Artificial Insemination

As in many other species, artificial insemination (AI) is the single most important technique to insure rapid genetic progress in camelidae. In addition to its use for genetic improvement, AI presents several other advantages including:
*    More efficient use of genetically superior males: Requests for breeding to a particular racing camel can reach up to 1500 females per year which would be impossible to accomplish by natural breeding without compromising the health and fertility of the male. The development of artificial insemination and especially the determination of the minimum dose of semen compatible with good fertility will allow the insemination of more females with a single ejaculate.
*    Prevention of diseases: Artificial insemination eliminates all contacts between the male and females, thus limiting the risks of transmission of diseases.
*    Elimination of the need for transportation of animals: The development of AI could lead to the elimination for the need of transporting animals (males and females), consequently reducing costs and the risks associated with transportation and boarding.
*    Increased fertility: Overall fertility of the herd can be expected to increase if artificial insemination is used because this technique requires frequent veterinary examinations and therefore allows early detection of reproductive problems and optimization of breeding.
*    Elimination of behavioral problems: Refusal of a male to breed a particular female is a frequent problem in naturally-bred dromedaries. We have noticed that some males are very aggressive and can bite some females even if they are ready to be bred (have a mature follicle and good uterine tone). AI will eliminate the risks associated with this behavior.
*    Insurance of the male: The reproductive life of a valuable male can be shortened by injuries or diseases. Routine collection and preservation of semen can extend the reproductive life. It also presents the advantage of preserving genetic material from particular strains of animals for future use even years after the death of the male.
*    Inter-species insemination: AI can also help managed cross-breeding programs between different species of camelidae. This eliminates problems due to size differences and behavioral problems. In addition males from wild species such as the vicuna can be used more efficiently.

The use of artificial insemination as a breeding technique has been reported in camelidae since the late 1960's.^{(39, 45, 46)} The first offspring from AI in camelidae was reported in a Bactrian camel inseminated with frozen semen collected by electroejaculation in 1961.⁽³⁹⁾ However, it is not until the last 2 decades that this breeding technique started to be used more frequently, especially in the Bactrian camel.^{(24-26, 89-91, 95, 97-99)} In the dromedary, many authors have studied semen preservation but actual artificial insemination trials are very rare.⁽⁶⁾ In the South American camelidae, artificial insemination with raw or diluted semen collected by artificial vagina or electroejaculation has been attempted in alpacas and vicuña-alpaca crosses with relatively good pregnancy rates (40 to 70%).

Semen is usually used raw, or after dilution. The use of raw semen offers only a few advantages such as in the case of behavioral problems or size incompatibility. More efficient use of artificial insemination requires dilution of the ejaculate and its preservation. Diluted semen can be used within minutes (fresh) or after short-term preservation (few hours) or long-term preservation (months or years).

Short-term semen preservation

Short-term preservation (a few hours) of camelidae semen has been attempted at different temperatures (30°C, 25°C or 4°C).

Semen extenders

Semen is collected by electroejaculation, artificial vagina, or flushing of the epididymis with heat-treated milk. Several extenders are used for dilution of freshly collected semen (Table 1). Most of these extenders are adapted from studies in other species and usually contain a source of energy (glucose or fructose), a protein for protection against cold shock (lipoprotein from egg yolk or casein from milk), a buffering system, and antibiotics.

In general, all extenders containing lactose or egg yolk are suitable for the short-term preservation of camelidae semen. However, there are no studies concerning the effect of physical and chemical properties (i.e. pH, ionic strength and osmotic pressure) of the extender on the motility and fertilizing ability of preserved semen.
 

Dilution

In the dromedary and Bactrian camels, semen is diluted at a ratio of 1:1 to 1:3 (semen:extender) ^{(6, 67)} depending on the concentration of the ejaculate. It is recommended to add the extender at a temperature of 30 to 35°C to the semen. It is very important to wait until the semen has completely liquefied before adding the extender in order to obtain a good mixture. Some authors suggest dilution to obtain a known concentration such as 50 x 10⁶/ml.⁽⁶⁾

Some authors have recommended keeping the semen at 37°C or at room temperature until insemination. This storage temperature is adequate only if the semen is to be used within a few minutes of collection.⁽⁶⁾ For longer preservation in a liquid form (up to 48 hours), semen should be cooled slowly to 4 or 5°C. Slow cooling of the semen can be achieved by placing the tube containing extended semen into a water bath at room temperature and placing it in the refrigerator. This system allows cooling of the extended semen to 5°C over one hour. If the semen is to be shipped, we have found that the Equitainer system used for chilled equine semen is suitable to keep good motility for at least 18 hours (Figure 11.1).

