Artificial Insemination (AI)
- Despite being widely used for long-term storage and dissemination of male genetic material in many species, semen cryopreservation and artificial insemination (AI) are not well-developed for camelids. The main reason for the delay in developing these technologies is the poor semen quality of camelids and the viscous nature of the seminal plasma, particularly in alpacas and llamas. An effective sperm cryopreservation and artificial insemination program would allow alpaca producers to cheaply and safely disseminate superior male genetics throughout the national herd and beyond. This report is aimed at all alpaca industry members and researchers in camelid reproduction and production. Alpaca farms are spread across Australia, but concentrated in the eastern states. All alpaca producers will find this report relevant. It will be of particular interest to those already incorporating assisted reproductive technologies (such as embryo transfer) into their husbandry or those wanting to increase the genetic diversity of their herd.
- Australia as a leader in the sheep industry has the experience, infrastructure, institutions, and assisted reproductive technologies required for genetic improvement of alpaca herds. With the development of artificial insemination, embryo transfer techniques, in vitro production of embryos (IVP) and the inauguration of a genetic improvement program for alpacas, it will be possible to develop a breed of extra fine Australian alpaca comparable to the alpaca kept by the Incas before the conquest in relatively few years.
Comparison of the Effect of Ovulation-Inducing Factor (OIF) in the Seminal Plasma of Llamas, Alpacas, and BullsWe have recently reported the presence of an ovulation-inducing factor (OIF) in the seminal plasma of llamas and alpacas—species characterized as induced ovulators. The study was designed to test the hypothesis that the seminal plasma of bulls will induce ovulation in llamas, and to compare the ovulation-inducing effect of seminal plasma of conspecific versus hetero-specific males. The seminal plasma of alpacas, a closely related induced ovulator (Lama pacos), and cattle, a distantly related ruminant species (Bos taurus) considered to be spontaneous ovulators, were compared with that of the llama (Lama glama). Ovulation and maximum corpus luteum diameter were compared by ultrasonography among female llamas (n = 19 per group) treated intramuscularly with 2 mL of phosphate buffered saline (PBS, negative control) and those treated with 2 mL of seminal plasma of bulls, alpacas, or llamas (conspecific control). The diameter of the preovulatory follicle did not differ among groups at the time of treatment. Bull seminal plasma induced ovulations in 26% (5/19) of llamas compared to 0% (0/19) in PBS group (P
- The aim of this project was to develop the technology for artificial insemination (AI) in alpacas by establishing efficient and reliable methods for the collection, processing, preservation and artificial insemination of alpaca semen, continuing the work of RIRDC project AAA-1A (publication 03/104).
Cryopreservation of Epididymal Alpaca (Vicugna pacos) Sperm: a Comparison of Citrate-, Tris- and Lactose-Based Diluents and Pellets and StrawsEpididymal spermatozoa were harvested from male alpacas and frozen after extension and cooling to 4°C in citrate-, Tris- and lactose-based diluents (Experiment 1) and as pellets in 0.25- and 0.5-mL straws on either dry ice or over liquid nitrogen vapour (Experiment 2) to determine the effects diluents and packaging on their motility and acrosome integrity. In Experiment 1, sperm motility was higher after cooling to 4°C and after freeze–thawing (0 but not 3 h post-thaw) for spermatozoa extended in the lactose- than the citrate- or Tris-based diluent (P < 0.05). Post-thaw acrosome integrity after cooling to 4°C and post-thaw (0 h) was reduced for spermatozoa frozen in citrate- compared with lactose- or Tris-based diluents, but was similar for all groups 3 h after thawing. In Experiment 2, sperm motility immediately after thawing was higher for pellet freezing than for 0.25- or 0.5-mL straws on dry ice or liquid nitrogen vapour (P < 0.05), although by 3 h post-thaw motility was similar for pellets and straws (P > 0.05). Acrosome integrity was similar for all groups immediately after thawing and 3 h post-thaw. Cryopreservation of epididymal alpaca spermatozoa is feasible, with retained motility and acrosome integrity post-thaw. Freezing as pellets in a lactose-based diluent is recommended.
