Studies on the cryopreservation of boar spermatozoa and its integration into assisted reproductive technologies

The aim of this thesis was to investigate the possibility of integrating frozen-thawed boar semen into reproductive technologies and into commercial production of pigs in Australia. This was to be achieved by establishing a semen freezing and AI regime that was of a standard acceptable to industry, and integrating the resultant frozen-thawed sperm into other reproductive technologies, such as flow cytometric sperm sorting and IVF. Initially, a protocol for freezing and thawing boar semen was established, based on the method described by Westendorf et al. (1975) and attempts were made to modify this protocol to improve the post-thaw sperm quality, as determined by in vitro assessment of motility, acrosome integrity and longevity. First, the egg yolk used in the freezing extenders was investigated, and the chicken yolk was replaced with either duck or quail yolk. It was shown that there was no benefit in substituting yolk from duck or quail for the chicken yolk traditionally used in freezing extender. Second, the effect of seminal plasma addition to the freezing extender, or seminal plasma addition to resuspension medium post-thaw was tested. Incorporating whole seminal plasma into the freezing extender at levels above 50% was found to be detrimental to post-thaw sperm quality. Reducing levels to 20% of the final volume improved acrosome integrity, but adversely affected motility of sperm. However, adding 20% seminal plasma to the resuspension medium used after thawing of boar semen had no significant influence on sperm quality compared with resuspension in medium without seminal plasma. The antioxidant catalase, and the iron chelator desferal added to the freezing extender, did not improve post-thaw sperm quality, nor was any benefit seen with addition of these substrates to the resuspension medium post-thaw. However, the bioactive phospholipid PAF and its regulating enzyme PAF:AH appeared to enhance post-thaw motility and acrosome integrity of sperm, respectively, when added to the semen pre-freezing. Unfortunately, due to the restrictions imposed on rPAF:AH as a research drug, it was not possible to test the in vivo effects at this time. After the in vitro experiments were completed, the in vivo fertility of frozen-thawed sperm was tested using the optimal freezing protocol and a novel technology, enabling non-surgical deep intrauterine insemination of sows. The aim was to establish the lowest possible dose of frozen-thawed sperm that could be used, without compromising fertility. Successful pregnancies were achieved with doses as low as 62.5 x 106 frozen-thawed sperm but the farrowing rates were too low to be practicable on a commercial scale. This is the first report of litters born after insemination of such a low dose of frozen-thawed sperm and using the novel DIU insemination technique. However, it was concluded that a double dose of 250 x 106 frozen-thawed sperm was the minimum dose required for maintaining acceptable fertility. Reduction in sperm numbers to such an extent made it possible to consider non-surgical insemination of sex-sorted, frozen-thawed semen. Previously, pregnancies had been achieved only after surgical insemination of sex-sorted boar sperm, or with DIU insemination of unfrozen sperm, immediately after sex-sorting. The low numbers of sex-sorted sperm available restricted the inseminate dose used here to 50 x106 motile sperm. A litter of 5 piglets was born after a low-dose, DIU insemination of sex-sorted, frozen-thawed sperm. This is the first report of piglets born after insemination with sex-sorted frozen-thawed sperm and non-surgical insemination. The low farrowing rate achieved in this experiment prompted the investigation of integrating sex-sorted, frozen-thawed boar sperm into IVF. Morulae were produced after IVF with sex-sorted, frozen-thawed sperm and successfully transferred using non-surgical techniques. This is the first report of pregnancy achieved with non-surgical transfer of embryos produced after IVF and IVC of IVM oocytes with sex-sorted, frozen-thawed boar sperm. Unfortunately, the pregnancy did not hold, and the embryos were lost prior to Day 32, but PCR of non-transferred embryos confirmed successful pre-selection of sex. Overall, this thesis demonstrated that it is still not economically feasible to incorporate frozen-thawed boar semen into the commercial production of pigs although it has considerable application in breeding programmes. However, the development of novel techniques enabling reduction in sperm dose, and for non-surgical transfer of embryos into recipient sows and incorporation of frozen-thawed semen into these technologies means that progress is being made with the integration of reproductive technologies and frozen-thawed semen into the pig industry