Effect of preservation on motility

Most of the studies on semen preservation in the camel use motility and acrosome integrity as the main parameters for the evaluation of semen.

Semen motility in camelidae is very difficult to evaluate, especially if the ejaculate has not completely liquefied. This is why in some studies semen motility seems to increase initially after dilution.⁽⁵²⁾

Several extenders have been tested to evaluate their suitability for liquid preservation of dromedary and Bactrian camel semen. The best results in terms of motility and acrosome integrity were achieved using the commercial bull-semen extender Laiciphos in both species.⁽⁷⁵⁾ In our laboratory, we have found that skim-milk extenders used for equine semen are also satisfactory for the preservation of dromedary semen in a chilled state.

Semen freezing

The best preservation technique for semen from any species is deep freezing. This technique has several advantages including:
*    Preservation for almost indefinite time
*    Extended use of semen, even long after the male's death
*    More efficient use of male
*    Ease of transportation and international exchange

Most of the scientific studies available at this time concern the freezing of semen from the Bactrian and dromedary camels. However, actual insemination of females with frozen-thawed semen has been carried out in the Bactrian camel only.⁽²⁷⁾

Extenders

A variety of extenders used for deep freezing of semen from other species have been adapted to dromedary and Bactrian camels (Table 2). The major difference between extenders used for the preservation of semen at the liquid state and those used for freezing is the presence of cryoprotectant substances. Although lipoproteins from egg yolk or milk provide some protection from the cold temperatures, semen cannot sustain the very low temperatures imposed by freezing if a cryoprotectant such as glycerol is not added to the extender. The final concentration of glycerol in the extender and the method of addition to the semen have been found to be critical for the survival of spermatozoa and maintenance of its fertilizing ability. However no studies have been carried out for the determination of these parameters in camelidae. Also, the effect of other factors on the freezability of semen are lacking in camelidae. Of particular interest is the effect of seminal plasma.

In a study comparing several freezing methods used for bull, ram, dog, stallion, and boar semen preservation, assessment of post-thaw motility and morphology showed that the best extender for freezing dromedary and Bactrian camel semen is a modified boar or stallion technique.⁽⁷⁵⁾ This technique uses two extenders (a cooling extender and a freezing extender). The cooling extender is added to the semen immediately after collection (Table 2). The freezing extender contains the cooling extender in addition to a cryoprotectant (glycerol) and an emulsifying agent (orvus paste) that plays a role in the stabilization of the sperm plasma membrane.
 

A similar study was conducted to compare tris-bull extender, sodium citrate-egg yolk extender, ram, stallion, and boar extenders with sucrose-based extenders (SYG extenders) for the freezing of Bactrian camel semen.⁽⁹⁷⁾ The best results as far as post-thaw motility and acrosome integrity are concerned were obtained using the sucrose extender containing 7% glycerol followed in order by stallion, swine, and sucrose extender with only 3.5% glycerol (Table 2).

Dilution rate

Dilution rates of semen vary from 1:1 to 1:8 (semen: extender) in the llama and from 1:2 to 1:3 in the dromedary and Bactrian camels.^{(27, 51, 97)}

Semen packaging and freezing procedure

Freezing procedure depends on the packaging method used (Figure 11.2). Semen can be packages as pellets,⁽⁵¹⁾ in plastic straws with different volumes (0.25 ml, 0.5 ml or 4 ml),^{(67, 75, 88)} or ampoules.^{(27, 97, 99)} The packaging method selected affects both the freezing and the thawing rates.

Freezing rate

Semen pellets are obtained by dropping a known volume (0.1 or 0.2 ml) of diluted semen into depressions made in dry ice. The pellet forms by freezing a few seconds after contact of semen with the dry ice. Therefore, the only way to vary the freezing rate of pellets is to modify the volume of semen used.⁽⁵¹⁾ This technique is rarely used today because of the difficulties in labeling the semen, the inability to modify the freezing rate, and the impossibility of properly labeling the semen.

Straws are usually frozen by placing them on a rack at known distances above the surface of liquid nitrogen. The volume of the straw has a great influence on the freezing process. The fastest freezing rates are obtained by the use of a small volume (0.25 or 0.5 ml). Freezing rates can be modified by adjustment of the elevation of the straws. More precise freeing rates can be achieved in semen packaged in straws by using a computerized freezer that can be programmed to follow a precise freezing curve. The samples are placed on a platform which is lowered stepwise toward the liquid nitrogen surface. The temperature inside the samples is monitored by a thermocouple which guides the movement of the platform.