- Degelification of highly viscous alpaca semen was attempted using two enzymes: trypsin and collagenase. Dilution effect on artificial insemination was determined in alpacas. Semen from 4 male alpacas was collected, degelified, diluted, and inseminated into 80 female alpacas. Degelification was achieved adding trypsin and collagenase enzymes to fresh semen samples. Semen was diluted with egg-yolk glucose citrate to give concentrations of 4, 8, and 12 million spermatozoa/mL. Females were induced to ovulate with human chorionic gonadotropin and then inseminated deep into the uterine horns. Analysis of variance was used to determine differences in the effect of trypsin and collagenase on sperm acrosome and on motility and live spermatozoa. The chi-square test was used to determine differences in pregnancy of artificially inseminated females. Semen was degelified with different concentrations of trypsin and collagenase. There were differences (p< .05) in the pregnancy rate of female alpacas inseminated with 4 million (53.3%), 8 million (66.7%), and 12 million sperm/mL (61.5%). Alpaca semen may be degelified using trypsin and/or collagenase. It seems that 8 million sperm/mL is adequate for artificial insemination in alpacas.
- The present study was designed to determine if the dose of purified ovulation-inducing factor (OIF) from llama seminal plasma required to provoke an ovulatory response is physiologically relevant in terms of the proportion present in a normal ejaculate and to test the hypothesis that corpus luteum (CL) form and function are affected by OIF in a dose-dependent manner. Female llamas were assigned randomly to five groups (n = 10 per group) and given a single i.m. dose of 500, 250, 125, or 60 μg of purified OIF (representative of the amount present in 1/25th to 1/200th of a normal ejaculate) or 1 ml of PBS (control). Ovulation and CL development were monitored by transrectal ultrasonography. Blood samples were taken to measure plasma progesterone concentrations and to determine changes in plasma concentrations of luteinizing hormone (LH). The high dose of OIF (500 μg) was associated with the highest incidence of ovulation (P < 0.05), the greatest maximum CL diameter (P < 0.05), and the largest day-to-day profiles of CL diameter (P < 0.05) and plasma progesterone concentrations (P < 0.01). A rise in plasma LH concentration was apparent in all llamas that ovulated and was most rapid and highest in the high-dose group (P < 0.01). The low dose of OIF (60 μg) was minimally effective for induction of ovulation and the least luteotrophic, as evidenced by the smallest maximum CL diameter and the smallest day-to-day profiles for CL diameter and plasma concentrations of progesterone and LH. Responses were intermediate for the middle-dose groups (125 and 250 μg). We conclude that OIF from llama seminal plasma has a dose-dependent effect on ovulation rate and CL form and function in llamas and that the biological effect of OIF is evident at physiologically relevant doses (i.e., as little as 1/100th of that present in an ejaculate).
