Predicting Fertility Potential In Male Alpacas Through Testicular Length

Background

Alpacas are bred in Australia primarily for their fine fleece. Generation intervals are long, and genetic gain is slow, because 60-80% of males reach puberty around 2 years of age, females reach puberty when at two-thirds of mature body weight which may take 1-3 years gestation length is around 342 days and twins are rare.

In addition, whilst embryo transfer has been commercialised in Australia, it is not yet possible to freeze alpaca semen or embryos on a commercial basis 8. Therefore, it is essential to select fertile males at a young age to minimise generation intervals to optimise genetic gain and fleece production in alpacas.It has been shown that mean fibre diameter can be extrapolated for an animal from the age of 1.5-2 years, it would therefore be advantageous to asses a superior animal (low mean fibre diameter and desirable coat colour) for potential fertility as early as possible also.

Customarily sires have been chosen based on early prepuce-penis freedom as yearlings. Just 8% of males have accomplished this by the age ofone 2. Sumar states that sires are also picked by their testicular size dependent on the assumption that there isa direct relationshipbetween testicular size and sperm production as observed in other production species. The urogenital tract, including testicular anatomy in camelids, has been described by others 3.

Progress has been made into the development of more advanced reproductive technologies in camelids around the world. The aim of this research is to act as an adjunctive and practical method for the early selection of high quality sires. The intention is to develop a method that can be used in the field by both vets and trained farmers. This will assist in optimising the selection of quality sires. Improving overall fertility potential and ultimately genetic gains in the Australian alpaca herd  by choosing desirable traits of fleece diameter and colour 4.

Two decades ago in SE Australia, approximately 70 male alpacas were examined before and after castration in an attempt to identify a field method of predicting when to use young male alpacas for breeding.  The study identified that mean testicular length correlated better with testicular weight and was physically easier to measure than total scrotal width. It was identified that all alpacas with a mean testicular length < 3 cm were azoospermic, because histologically no spermatogenic tubules in any males contained elongated spermatids. Two-thirds of males with a mean testicular length of 3-4 cm produced no or few spermatozoa and only one-third of males with a mean testicular length of 4-5 cm had reached the mature pattern of spermatogenesis.

Methodology

The study showed that at any age, there is great variation in the development of spermatogenesis. Nevertheless, under adequate climatic and nutritional conditions, alpaca males that have failed to achieve pregnancies by 3 years of age are regarded as sub-fertile, and not an asset to any breeding herd. Testicular weight was also measured in the 2000 study by Galloway, he found that a minimum weight of 30grams was necessary to be fully functional (100% of seminiferous cross sections containing long spermatids) 1. This suggests that the longer the testicle, and therefore the heavier, potentially correlates to more mature sperm cells being present.  

More recently, a Swedish study has found that as a single measure, testicular length was a good predictor of the presence of long spermatids and that alpacas with a mean testicular length ³ 3.8 cm would be producing sperm 1.but that azoospermia occurs when mean testicular length is < 1.7 cm. The results in this study agreed that testicular size was a better attribute to predict the chances of sperm production rather than selection of alpacas by age 6. These findings indicated that further measurement of reproductive indices in Australian male alpacas would be useful to further assist Australian alpaca breeders with early selection of fertile males for breeding.

Castration of 80 entire animals took place in winter on a property in North-East Victoria, Australia. The ear tag number, birth date, body weight, thoracic circumference, whithers height and body condition score was recorded for each animal. It was ensured that both testicles were present and then the length and width of both the right and left testicles were measured – both length and width using digital callipers.  

Anaesthesia and analgesia –1mg/kg of meloxicam (Ilium Buccalgesic^Ò, Troy Animal Healthcare) with 0.5mg/kg xylazine hydrochloride added (Ilium Xylazil-20^Ò, Troy Animal Healthcare) administered orally. Overall concentration once mixed were 8mg/ml of meloxicam and 4mg/ml of oral xylazine.

Standing, closed surgical castration, consisting of two scrotal incisions parallel to and 1cm lateral to the median raphe. Incision length was sufficient to remove the testicle and to allow for good post-operative drainage. Closed castration method, removing poorly vascularised tunica albuginea and adipose tissue from the wound to minimise the risk of infection or delayed healing. An angiotribe was applied for one minute prior to testicle removal, to guard against the risk of pulling the testicular artery off the aorta. If it was deemed that the oral analgesia was not effective enough for some individuals, 1ml lignocaine was injected into each spermatic cord.

Results

Local anaesthetic and antiseptic gel containing lignocaine, bupivacaine, adrenaline, and cetrimide (Trisolfen^Ò, Bayer) was sprayed onto/into the surgical incisions after castration.