A Coding Variant in the Gene Bardet-Biedl Syndrome 4 (BBS4) Is Associated with a Novel Form of Canine Progressive Retinal Atrophy

Progressive retinal atrophy is a common cause of blindness in the dog and affects >100 breeds. It is characterized by gradual vision loss that occurs due to the degeneration of photoreceptor cells in the retina. Similar to the human counterpart retinitis pigmentosa, the canine disorder is clinically and genetically heterogeneous and the underlying cause remains unknown for many cases. We use a positional candidate gene approach to identify putative variants in the Hungarian Puli breed using genotyping data of 14 family-based samples (CanineHD BeadChip array, Illumina) and whole-genome sequencing data of two proband and two parental samples (Illumina HiSeq 2000). A single nonsense SNP in exon 2 of BBS4 (c.58A > T, p.Lys20*) was identified following filtering of high quality variants. This allele is highly associated (P-CHISQ = 3.425e(-14), n = 103) and segregates perfectly with progressive retinal atrophy in the Hungarian Puli. In humans, BBS4 is known to cause Bardet-Biedl syndrome which includes a retinitis pigmentosa phenotype. From the observed coding change we expect that no functional BBS4 can be produced in the affected dogs. We identified canine phenotypes comparable with Bbs4-null mice including obesity and spermatozoa flagella defects. Knockout mice fail to form spermatozoa flagella. In the affected Hungarian Puli spermatozoa flagella are present, however a large proportion of sperm are morphologically abnormal andPeer reviewe

Vaginal and vestibular electrical resistance as an alternative marker for optimum timing of artificial insemination with liquid-stored and frozen-thawed spermatozoa in sows

Abstract Development of a pen-side test to objectively determine the ideal time for artificial insemination (AI) in the sow would save producers time and money. Current processes rely on identification of oestrus via subjective behavioural and physiological markers that are indicative of high blood oestrogen concentrations. This study attempted to use measurements of electrical resistance (ER) in the cervical mucus to pinpoint timing of AI accurately enough to lead to equivalent pregnancy rates as a natural mating. Thirty-six sows were divided into 3 groups and observed for signs of oestrus. Seven sows did not show any oestrus behaviour and were excluded from the study. The remaining 29 sows were inseminated via natural mating and conventional oestrus detection (n = 14), or inseminated artificially with either liquid-stored semen (n = 8) or frozen-thawed semen (n = 7) according to timing indicated from electrical resistance measurements in the vagina and vestibule. Sows that were artificially inseminated on the basis of the electrical resistance readings had a lower pregnancy rate (P = 0.034) and less piglets born alive per litter (P < 0.05) than those that were naturally mated according to a conventional oestrus detection regime. However, the pregnancy rate and total piglets born alive did not differ between the two groups that underwent artificial insemination. Change in electrical resistance in the vagina has the potential to accurately predict ovulation timing, but more work is required to refine the timing of AI in relation to the readings before the technique can be adopted by industry

Defining &ldquo;Normal&rdquo; in Pig Parturition

Animal production industries rely on efficient and successful reproductive outcomes, with pigs being no exception. The process of parturition in pigs (farrowing) can be especially prolonged, due to the large numbers of piglets being born (on average, approximately 13 piglets per litter in Australian conditions). Difficulties in farrowing (dystocia) lead to poor piglet outcomes and health problems in sows, in turn, causing economic loss for producers and welfare concerns for the animals. Despite the importance of this topic and publications in the area stretching back nearly 50 years, there is still no consensus on the prevalence of dystocia in pigs nor on how to identify a pig experiencing the condition. Understanding the process of parturition and the factors that influence its success is a crucial step towards the early identification of sows undergoing dystocia and development of best practices to assist them. This article describes the key factors that contribute to successful farrowing and identifies areas in which more research is required before the parturition process in the pig can be fully understood

Adjusting cryodiluent composition for improved post-thaw quality of rabbit spermatozoa