Effect of Glycerol Concentration, Equex STM® Supplementation and Liquid Storage Prior to Freezing on the Motility and Acrosome Integrity of Frozen-Thawed Epididymal Alpaca (Vicugna pacos) SpermTwo experiments were conducted to determine the effects of glycerol concentration and Equex STM® paste on the post-thaw motility and acrosome integrity of epididymal alpaca sperm. In Experiment 1, epididymal sperm were harvested from male alpacas, diluted, and cooled to 4 °C in a Lactose cooling extender, and pellet-frozen in a Lactose cryodiluent containing final glycerol concentrations of 2, 3, or 4%. In Experiment 2, epididymal sperm were diluted in Biladyl®, cooled to 4 °C, stored at that temperature for 18–24 h, and further diluted with Biladyl® without or with Equex STM® paste (final concentration 1% v:v) before pellet freezing. In Experiment 1, sperm motility was not affected by glycerol concentration immediately (2%: 16.1 ± 4.6%; 3%: 20.5 ± 5.9% and 4%: 18.5 ± 6.6%; P > 0.05) or 3h post thaw (< 5% for all groups; P > 0.05). Post-thaw acrosome integrity was similar for sperm frozen in 2% (83.6 ± 1.6%), 3% (81.3 ± 2.0%) and 4% glycerol (84.8 ± 2.0%; P > 0.05) but was higher 3h post-thaw for sperm frozen in 3% (75.7 ± 3.8%) and 4% (77.2 ± 4.1%) than 2% glycerol (66.9 ± 2.7%; P < 0.05). In Experiment 2, sperm motility was higher immediately after thawing for sperm frozen in the presence of Equex STM® (Equex®: 21.5 ± 3.5%; control: 14.4 ± 2.1%; P < 0.05) but was similar at 3h post-thaw (P > 0.05). Acrosome integrity was similar for sperm frozen with or without Equex STM® paste immediately (control: 89.6 ± 1.2%; Equex®: 91.1 ± 1.4%; P > 0.05) and 3 h post-thaw (control: 69.3 ± 3.7%; Equex®: 59.9 ± 5.8%; P > 0.05). Sperm cryopreserved in medium containing 3–4% glycerol and 1% Equex STM® retained the best motility and acrosome integrity, even after liquid storage for 18–24 h at 4 °C prior to cryopreservation.
- Semen characteristics of alpacas were studied after repeated collections. Twelve adult males were divided into three groups of four each for semen collection once, twice, or three times every other day. The duration of copulation; volume of ejaculate; pH; motility; sperm concentration (number of sperm/milliliter semen); total number of sperm per ejaculate; and percentages of live, normal, and abnormal spermatozoa were analyzed by regression analysis. Semen color and consistency were analyzed by the chi-square test. Between the first, second, and third ejaculations, there were differences (p < 0.05) in sperm concentration; percentages of normal spermatozoa and abnormal spermatozoa; sperm with abnormal heads and abnormal tails; and consistency (viscous, viscous, and semi-viscous). There were no differences (p > 0.05) in ejaculated volume, percentage of live spermatozoa, pH, percentage of cytoplasmic droplets, and duration of copulation. Some males from which semen was collected on the three-mating schedule ejaculated only seminal plasma during the second and third copulation starting on Day 10 of the study. There were differences between males (p < 0.05) for most of the characteristics studied. In sum, frequency of mating affected some semen characteristics that may be important determinants of the fertility of male alpacas.
Effects on the Quality of Frozen‐Thawed Alpaca (Lama pacos) Semen Using Two Different Cryoprotectants and ExtendersAim: To evaluate two extenders and two cryoprotectant agents (CPA) for alpaca semen cryopreservation. Methods: Semen samples were obtained from four adult alpacas (Lamapacos) and frozen using extender I (TRIS, citrate, egg yolk and glucose) or extender II (skim milk, egg yolk and fructose), each containing either glycerol (G) or ethylene glycol (EG) as CPA. Consequently, four groups were formed: 1) extender I‐G; 2) extender I‐EG; 3) extender II‐G; and 4) extender II‐EG. Semen was diluted in a two‐step process: for cooling to 5 °C (extenders without CPA), and for freezing (extenders with CPA). Viability and acrosome integrity were assessed using trypan blue and Giemsa stains. Results: When compared, the motility after thawing was higher (P < 0.05) in groups II‐EG (20.0 %± 6.7 %) and II‐G (15.3 %± 4.1 %) than that in groups I‐G (4.0 %± 1.1 %) and I‐EG (1.0 %± 1.4 %). Viable spermatozoa with intact acrosomes in groups II‐EG (18.7 %± 2.9 %) and II‐G (12.7 %± 5.9 %) were higher than that in groups I‐G (5.7 %± 1.5 %) and I‐EG (4.0 %±1.0 %). Conclusion: The skim milk‐ and egg yolk‐based extenders containing ethylene glycol or glycerol to freeze alpaca semen seems to promote the survival of more sperm cells with intact acrosomes than the other extenders.
