a cytoplasmic droplet, indicating a lack of sperm maturation. The other morphology characteristics—abnormal heads and abnormal tails—are comparable to those collected by vaginal aspiration.
3. Semen characteristics collected from the reproductive tract of the female
In South American camelids, semen is deposited some hours before ovulation, i.e., 24 h after ovulation. The place of ejaculation is near the utero-tubal junction, within the uterine horns, the uterus corpus, and the vaginal fundus, close to the external cervical os. This is due to continuous ejaculation starting 5 min after penis intromission into the uterus and during the change of the penis within the two uterine horns. As the male is ejaculating, he positions his penis in both uterine horns and the vaginal fundus as he changes from one uterine horn to the other. In addition, since copulation induces ovulation, the waiting time for spermatozoa in the oviducts for the ovum is longer than in other livestock species. In this regard, spermatozoa from rams are present in 3–5 min in the oviduct [8] and 4 h in the mare [9] from ejaculation at the external os of the cervix. In camelids, spermatozoa are entrapped within a thick seminal plasma product from the bulbourethral glands, and motility is non-progressive. Instead, it is only in-place motility and only with their tail movement. Thus, these precluded situations are present, What are the semen characteristics within the reproductive organs of the female?
First, motility decreases temporarily (Figure 4). From an initial 60% at ejaculation, it drops to 10% by 24 h, then increases to 70% and is activated and progressive. This increase in motility is accentuated in the oviduct, where fertilization will happen. In fact, by 36 and 48 h, spermatozoa motility in the oviducts is 20% higher than in the uterus. This is functionally different, even though spermatozoa are localized within the oviductal crypts. Spermatozoa activation was present from 36 to 48 h at the end of this study. In addition, sperm morphology was simultaneously different at 24 h in the uterus than in the oviduct. What is striking is the close-to-zero spermatozoa without abnormalities in the head (0.3%) and cytoplasmic droplet (0.2%) in the oviduct than in the uterus 9% and 3%, respectively. This last suggests, to be proven, a selection process at the level of the utero tubal junction leaving only good spermatozoa in the uterus and an active phagocytic process of abnormal spermatozoa in the uterus. This has been demonstrated in the rabbit, another induced ovulatory [10].
Figure 4.
Different lengths of semen vicosity in alpacas and llamas. Thickness was measured by pulling a drop of semen, and the thread length was compared to a ruler.
Second, glucose and fructose are the carbohydrates used as the energy sources for spermatozoa within the female reproductive tract. Even though there are minute amounts of these two carbohydrates, there are 20 times more fructose than glucose between 0 and 48 h; however, glucose is used more than fructose. Glucose concentrations are the lowest within 24 h of spermatozoa living in the oviduct. This drop is concomitant with depressed motility within 24 h of spermatozoa being in the reproductive tract of females.
Third, the concentration of acid phosphatase is greater at the time of semen deposition, especially in the uterus, than at any other time or in the oviduct. This may represent a reaction to the ejaculation in the uterus.
As mentioned above, glucose and fructose are essential for spermatozoa metabolism. Lately, with the determination of glucose and fructose in the uterine and oviductal fluid of the female, further analysis of these two carbohydrates has become mandatory. First, the concentrations of glucose and fructose in the prostate and bulbourethral glands indicate three times higher glucose concentrations than fructose in both the prostate and bulbourethral glands [11]. Second, in the ejaculate, glucose is 4–8 mg/dL [12]. Third, the amount of glucose used in-vitro and with spermatozoa collected with an artificial vagina. In this sense, both carbohydrates were used. By 2 h of incubation, more glucose was used than fructose. Glucose concentrations were 50% of what was determined in the ejaculate. By contrast, only 25% of fructose was used. From 2 to 4 h, neither glucose nor fructose was used (Figure 5). This early work suggested more affinity to use glucose than fructose [13]. Fourth, more recent work [14] from uterine and oviductal fluid and with spermatozoa present indicates that fructose is generally four times more than glucose in the uterus and oviduct from copulation to 48 h after. Although fructose concentrations appeared unchanged, a significant decrease was observed at 36 h. In addition, glucose concentrations were at their lowest by 24 h, which is concomitant to a reduction of sperm motility simultaneously. Something necessary to mention is that glucose was down by 48 h; however, fructose always remained high. This in-vivo report reaffirms what was described in-vitro earlier, even by 4 h. Glucose concentrations were less than fructose. Thus, there was fructose to be converted into glucose at the time of ovulation. More work is needed with these two carbohydrates by the time of fertilization (Figure 6).
Figure 5.
Spermatozoa motility in the uterus and oviducts at different times after copulation in the female alpaca.
Figure 6.
Glucose and fructose concentraions for up to 4 h of ejaculated spermatozoa maintained in-vitro conditions.
Using spermatozoa from the epididymis for in-vitro fertilization and other advanced reproductive technologies is the norm; however, due to the extended copulatory period of camelids, ejaculated sperm is an alternative. On the same aspect, the size of the epididymis may hinder the number of spermatozoa ready for in-vitro fertilization. A trial of culturing ejaculated sperm [15] may be helpful and considered. More spermatozoa were alive and improving their motility at 48 h of culture when a simple semen extender was used as a vehicle rather than a swine extender. The results of this trial are presented in Table 2.
Semen trait
Males with Preñatec
Males with Catosal
Control males
Motility (%)
50
33
24.4
Concentration (million/mL)
192c
82b
59.8a
Viability (%)
86
80
88
Normal spermatozoa (%)
72
70.2
69.1
Table 2.
The influence of injectable nutritional supplementation on semen characteristics in male Huacaya alpaca (n = 6, per treatment).
Male llamas and alpacas in South America are not subjected to nutritional improvement, and instead, they are destined to the poorest pastures for a significant part of the year. In addition, they have testicles that are only 16–20 g, which is far less than 500 g in the ram. Consequently, spermatic concentration is 30–70 million rather than billions for the ram. For this reason, one limiting factor in the application of artificial insemination is the spermatic concentration.
Supplementing alpaca sires for 60 days before breeding time has a positive effect. The supplement was administered subcutaneously every 2 weeks for 2 months before the breeding season [16]. Two commercial products, Preñatec1 and Catosal,2 were used. The results are presented in Table 2. Spermatic concentration was the trait that improved the most, and it was two times higher than the other supplement.
The effects of nutritional supplement in females has also been determined in the same work as in males. Many females, 240, were also supplemented as the males did. The premise is that if the effect is on the quality of semen, such impact should also be translated in females, such as ovulation and pregnancy. The product was strikingly prominent in females receiving Preñatec and bred in males receiving Preñatec. The result on the female side is presented in Table 3.
This chapter was devoted to domesticated South American camelids’ primary and practical reproductive aspects. Work on semen characteristics from ejaculates collected by vaginal aspiration is a helpful tool that gives information, and those semen samples are perfectly normal. It is also a revolutionary technique to assess semen quality. Basic information is provided about the physiology of spermatozoa in the uterus and the oviduct before ovulation. One salient feature of this chapter is the use of glucose and fructose within the uterus and the oviduct. A small section is given to the culture of ejaculated spermatozoa as an alternative to epididymal spermatozoa for in-vitro fertilization. The final section is about the nutritional effect on the semen of the male and the ovulation and pregnancy rate of the female.
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Notes
Preñatec, from TQC, Tecnología Química y Comercio.
Catosal, from Bayer.
Written By
Pedro Walter Bravo Matheus
Submitted: 06 June 2023Reviewed: 11 August 2023Published: 16 October 2023
Open access peer-reviewed chapter - ONLINE FIRST
