History and Initial Clinical Evaluation

The male alpaca or llama reaches sexual maturity at age 4 years, with a wide range of variability. Puberty is defined as the ability of a male to exhibit breeding behavior and to have sufficient spermatogenic development to get a female pregnant. Young male camelids may demonstrate mounting behavior as young as age 6 months, although preputial attachments will prohibit full erection and intromission.¹ In pasture management systems, crias should be segregated on the basis of sex, especially after weaning, to minimize potential unplanned early matings.

Often a producer will want to start using a male for breeding once the animal reaches age 2 years. A full breeding soundness examination performed by the veterinarian will dictate if the male is suitable for breeding and is mature enough to be used for this purpose, and the veterinarian will propose a plan for regularity of use. Young males demonstrate their immaturity in terms of breeding behavior, breeding capacity, and sperm morphology, and their use for breeding should be severely limited during this time of maturation.

Producers should be encouraged to present any candidate males as herd sires at a young age (12 to 24 months) for the first reproductive examination. A full history of the male should be obtained, including health and vaccination status as well as travel history. Males of the camelid species are extremely sensitive to heat stress, so travel during times of high ambient temperature or humidity should be minimized. The first reproductive examination serves as a baseline for all future breeding soundness examinations. Frequently, these first examinations are overlooked by producers and when a male develops infertility, and often no baseline measurements exist for comparison.

The examination is tailored to the age and maturity of the male. Very young males will be evaluated on physical parameters only. A general physical examination is performed, noting any musculoskeletal abnormalities that may impair breeding behavior in the future. The external genitalia are examined for any congenital defects. The scrotum is evaluated for two descended testes, which should be of the same size, with up to 15% variation.² Often the testes are offset slightly in the dorsoventral orientation. The testes are measured using calipers or with ultrasonography and the length and width recorded in millimeters.^3,4 Ultrasonographic examination of the testes is performed using a linear 7.5 megahertz (MHz) transducer. The parenchyma is evaluated in the longitudinal (sagittal) and transverse orientations (Figure 19-1). Any abnormalities, including the presence of rete testis cysts, should be noted. Any cysts should be measured at the widest diameter. The presence of small cysts should not exclude the male from being used as a sire, but regularly scheduled evaluation (every 6 to 12 months) with ultrasonography helps identify any changes.⁵ The testicular dimensions in a young male continue to increase as the animal attains sexual maturity. The accessory sex organs are not routinely examined. However, the prostate and bulbourethral glands are easily assessed for size and general appearance by using ultrasonography. The bulbourethral gland is imaged just cranial to the anal sphincter by slightly rotating the transducer from middle at a 40-degree angle (Figure 19-2, A). The gland is homogeneous until stimulated. The examination continues along the midline to image the pelvic portion of the penis (see Figure 19-2, B). The prostate is wrapped around the cranial border of the pelvic penis and is recognized as an oblong bilobed homogeneous body (see Figure 19-2, C). Determination of the depth of the prostate is important for positioning the electrode of the electroejaculator during semen collection with this technique.

A mature adult male alpaca or llama will undergo the same physical examination, with the addition of semen collection and evaluation. Collection by using an artificial vagina has been reported in alpacas and llamas, but often these animals require extensive training to be receptive to this equipment. The most often used methods are postbreeding aspiration from the female or electroejaculation under general anesthesia. Semen should be evaluated for mass activity and individual progressive motility after liquefaction and stained for morphologic and cytologic evaluations under phase-contrast microscopy. These examination protocols are discussed in detail elsewhere in this text. Young males demonstrate a high percentage of morphologically abnormal spermatozoa, most notably cytoplasmic droplets. Cytology demonstrates the presence or absence of germinal cells, bacteria, and white blood cells in the ejaculate.

All parameters should be recorded, and males should be examined at least yearly. Males should also be presented for evaluation if the conception rate of bred females drops acutely or if any physical abnormalities of the male are noted. Additionally, any signs of systemic illness or fever must be promptly addressed to minimize any potential effects on spermatogenesis. Lastly, males should be maintained on proper nutrition with adequate trace mineral supplementation.

