Production of Lambs After the Transfer of Fresh and Cryopreserved in vitro Produced Embryos from Prepubertal Lamb Oocytes and Unsorted and Sex-sorted Frozen-thawed Spermatozoa

Cumulus-oocyte complexes from hormone-stimulated 3-4-week-old (n=43) and 6-7-week-old (n=12) prepubertal lambs were matured in vitro and incubated with unsorted, or X- or Y-spermatozoa separated with a high-speed cell sorter (SX MoFlo)frozen-thawed. Presumptive zygotes were then cultured to the blastocyst stage, and transferred to recipients fresh or after cryopreservation (frozen). Oocyte cleavage was higher (p <0.05) with unsorted (515/926, 55.6%) than X- or Y-spermatozoa (261/672, 38.8% and 229/651, 35.2%, respectively) and blastocyst formation (% zygotes) by Day 9 of in vitro culture was lower (p <0.05) for X- (102/261, 39.1%) than unsorted spermatozoa (249/515, 48.3%), but did not differ between Y-spermatozoa (103/229, 45.0%) and unsorted spermatozoa, or between X- and Y-spermatozoa (p >0.05). For fresh embryos, survival to term was 50.0% (3/6) for unsorted, 0.0% (0/6) for X- and 16.7% (1/6) for Y-spermatozoa-derived embryos (p >0.05), and for frozen embryos was 4.0% (2/50) for unsorted, 9.1% (2/22) for X- and 2.9% (1/34) Y-spermatozoa-derived embryos (p >0.05). Of the two lambs born from X-spermatozoa-derived embryos, one was female (50%), and from the two Y-spermatozoa-derived lambs, both were male (100%), demonstrating that lambs can be produced after the transfer of fresh and cryopreserved IVP embryos derived from prepubertal lamb oocytes and frozen-thawed sex-sorted sperm.     

Device Applications of Side-Chain Ferroelectric Liquid Crystalline Polymer Films

Side-chain ferroelectric liquid crystalline polymers are currently used in a number of applications, including displays and electrical sensors. Comparisons with existing technologies and materials indicate that relative to ceramics, such polymers have lower figures of merit but offer greater durability in sensor applications.     

Impact of Distillery Effluent on Soil Carbon and Nitrogen Dynamics in a Vertisol of Central India

Distilleries produce a huge quantity of effluents, popularly known as spent wash (SW), which when bio-methanated produce post-methanation effluents (PME). A field experiment on soybean–wheat system was conducted for five consecutive years in a Vertisol of central India to evaluate the effect of distillery effluent (DE) on soil carbon and nitrogen dynamics. Ten treatment combinations consisting of control, 100% NPK + Farmyard Manure (FYM), and graded level of SW and PME were applied. Total carbon content of soil increased significantly with applications of FYM and DE. SW was found superior in enhancing carbon content of soil in comparison to PME. Farmyard Manure contributed more carbon toward the recalcitrant pool, whereas DE contributed more carbon toward the active and slow pool. Nitrogen (N) availability was significantly improved with the application of DE. Balanced application of DE may act as amendment for increasing C and N stocks in Vertisol.     

Isolation and molecular characterisation of Neospora caninum in cattle in New Zealand

AIM: To isolate Neospora caninum from the brains of naturally infected cattle and use molecular techniques to characterise the isolates.

METHODS: Neospora caninum tachyzoites were isolated in Vero cell culture from the brains of a cow and two calves. The isolates were characterised using polymerase chain reaction (PCR) methods, DNA sequencing, an immunofluorescent anti-body test (IFAT), transmission electron microscopy (TEM), and immunohistochemistry (IHC). The brains of the three cattle were subjected to histopathological examination. A pathogenicity study was conducted in 120 BALB/c mice.

RESULTS: Neospora caninum tachyzoites were isolated from all three cases and first observed in vitro between 14 and 17 days post-inoculation. Parasites were sub-cultured and maintained in Vero cell culture for more than 6 months. PCR products were generated for all three isolates, using two different primers. Sequencing of the PCR products and a subsequent BLAST search identified the isolates as N. caninum. In addition, the isolates tested positive using IFAT and IHC, and ultrastructure revealed by TEM was characteristic of N. caninum. Histopathological examination revealed lesions characteristic of N. caninum in 1/3 brains. In the pathogenicity study using BALB/c mice, the mortality rate was 3–7%.

CONCLUSION: This was the first successful isolation of N. caninum in New Zealand confirmed using molecular characterisation tests.     

The Ethics and Role of AI with Fresh and Frozen Semen in Dogs

The use of artificial insemination (AI) with fresh semen has resulted in many benefits for the management of dog breeding, but there are disadvantages that can sometimes be overlooked. Furthermore, poorer quality semen arising as a result of cryopreservation necessitates uterine insemination, which raises the potential for surgical insemination. A number of significant ethical concerns have been raised by key stakeholders (such as The Kennel Club and the Royal College of Veterinary Surgeons) about AI per se , but particularly about the use of surgical insemination. This paper discusses the technological development of AI and explores a number of the ethical issues raised by its application to dog breeding. An Ethical Matrix method is used to map the potential ethical issues for key interest groups, namely dogs, breeders, owners, veterinarians and wider society. There are national variations in the way in which institutions have evaluated potential ethical impacts, and this is reflected in the different regulatory frameworks governing the use of AI in dogs. In order to facilitate decision-making and reduce some of the ethical risks associated with this technology, the veterinary research community could take several proactive steps including: (i) clarifying clinical decision-making processes, (ii) enhancing informed choice among clients and (iii) increasing the knowledge-base of potential impacts of AI.     

Quantification of the DNA Difference,and Separation of X‐ and Y‐Bearing Sperm in Alpacas (Vicugna pacos)

Sperm sexing is an emerging reproductive technology which has been successfully used to produce offspring of a pre‐determined sex in domestic and wildlife species but has yet to be applied to New World camelids. The aims of the present study were to (i) optimize the Hoescht 33342 (H33342) staining concentration for the flow cytometric separation of X and Y chromosome‐bearing alpaca (Vicugna pacos) sperm nuclei, (ii) separate alpaca sperm nuclei into high purity (>90%) populations bearing the X‐ and Y‐chromosome and (iii) determine the DNA difference between X‐ and Y‐bearing sperm in alpacas. Semen was collected from alpacas and sperm nuclei stained with H33342, incubated and analysed using a high‐speed cell sorter (SX‐MoFlo^®). H33342 staining concentrations of 36, 54, 72 or 90 μm did not affect the proportion of correctly oriented sperm nuclei (43.3 ± 3.9, 46.4 ± 3.7, 44.5 ± 4.0 and 51.1 ± 2.5% respectively) nor the speed of sorting (1381 ± 160, 1386 ± 123, 1371 ± 133 and 1379 ± 127 sperm nuclei/s). Sort reanalysis determined high levels of purity for X‐ and Y‐enriched populations (96.6 ± 0.7% and 96.1 ± 1.1% respectively). The DNA difference, based on fluorescence intensity (determined by the SX‐MoFlo^®), was 3.8 ± 0.06%. These data demonstrate for the first time that alpaca sperm nuclei can be separated into high purity populations and the potential for applying sperm sexing technology to New World camelids.