Improved fertility following artificial insemination with frozen-thawed spermatozoa would offer rabbit producers faster genetic improvement. Previous work investigating cryoprotectants for rabbit spermatozoa have reported inconsistent results. Semen was collected from three rabbit bucks by artificial vagina and frozen using a standard procedure with varied cryodiluent components. Post-thaw analysis encompassed motility, sperm kinematic parameters and acrosome and membrane integrity. Spermatozoa were evaluated at 0, 2 and 4 h after thawing. Experiment 1 compared diluents with 3.5% dimethyl sulfoxide (DMSO), 1.5% acetamide, 1.75% DMSO + 0.75% acetamide or 3.5% DMSO + 1.5% acetamide. The treatment that resulted in the highest post-thaw motility (P<0.001) and acrosome integrity (P<0.001) was DMSO alone. Experiment 2 compared 3.5, 7 and 10% DMSO in the cryodiluent. The best post-thaw sperm motility (P<0.001) and linearity (P=.002) was in 3.5% DMSO, while 10% DMSO afforded higher acrosome/membrane integrity at this last time point (P<0.05). Experiment 3 varied the cryodiluent to contain either 9 or 17% egg yolk or 9 or 17% low density lipoproteins extracted from whole egg yolk. The treatment with the best post-thaw result was 17% egg yolk (motility, P=0.01; acrosome/membrane integrity, P<0.001). Experiment 4 compared different carbohydrates in the cryodiluent; 50 mM glucose (TCG), 25 mM glucose with 25 mM sucrose (TCGS low), or 50 mM glucose with 50 mM sucrose (TCGS high). When data were pooled across time points, TCG had significantly higher motility than TCGS high (P=0.021), but was not different from TCGS low. However, TCG had fewer spermatozoa with intact acrosomes and membranes than both TCGS low and TCGS high (P=.002). Put together, these results indicate that the best cryodiluent for rabbit spermatozoa frozen under the conditions used in this paper is with 7% DMSO and 17% egg yolk in a base medium containing 25 mM glucose and 25 mM sucrose

Total motility and flow cytometric results from experiment 4.

<p>Post-thaw motility (A) and proportion of spermatozoa with intact membranes and acrosomes (B) from data pooled across 0, 2 or 4 hours post-thaw with incubation at 37°C of rabbit spermatozoa frozen in diluent containing 50 mM glucose as the sole carbohydrate source (Control; TCG; ●), a combination of 25 mM glucose and 25 mM sucrose (TCGS low; ■), or a combination of 50 mM glucose and 50 mM sucrose (TCGS high;▲). Each point represents datum from a replicate and the horizontal lines represent range around the mean. Superscripts represent significant differences within time points between treatment groups.</p

Motility kinematics from experiment 1.

<p>Motility kinematics from experiment 1.</p

Artificial insemination with fresh, liquid stored and frozen thawed semen in dromedary camels.

This study was conducted to evaluate various factors affecting fertility following insemination of dromedary camels. In experiment 1, camels were either bred by natural mating (NM) or inseminated in the body of uterus with whole, split (50:50) or 1 mL of undiluted ejaculate. In experiment 2, camels were inseminated with fresh diluted semen either in the body of the uterus or tip of the uterine horn and at either the time of ovulation induction (0 h), 24 or 30 h later. In experiment 3, camels were inseminated at the tip of the uterine horn with different doses of fresh diluted semen (75, 150 or 300 x 106 motile spermatozoa) or with 150 x 106 motile spermatozoa diluted with different extenders (Green buffer, Optixcell or Triladyl). In experiment 4, camels were inseminated in the tip of the uterine horn with diluted (Triladyl or Optixcell) liquid-stored semen or diluted (Triladyl) frozen-thawed semen consisting of either 300 or 500 x 106 motile spermatozoa. The pregnancy rate in camels bred by NM was similar to camels inseminated with whole undiluted ejaculates whereas insemination with 1 mL undiluted ejaculate resulted in lower pregnancy compared to whole and split undiluted ejaculates (P 0.05). No pregnancies were obtained with frozen thawed semen. In conclusion, this study demonstrated that the success of camel AI is highly dependent on sperm dose, location of semen deposition, timing of insemination and semen type. Further studies are required to determine the reason for the compromised fertility of preserved semen despite apparent high in vitro quality