The freezing procedure used for dromedary and Bactrian camel semen packaged in 4-ml straws can be summarized as follows: ^{(67, 75, 88)}
*    Equilibration of raw semen at 25°C to 30°C until liquefaction
*    Initial dilution with the cooling extender (1 volume semen : 1 volume extender), then cooling to 15°C over a 2 hour period
*    Second dilution with the freezing extender to a concentration of 150 x 10⁶/ml then cooling to 5°C over a period of 1.5 hours
*    Final dilution with the freezing extender to a concentration of 100 x 10⁶/ml and packaging in large straws (4 ml)
*    Freezing by placing the straws in liquid nitrogen vapors for 20 minutes (allowing them to reach a temperature of -120°C then plunging into liquid nitrogen)

We use a simplified technique of freezing of semen packaged in 0.25 or 0.5 ml straws. Dilution is completed as soon as the semen is liquefied. The diluted semen is cooled down to 5°C over one hour and maintained at this temperature for 2 hours before packaging in straws. The straws are placed on a rack 4 cm above the liquid nitrogen surface for 10 minutes then transferred directly into liquid nitrogen (Figure 11.3).

Packaging in 1.5 ml ampoules is the most commonly used technique for freezing Bactrian camel semen in China.^{(23, 97, 99)} The technique consists of the following steps:
*    Equilibration at 37°C for 10 minutes
*    Equilibration at 20°C for 10 minutes
*    Equilibration at 10°C for 10 minutes
*    Equilibration at 4°C for 4 hours
*    Freezing on a wire grid placed above liquid nitrogen in four (4) steps:
    *    Step 1) 3 minutes at 3 cm, corresponding to -5°C
    *    Step 2) 2 minutes at 2 cm (-75°C)
    *    Step 3) 1 minute at 1 cm ( -175°C)
    *    Step 4) plunging into liquid nitrogen (-196°C)
 

Thawing rate

Thawing rates vary according to packaging technique used. Pellets are usually thawed by dropping them into heated receptacles or by mixing in a warm thawing extender. Semen frozen in ampoules is thawed out by placing them in a water bath set at 45 to 55°C for 30 seconds to 1 minute.^{(27, 97)} Small straws are thawed in a water bath at 37°C for 30 to 40 seconds or 40°C for 8 seconds. Large straws are thawed by continuous agitation in a water bath at 40°C for 50 seconds.

Post-thaw semen quality

Llama semen frozen in pellets and thawed in warm extender showed 45% post-thaw motility (pre-freezing motility 50%). Motility of frozen semen remained acceptable even after 8 years of storage.⁽⁵¹⁾

Similarly, we have found in the dromedary that if the initial semen motility is very good, very little loss is caused by freezing. Semen frozen in lactose, egg-yolk extenders has maintained the same post-thaw motility after 6 years of storage in liquid nitrogen.

In the Bactrian camel, it is recommended that only semen showing 30% or more motility should be used for insemination.⁽²⁷⁾ Motility usually remains unchanged after dilution but decreases after equilibration and freezing. Motility before freezing ranges from 65 to 85% and decreases to 67 to 79% after equilibration in SYG1 and SYG2 extenders respectively. Post-thaw motility of semen frozen in ampoules is on the average 43% and 64% for SYG1 and SYG2 extenders respectively.^{(27, 97, 99)} Survival of spermatozoa after thawing is also tested by incubation of the thawed semen at 37°C or 4°C. Survival rates at these temperatures are very variable from one male to another.⁽²⁷⁾ in addition to post-thaw motility, acrosome integrity should be evaluated in all frozen-thawed samples (cf. Semen evaluation).^{(75, 97)} Factors that influence the quality of semen after freezing and thawing have not yet been thoroughly investigated. Post-thawing semen quality and survival of spermatozoa are highly variable from one male to the other, even after using the same freezing technique.

Artificial Insemination

The most challenging aspect of artificial insemination in camelidae is its timing in relation to ovulation. Ovulation in these species is induced by copulation and/or semen deposition in the female genital tract (cf. Physiology). Studies in the Bactrian camel showed that a GnRFI-like factor present in semen is responsible for the induction of ovulation. This means that a sufficient amount of semen has to be deposited in the uterus at insemination in order to obtain ovulation. In the Bactrian camel, at least 1 ml of semen is needed in order to achieve ovulation. This is not a viable solution because using a high volume of semen will reduced the number of females inseminated per ejaculate and can also compromise the freezability of semen by interaction with seminal plasma. The best alternative is to inseminate at known intervals following induction of ovulation by hormonal treatment (hCG, GnRH).