- Camelids are induced (reflex) ovulators. We have recently documented the presence of an ovulation-inducing factor (OIF) in the seminal plasma of alpacas and llamas. The objective was to test the hypothesis that OIF exerts its effect via a systemic rather than a local route and that endometrial curettage will enhance the ovulatory response to intrauterine deposition of seminal plasma in alpacas.
- Ovulation in mammals involves pulsatile release of GnRH from the hypothalamus into the hypophyseal portal system with subsequent release of LH from the anterior pituitary into systemic circulation. Elevated circulating concentrations of LH induce a cascade of events within the mature follicle, culminating in follicle rupture and evacuation. The broad classification of species as either spontaneous or induced ovulators is based on the type of stimulus responsible for eliciting GnRH release from the hypothalamus. In spontaneously ovulating species (e.g., human, sheep, cattle, horse, pigs), release of GnRH from the hypothalamus is triggered when, in the absence of progesterone, systemic estradiol concentrations exceed a threshold. In induced ovulators (e.g., rabbits, ferrets, cats, camelids), release of GnRH is contingent upon copulatory stimuli; hence, ovulation is not a regular cyclic event. Since a classic 1970 Peruvian study, dogma has maintained that physical stimulation of the genitalia during copulation is the primary trigger for inducing ovulation in alpacas and llamas. Exciting results of recent studies, however, provide direct evidence for the existence of an ovulation-inducing factor (OIF) in semen, and compel us to re-examine the mechanism of ovulation in both induced and spontaneous ovulators. Ovulation-inducing factor in seminal plasma is a potent stimulant of LH secretion, ovulation and luteal gland development, and acts via a systemic rather than a local route. OIF is a protein molecule that is resistant to heat and enzymatic digestion with proteinase K. It has a molecular mass of 14 kDa, and may be part of a larger protein complex or pro-hormone. The effect of OIF is dose-related and evident at physiologically relevant doses (i.e., as little as 1/100th that present in the ejaculate), and is mediated, in whole or in part, at the level of the hypothalamus in vivo. The factor exists in the seminal plasma of every species in which it has been examined thus far, including Bactrian camels, alpacas, llamas, cattle, horses, pigs, and koalas. Seminal plasma OIF does not appear to be a phylogenetic vestige in spontaneous ovulators since it (1) induced ovulation in pre-pubertal mice, (2) altered ovarian follicular wave dynamics in cows, and (3) elicited LH release in vitro from primary pituitary cell cultures of rats, mice, guinea pigs, rabbits, llamas and cows.
- Alpacas have become more popular during the last decades. The herds have been built up by importing live animals since reproductive biotechnologies, for example artificial insemination and semen preservation, are not well-developed in this species. A major problem is the viscosity of the seminal plasma which hinders processing or evaluation of the semen. Enzymes have been used to deal with the viscous seminal plasma but they may damage spermatozoa or render them incapable of fertilization. The use of reproductive biotechnologies would permit the introduction of new genetics without the need to import live animals, thus improving animal welfare and reducing the risk of spreading diseases. Therefore, our aim was to improve reproductive biotechnologies to help develop the Swedish alpaca breeding industry. Laboratory techniques were performed to select the best spermatozoa with Single Layer Centrifugation (SLC), in order to improve cryopreservation. These techniques were developed first using bull semen. There was an improvement in sperm quality in the SLC-selected samples, particularly from poor quality semen. In addition, the SLC technique could be modified to process small volumes. Alpaca epididymides were obtained after routine castration for husbandry purposes, with the intention of comparing semen extenders using extracted epididymal spermatozoa. Most of the organs came from pre-pubertal animals and therefore did not contain spermatozoa. Nevertheless, a decision-making tool for alpaca husbandry under Swedish conditions was developed. We suggest a combination of testicular size and body condition score as a tool for decision-making in the selection of potential sires for animal husbandry under Swedish conditions. A phantom was designed and built to collect semen samples in Sweden, and semen collection trials were also performed in Perú. The advantages and disadvantages of different semen collection techniques were evaluated. However, the problem with semen viscosity still has to be solved. Therefore a semen collection method should be established so that semen handling methods can be developed. We conclude that a phantom could be the best method to use for semen collection in Sweden, since it is a fairly simple technique and, as far as we are aware, there are no animal welfare concerns.
