The development of assisted reproductive technologies in camelids, especially the alpaca (Vicugna pacos)

Abstract

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 reasons for the delay in developing these technologies are the poor in vitro semen quality of camelids and the viscous nature of the seminal plasma. Though high fertility is routinely achieved with natural mating on commercial alpaca farms, the low sperm concentration and high viscosity makes semen dilution and cryopreservation challenging. 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. The recent discovery that alpaca semen viscosity is caused by the protein mucin 5B (Kershaw-Young and Maxwell 2012b) and that this protein can be effectively broken down with the protease papain without affecting sperm quality (Kershaw-Young et al. 2013) has facilitated further attempts to cryopreserve alpaca spermatozoa and utilise cryopreserved spermatozoa in artificial insemination. The aims of the following thesis were to: 1) measure the effect of varying doses of β-NGF on the timing of ovulation, plasma progesterone (P4) concentration and corpus luteum (CL) size in female alpacas 2) determine the effects of diluent type, cryoprotectant concentration, storage method and freeze/thaw rates on the post-thaw survival of cryopreserved alpaca spermatozoa. 3) determine the effects of the addition of varying concentrations of seminal plasma pre-freeze and post-thaw on the function of cryopreserved alpaca spermatozoa 4) compare the fertility of fresh, papain-treated and frozen-thawed spermatozoa using artificial insemination. 5) validate a heterologous oocyte-binding assay for the assessment of alpaca sperm quality This project comprised both in vitro and in vivo experiments. The in vitro studies focused on the development of a semen cryopreservation protocol. Several key aspects of cryopreservation were optimised – the diluent type, concentration of cryoprotectant, storage vessel and freezing and thawing rates. Addition of seminal plasma both before and after freeze-thawing was also investigated. Sperm quality was assessed by subjective motility assessment, fluorescent staining to determine acrosome, membrane and DNA integrity. The in vivo studies determined the timing of ovulation, subsequent size of corpora lutea (by transrectal ultrasound) and plasma progesterone concentration (by radioimmunoassay of blood plasma) following treatment of females with either β-NGF or buserelin. In Chapter 5, the ovulation timing finding and those of the in vitro sperm cryopreservation experiments were used to conduct a field trial in which females were artificially inseminated with either fresh, papain-treated or frozen-thawed spermatozoa to establish fertilising potential of spermatozoa after having undergone these treatments. The major findings of these studies were as follows: 1) The time to ovulation after induction treatment was 26.2 ± 1.0 h. 2) The minimum dose of β-NGF for effective induction of ovulation was >0.1mg. 3) The post-thaw motility and viability of ejaculated alpaca spermatozoa has been greatly improved by optimising cryopreservation protocols. The best in vitro results were obtained when alpaca spermatozoa was frozen in an 11% lactose diluent, supplemented with 5% (v:v) egg yolk and 2-5% (v:v) glycerol in straws 2 cm above liquid nitrogen for 4 minutes, then thawed at 37˚C for 1 minute, without additional homologous seminal plasma. However, these freezing conditions did not result in any pregnancies when inseminated into 28 synchronised female alpacas. Thus, further studies are required to investigate the reason for the in vivo infertility and improve the fertility of cryopreserved spermatozoa. 4) The first pregnancy using papain-treated alpaca semen was obtained, allowing future studies to build upon this preliminary result. 5) The first steps in the development of a heterologous oocyte-binding assay for in vitro fertility assessment for alpaca spermatozoa were conducted, with promising results.