Evaluation of Infertility or Subfertility

A male presented for infertility, subfertility, or any physical abnormalities of the reproductive tract should be thoroughly examined by the same methods detailed above. Additionally, the producer should provide all breeding records, including dates and lengths of matings, travel schedule, and any records of fever or systemic illness. Vaccination dates, parasitology (fecal) evaluations, and dates of deworming should also be provided. A thorough physical examination followed by testicular imaging and measurements is required. In some cases, heavy sedation is warranted for examination of the penis, especially in cases of physical trauma. Additional diagnostic tests may include imaging of the accessory sex glands, testicular biopsy, complete blood count and serum biochemistry evaluation, serum trace mineral evaluation, and serology for titers to select infectious diseases. These will be discussed in the following sections.

Testicular Biopsy

Testicular biopsy in the male is not a commonly employed diagnostic tool but may be invaluable in cases of azoospermia or oligozoospermia when no other causes are identified. Additionally, biopsy may be diagnostic for neoplastic or degenerative changes and is warranted in cases of testicular asymmetry or abnormal testicular ultrasonography. In cases in which testicular hematoma or orchitis is suspected, testicular biopsy is contraindicated.⁶

Several methods of testicular biopsy have been used in alpacas and llamas, with varying results and complications. These methods include a wedge biopsy, Tru-cut needle biopsy, core self-firing biopsy, or fine-needle aspiration.⁶

Wedge biopsy, also called incisional or open biopsy, requires the use of general anesthesia. The scrotum is aseptically prepared, and a small incision is made over the selected testicle through the skin, parietal vaginal tunic, and the exposed tunica albuginea, taking care to avoid vascularized areas. The testicular tissue will protrude through this incision in the tunica albuginea, and a scalpel blade is used to remove this section. The tunica albuginea, parietal vaginal tunic, and scrotal skin are closed with absorbable sutures. This type of biopsy provides a good sample of tissue for evaluation of the architecture of the seminiferous tubule and spermatogenesis, but its use has diminished because it is unsafe and has a very high rate of complications, including hematoma, hemorrhage, formation of testicular adhesions, inflammation, infection, and immune-mediated reactions. Additionally, degeneration of the germinal epithelium and seminiferous tubules may occur, and a transient decrease in spermatogenesis and spermatozoa output may be seen for several months.⁶

A Tru-cut needle may be used to obtain a sample of the testicular parenchyma either under general anesthesia or heavy sedation and physical restraint. The scrotum is aseptically prepared, and the scrotal skin incised with a scalpel blade. The Tru-cut needle is introduced through the tunica albuginea. Once placement of the needle is adequate, care should be taken to not further insert it when taking the biopsy sample. The scrotum is closed with either absorbable or nonabsorbable sutures. Sutures may be removed 10 to 14 days later. The Tru-cut technique results in a significant decrease in complications compared with incisional biopsy, but hematoma formation is still quite common. The sample obtained is adequate for histologic evaluation of the architecture of the seminiferous tubules and assessment of spermatogenesis.

A core self-firing biopsy instrument (Bard Biopsy Systems, AZ) is the best choice for testicular biopsy (Figure 19-3). The self-firing mechanism decreases the amount of testicular damage inflicted during the procedure, with hematoma formation as the primary complication. As with the Tru-cut biopsy, the scrotum is aseptically prepared, the scrotal skin incised with a scalpel, and the biopsy sample obtained. The tip of the instrument (14- to 18-gauge, depending on the male) is placed against the tunica albuginea and the instrument fired. The scrotum is left open. Samples are adequate for histologic evaluation (Figure 19-4). In our practice, males undergoing this procedure are given a single dose of long-acting ceftiofur preparation and flunixin meglumine for 2 days. No complications have been observed in our practice.

Another technique for core biopsy sampling of the testicle uses just a straight 14-gauge needle (Dr. LaRue Johnson, personal communication).