All castrated animals were turned out onto a large clean, 20cm tall pasture to maximise exercise and minimise wound contamination. Alpacas were walked around by management for five minutes twice a day for three days to keep wounds open and draining well and provided opportunity to detectany reluctance to move.Animals were observed at 24 and 48 hours post-surgery, then again at 10 and 24 days post-surgery by the local veterinarian. All animals recovered uneventfully.

Immediately after removal of both testes from each male, a 5 mm thick cross-section of tissue from both left and right testes was collected using a new scalpel blade.The testicular specimens were fixed in Bouin’s fixative rather than 10% neutral buffered formalin because the acetic acid component of the former penetrates tissues rapidly to coagulate nucleic acids, thereby optimising the morphological appearance of the nuclei and cytoplasmic components of the cells in stained histological slides.    

After fixation and standard post-fixation rinsing in water and immersion in 70% alcohol, the specimens were trimmed into labelled cassettes and processed overnight in an enclosed, fluid-transfer, automated tissue processor (with the routine sequence of tissue dehydration in increasing concentrations of ethanol, clearance by xylene and thence infiltration with paraffin wax).  The paraffin-embedded tissue blocks were sectioned at a thickness of 3 μm and stained with haematoxylin and eosin for light microscopic examination.  

Slides were examined at x400 magnification. Cellular stages of spermatogenesis of 40 round cross-sections of seminiferous tubules of left and right testes (80 per animal) were recorded for each male in a similar manner to Galloway (2000). Stages were:sertoli cells only, spermatogonia, spermatocytes, round spermatids and elongated spermatids. A mature pattern of spermatogenesis was defined as > 60% spermatogenic tubules containing elongated spermatids (Galloway, 2000).

Descriptive statistics and regression analyses were generated using Microsoft^â Excel for Mac (Version 15.31). Results are presented as means ± standard error of the mean (sem).

The data was split into two age groups, animals below three years and those equal to 3 years and above (see table 1). Every alpaca >3.2 years (n=25) had long spermatids present. Within the older group of alpacas, 80% of animals have over 70% long spermatids (71.25%-93.75% ave=82.86%). Every male with mean testicular length > 3.38 cm was producing long spermatids.

There were possibly 10 animals with unilateral hypoplasia (8% with at least 0.5 cm [range between 0.5-0.7 cm] variation between right & left testes length). This result is the same percentage as found by Galloway in 2000.

Interpretation of Results

Table 1. Mean age, body weight, testicular length and percentage of spermatogenic tubules containing elongated spermatids (±sem) and range in males depending on whether more or less than 3 years of age. *standard error of the mean

Testicular length was chosen as the best measurement due to there being more variation with age. The width is not as variable between age groups. (1.59cm is the difference between the mean testicular length age groups, 0.44cm is the difference between the mean testicular width age groups) There was a stronger correlation between testicular length and the presence of long spermatids (R²=0.49).No males less than 2 years of age exhibited a mature pattern of spermatogenesis (>60% tubules with long spermatids). There was considerable variation in percentage of spermatogenic tubules containing elongated spermatids in males 2-3 years of age (Figure 1). However, males ³ 3 years of age exhibited a mature pattern of elongated spermatids or zero elongated spermatids 2.

Figure 1. Comparison of percentage of spermatogenic tubules containing elongated spermatids and age (years; ?) with data collected by Galloway.

To attempt to further equip farmers with practical advice in choosing the best sires they can. It will assist in narrowing in on the best genetic gains for alpacas in Australia by deciding for desirable traits for fleece diameter and colour and also males with higher fertility potential. Due to the relatively slow genetic increase in this species, sire choice is paramount 5.

Galloway’s study suggested that the maturation of testicles in alpacas was not dependent only on age. Our study upheld this finding, age cannot be used as a significant determinant in the case of determining and advancing reproductive techniques in male alpacas(see figure 2) . This study also shows that there is no sperm production in scrotums less than 3 cm in length. A significant part of his study is finding 90% long spermatids in scrotums measuring over 5cm 7.

Figure 2. Simple polynomial regression of mean testicular length (cm) and age (years; ?; y = -0.1571x² + 1.2764x + 1.4819, R² = 0.240; n=80) compared with data collected by Galloway (2000; ?; n=50).

Regardless of age, when mean testicular length was < 3 cm, < 10% of spermatogenic tubules contained elongated spermatids (Figure 3). Regardless of age, when mean testicular length > 4 cm, all males in this study showed a mature pattern of spermatogenesis 9.