- Semen preservation and artificial insemination in South American camelids are reviewed giving emphasis to work done in Peru and by the authors. Reports on semen evaluation and the preservation process indicate that semen of alpacas and llamas can be manipulated by making it liquid first. Collagenase appears to be the best enzyme to eliminate viscosity. Tris buffer solution maintains a higher motility than egg-yolk citrate, phosphate buffered saline (PBS), Triladyl, and Merck-I extenders. Cooling of semen took 1h after collected, and equilibrated with 7% glycerol presented a better motility and spermatozoa survival at 1,7,15 and 30 days after being slowly frozen in 0.25mL plastic straws. Trials of artificial insemination with freshly diluted semen and frozen–thawed semen are encouraging and needs to be tested extensively under field conditions. Recently, fertility rates varied from 3 to 67%. Semen preservation and most important, artificial insemination appear to be a reality, and could be used to improve the genetic quality of alpacas and llamas.
Sire Genetics, Protein Supplementation and Gender Effects on Wool Comfort Factor in Australian Crossbred SheepAims: To investigate the effects of sire genetics, nutrition, level of supplementation, gender and their interactions on wool comfort factor (CF) and its correlation with other wool quality traits in crossbred sheep either grazing or supplemented with dietary protein. Study design: A 5 x 2 x 2 x 2 factorial experimental design comprising five sire breeds, two dietary protein sources, two supplementation levels and two sexes respectively, was utilized. Place and Duration of Study: University of Tasmania Farm, Cambridge, Hobart, Tasmania, Australia, between April 2008 and November 2010. Methodology: Texel, Coopworth, White Suffolk, East-Friesian and Dorset sires were joined with 500 Merino ewes at a mating ratio of 1:100 in individual paddocks. Five hundred of the crossbred progeny were raised on pastures until weaning at 12 weeks of age. Forty of the weaners with an initial body weight (BW) range of 23-31 kg (average of 27 ± 3.2 kg) were fed with lupins or canola at 1 or 2% BW for 6 weeks in individual metabolic crates. CF and other wool quality traits were commercially measured at the Australian Wool Testing Authority, Melbourne. The data were analyzed in SAS using MIXED model procedures with sire fitted as a random effect, while sire breed, nutrition, supplement, level of supplementation and gender and their interactions were fitted as fixed effects. Results: CF was significantly correlated with fiber diameter (-0.89), spinning fineness (-0.95) and wool curvature (0.33). Grass-fed sheep produced wool with significantly higher comfort factor (93.1±0.3%) than supplemented sheep (CF=85.9±1.1%). Sire genetics was a significant source of CF variation; White Suffolk crosses had the highest CF (90.1±8.7) and East-Friesian crosses the least (81.5 ± 10.1%). Males fed canola at 1%BW had the highest CF (90.8 ± 7.0%), while females fed lupins at 1% BW had the least (81.1±10.8%). Conclusion: From a practical point of view, sheep farmers engaging in prime lamb production with wool comfort factor as an additional breeding objective should concentrate their effort on grass-feeding White Suffolk x Merino wethers. During the winter feed gap, supplementing the wethers with canola at 1% BW will not compromise wool CF.