The safest method of testicular biopsy is also the least diagnostic.³ Fine-needle aspiration may be performed without the use of general anesthesia. A 20-gauge 1-inch needle is fitted to a 12-mL syringe and introduced into the testicular parenchyma, taking care to avoid the epididymis. Tissue is aspirated, redirecting the needle several times. The cells are then injected onto slides, which are stained with DiffQuick or Wright-Giemsa stain. A representative sample of cells may be obtained; however, the cellular architecture of the seminiferous tubules cannot be evaluated. Interpretation of the sample, therefore, is based on the amount of cellular material and presence of specific cell types (spermatogenic, Sertoli cells, Leydig cells) and is scored from 0 to 3 (Box 19-1; Figure 19-5). Spermatogenic cells are scored from 1 to 5 (see Box 19-1). Twenty-five percent of aspirated samples are nondiagnostic based on the cellular population obtained. Fine-needle aspiration has a low rate of complications, but the male should still be monitored for signs of inflammation or infection.

All males that undergo testicular biopsy should be treated with several days of systemic broad-spectrum antimicrobial therapy (starting prior to the procedure) and at least one dose of systemic antiinflammatory medication. The male should be monitored for signs of complications, including hematoma formation, fever, infection, increased testicular sensitivity, or discharge.

Trace Mineral Deficiency

Trace mineral imbalance of copper, zinc, and selenium are associated with derangements in spermatogenesis and infertility.14–18

Serum analysis of trace mineral levels in animals that are presented for infertility may not be reflective of stored levels in tissue (mainly liver); however, animals whose serum demonstrates low trace mineral levels may benefit from supplementation, especially those living in areas of known soil deficiencies. The producer should be aware that correction of trace mineral imbalances may not restore fertility, especially in cases of azoospermia or heat shock, but may help improve the motility and morphology of produced spermatozoa and may improve other systemic health aspects of the animal or the herd.

Heat Stress

Fevers caused by infection may reach extremes of 106°F (A. Tibary, personal observations) and last for several days. This elevation in body temperature will elevate scrotal and testicular temperature to the point of arresting spermatogenesis (heat shock). Other causes of heat shock include high ambient temperatures, combined with lack of shearing or trailering for shows or other travel purposes. In some cases, heat shock results in temporary oligozoospermia or azoospermia, but in cases of high fever or high ambient temperature for longer than 3 days, often the effects on spermatogenesis are long lasting and potentially irreversible. It has been demonstrated that in times of high ambient temperatures, shorn animals will dissipate heat from the entire body, whereas unshorn animals will increase the respiratory rate to maintain temperature homeostasis.¹⁹ However, despite maintaining normal body temperature, animals subjected to high ambient temperatures had significant decreases in both ejaculate volume and spermatozoa concentration, with histologic changes in the seminiferous tubule architecture and derangements in spermatogenesis.^19,20

Severely affected animals demonstrate the systemic effects of heat stress, including hyperthermia, depression, ataxia, weakness, dehydration, ketosis, hepatic lipidosis, and dyspnea.²¹ Serum electrolyte levels often demonstrate hypophosphatemia, hypocalcemia, hypomagnesemia, hyponatremia, hypochloridemia, and hypo- or hyperkalemia. Other derangements include hyperglycemia, elevations in alanine aminotransferase (AST) and creatine kinase (CK), anemia, and inflammatory leukogram. Prolonged derangements in untreated animals, especially in electrolyte levels, may lead to damage of the thermoregulatory center of the hypothalamus, which subsequently leads to multiorgan failure and death.²¹

The aim of the initial treatment is to cool and stabilize the animal with shearing, cold water, fans, and fluid therapy with added electrolytes to correct any imbalances identified in the serum biochemistry assay. Fluid rates should be monitored to prevent pulmonary edema. Dyspneic animals should be supplemented with intranasal oxygen. Additional treatments include correction of metabolic acidosis, antiinflammatory medications, antioxidants such as selenium and vitamin E, and broad-spectrum antibiotics to prevent secondary infection.²¹ Corticosteroids may be used in cases of severe inflammatory reactions but not in pregnant females because of risk of abortion. Scrotal and preputial edema should be monitored for resolution. Increased durations of increased scrotal temperature often lead to higher risks of spermatogenic arrest and subsequent infertility.