Behavioral signs of estrus should be combined with assessment of ovarian activity by rectal palpation or ultrasonography before insemination.⁽⁶⁾ In the dromedary, some authors have suggested that insemination is to be carried out when the follicle has reached 15 to 30 mm in diameter and that the optimum time for insemination is the first day of behavioral estrus.⁽⁶⁷⁾ In our experience, the best time for insemination can only be determined by ultrasonography and palpation of the uterus and the ovaries. Using these techniques, we have found that insemination should be carried out in the dromedary when the follicle is between 12 and 18 mm in diameter and the uterus has a good tone. In the Bactrian camel, insemination is carried out when the follicle reaches 12 mm in diameter.⁽²⁷⁾

Semen deposition technique

Semen is deposited into the uterus by using an insemination gun or a pipette in the same manner described for the bovine or equine species. Although the instrument can be guided directly into the cervix by vaginal manipulation, we prefer to use the rectovaginal technique in order to eliminate the risk of contamination. Rectovaginal manipulation is easier in the estrous female because the uterus is contracted and the cervix is open/6) Semen should be deposited just cranial to the internal cervical os. In the Bactrian camel, insemination has to be done using a porcine rubber insemination tube heated to 38°C to prevent cold shock.⁽²⁷⁾ When frozen semen is used, a second insemination is performed 24 hours after the first one. We avoid using large diameter pipettes because they can cause a loss of semen.

Dosage

No study has been conducted to determine the minimal number of spermatozoa to be used for insemination in camelidae. Chinese authors suggested the use of 400 million spermatozoa in the Bactrian camel.^{(26, 27, 96, 97, 99)} However, it is almost certain that these doses can be reduced if ovulation is induced by hormonal treatment and the timing of insemination is determined with precision.

Fertility

Fertility rates obtained with fresh semen are variable. A pregnancy rate of 2.3% was obtained in alpacas following intracervical insemination with vicuna semen. This low fertility rate was explained by the poor quality of the semen used.^{(42, 46)} Deposition of semen by rectovaginal manipulation resulted in a pregnancy rate of 38%. In the South American camelidae, its was found that fertilization rates are highest when insemination is carried out 35 to 45 hours after treatment for the induction of ovulation (breeding to vasectomized males or injection of hCG (500 to 1000 IU IV or IM) or GnRH (50 mg IM)).⁽⁴⁶⁾

Pregnancy rates obtained with fresh semen in the dromedary camel vary from 30 to 80% and depend on the male, ovulation treatment used, and timing of insemination ^{(6, 62, 63)}(Table 3).

Artificial insemination trials using frozen semen are available only for the Bactrian camel. In this species, a double insemination at 24 hour intervals or a single insemination of 400 million sperm cells after hCG treatment resulted in very high conception rates (80 to 90%) ^{(94, 96, 97, 99)} (Table 3).

Problems with development of AI in camelidae and needed research

The use of artificial insemination in camelidae is limited at this time to experimental trial. With the exception of the Bactrian camel, no data is available on the fertility of females after insemination with frozen semen. This shows that many problems concerning this technology have yet to be resolved. These problems can be summarized as follows:

*    Lack of adequate methods for semen collection. Semen collection techniques used so far yield variable results. New, improved techniques are very promising and should allow a more reliable semen collection (cf. Semen collection).⁽⁵⁶⁾
*    Poor post ejaculation sperm motility: This is mainly due to the gelatinous nature of camelidae semen. Liquefaction of semen can take several hours.^{(50, 52)}
*    Lack of standard techniques for freezing semen
*    Difficulty in transcervical pipette passage, especially in South American camelidae: This can be overcome by adapting some techniques used in the sheep and goat, such as direct cervical handling and laparoscopic insemination.

The areas where more research is needed in order to develop artificial insemination in camelidae are as follows:
In the area of initial semen quality:
-    Develop reliable semen collection techniques.
-    Define procedures for semen handling.
-    Determine the appropriate frequency of collection.
-    Develop reliable semen evaluation techniques.
 

In the area of gamete physiology:

-    Study the biochemistry of semen, especially with reference to liquefaction.
-    Study the effect of seminal plasma on freezing and interaction with extenders.
-    Determine life span of ejaculated and preserved spermatozoa in the genital tract.
-    Study the biology of capacitation and acrosome reaction of camelidae spermatozoa.
-    Study the mechanisms of oocyte maturation in camelidae and life span of the ovum.

In the area of semen preservation:
-    Determine properties of suitable extenders: pH, osmotic pressure, glycerol concentration, ionic strength.
-    Determine optimal semen freezing procedure - cooling rate, equilibration time, method of glycerolization, freezing rate, and thawing rates.

In the area of management of insemination:

-    Determine the best time for induction of ovulation.
-    Determine the optimal timing of insemination in relationship to ovulation.
-    Determine the optimal number of live normal spermatozoa per insemination.

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