There was considerable variation in percentage of spermatogenic tubules containing elongated spermatids when mean testicular length was 2-3 cm in males less than 3 years of age. However, when mean testicular length was ³ 3.4 cm, males ³ 3 years of age all showed a mature pattern of spermatogenesis.

Implications for Breeding and Selection

Figure 3. Comparison of percentage of spermatogenic tubules containing elongated spermatids and mean testicular length (cm) in males < 3yo (?) and males ³ 3yo (?), with data collected by Galloway (2000; ?).

Testicular length was chosen as the more reliable measurement due to there being more variation with age than testicular width. Testicular length is shown to be more reliable than age as a predictor of stage of spermatogenesis.

The intention of this research was to discover a good practical method for predicting the fertility potential in male alpacas. While there was a correlation between testicular width and the percentage of long spermatids visualised, the correlation between testicular length and percentage long spermatids was stronger. The correlation was still not as strong as expected 11.   

After reviewing the data, it was found that alpacas are much more varied than other production animals when it comes to body size and testicular size relating to each other or age or to the types of cells present in the testicles. This study has found the best correlation to be between testicular length and percentage of long spermatids. Some general cut off measurements can be used to make management decisions. This is useful, as just with rams and bulls, alpaca testicles can be measured as part of a breeding soundness exam. It was demonstrated that all animals with testicular length exceeding 3.38cm had long spermatids.  Further in this cohort, all animals over 3.2 years of age were producing long spermatids, supporting historical choices to mate 3 year old males Ideally, young alpacas with longer testicles should be selected for breeding. This is a practical in vivo measurement which can be used by veterinarians and experienced alpaca farmers 10.

Another study has confirmed that measurement of external testicular dimensions with calipers is useful to evaluate testicular size but discrepancies between caliper and ultrasonographic measurements were significantly greater in summer  so castrations in this study occurred in winter.

Studies have found, including this one, that potential early fertility is difficult to predict by age or weight. Testicular length at a young age is the best measure found, but even then it is not as strong a correlation as was expected.

This should be useful to further equip farmers with practical advice in choosing the best sires they can, in turn assist in narrowing in on the best genetic gains for alpacas in Australia by deciding for desirable traits for fleece diameter and colour and also males with higher fertility potential. Due to the relatively slow genetic increase in this species, sire choice is paramount.

Alpacas prove to be a unique production species that make it difficult to designate a single attribute to predict fertility in males. This suggests that perhaps one attribute isn’t enough in the selection of superior sires. Several attributes, including long testicles should be looked at together in making management decisions about which males will be retained for breeding purposes. This is particularly the case where not only fertility is the aim of selection, but also phenotype especially pertaining to fleece colour and diameter 12.

Further research into reduction of generation times and desirable selection traits will go on to aid in better alpaca management and advancement in the Australia fibre market. The areas where alpacas are currently raised call for a more local perspective than some other production systems. The environmental, nutritional and general management systems vary too much to compare reliably. While age proves to be unreliable in predicting future fertility there may be an important nutritional element.

References

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2. Al-Bulushi S, Manjunatha B, de Graaf S et al. Reproductive seasonality of male dromedary camels. Animal Reproduction Science2019;202:10-20.
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4. Cruz A, Cervantes I, Burgos A et al. Estimation of genetic parameters for reproductive traits in alpacas. Animal Reproduction Science2015;163:48-55.
5. McGregor B, Butler K. Sources of variation in fibre diameter attributes of Australian alpacas and implications for fleece evaluation and animal selection. Australian Journal of Agricultural Research2004;55:433.
6. Rashid M, Beveridge I, Vaughan J et al. Worm burdens and associated histopathological changes caused by gastrointestinal nematodes in alpacas from Australia. Parasitology Research2019;118:1031-1038.
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8. Tomlinson M, Jennings A, Macrae A et al. The value of trans-scrotal ultrasonography at bull breeding soundness evaluation (BBSE): The relationship between testicular parenchymal pixel intensity and semen quality. Theriogenology2017;89:169-177.
9. Vaughan J. Ovarian function in South American camelids (alpacas, llamas, vicunas, guanacos). Animal Reproduction Science2011;124:237-243.
10. LENGHAUS C, O’CALLAGHAN M, ROGERS C. Coccidiosis and sudden death in an adult alpaca (Lama pacos). Australian Veterinary Journal2004;82:711-712.
11. Vaughan J, Mihm M, Wittek T. Factors influencing embryo transfer success in alpacas—A retrospective study. Animal Reproduction Science2013;136:194-204.
12. Rankin A, Hosking K, Roush J. Corneal sensitivity in healthy, immature, and adult alpacas. Veterinary Ophthalmology2011;15:31-35.

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