Technique and Application of Sex-Sorted Sperm in Domestic Farm Animals
The Food and Agriculture Organization of the United Nations has recognised that the production of pre-sexed livestock by sperm or embryo sexing as a useful breeding tool to increase production efficiency, especially for traits that are sex-related. In this chapter, we briefly explain sex determination in mammals, review approaches to identifying X and Y chromosome-bearing sperm and their practical implications for semen handling and artificial insemination (AI) and compare their importance and success in the main farm animal species. The problems associated with current technology for sperm sexing, as reflected in the damage caused to mammalian sperm are then considered, followed by an assessment of the potential for replacing this technology by other methods.
In mammals, the most efficient method to bias sex ratios in offspring is to separate X and Y chromosome-bearing sperm by flow cytometry before insemination. Numerous other techniques purporting to alter the sex ratio have been proposed or discussed. None of these were able to produce significant separation of fertile X and/or Y sperm populations or were not repeatable. Only quantitative methods, which differentiate between X and Y sperm on the basis of total DNA and then apply flow cytometric sorting, have been able to separate the two sperm populations with high accuracy. Sperm are labelled with a DNA fluorescent dye. After recognition and electric charging, droplets containing single sperm are deflected and pushed into a collection medium from which they are further processed. This set-up allows the identification and selection of individual sperm into populations with sort purities above 90% of the desired characteristics. A critical point is the orientation of sperm in front of a UV laser, requiring modifications of a standard flow cytometer. A specially designed nozzle assembly hydrodynamically focusses the sperm-containing laminar core stream by means of a sheath fluid and the specific geometrics of the internal assembly parts.
Sperm sorting requires special liquid media. For example, a system based on Tris extender has been developed for bull and ram semen. Besides TRIS and other ingredients, the medium contains antioxidant scavengers to combat reactive oxygen species (ROS) and the Hoechst dye 33342. Porcine semen is handled in a similar way, except that the sample fluid is based on TRIS-HEPES. The sample fluid for stallion semen is generally based on skim milk, INRA 96 or Kenney’s modified Tyrode (KMT). Sorted samples are collected in tubes pre-filled with collection medium. The composition of this medium is, in most cases, a TEST-yolk extender, supplemented with seminal plasma in order to decapacitate the collected sperm.
In the animal industries, changing the sex ratio of offspring can increase genetic progress and productivity. Animal welfare can be improved, for example, by decreasing obstetric difficulties in cattle and minimising environmental impacts by eliminating the unwanted sex. Sexed sperm has been most widely applied in the dairy industry, and it is likely that this will continue, dependent on the market situation. For US dairy farmers, milk production and the sale of surplus calves and cull cows are as important as the production of replacement heifers on-farm. Outside the USA, at least in Europe and Australia, the demand for sexed sperm is potentially high for milk producers to optimise herd management. In these countries, the genetically superior cows will be bred with X chromosome-bearing sperm to produce genetically superior females with high milk yield and for (female) pregnant heifer export to other countries. Besides AI, embryo transfer (ET) can be performed after insemination with sex-sorted sperm. The combination of sex-sorted sperm with in vitro embryo production (IVEP) is advantageous, but much more difficult than ET, and depends on species, individual semen donor and composition of media used for in vitro maturation, in vitro fertilisation (IVF) and in vitro culture.
Commercialisation of sex-sorted ram sperm has, to date, been restricted by the dearth of commercial sorting facilities in Australia and New Zealand, although sheep are the only species in which sex-sorted frozen-thawed sperm have been shown to have comparable, if not superior, fertility to that of non-sorted frozen-thawed controls. Moreover, there has been little incentive to take up the technology due to low rates of adoption of genetic improvement programmes and/or artificial breeding technology.
In pigs, apart from economic benefits from faster growth rates, sex-sorted sperm would provide major welfare advantages through the elimination of surgical castration. However, the current method of individual sperm sorting is not efficient enough to satisfy the potential demands of the porcine AI industry, due to the high number of sperm required for each insemination. For special applications, such as building up nucleus herds or for research, sexed boar sperm can be utilised in combination with specially adapted insemination strategies. A significant reduction in the total sperm dose, maintaining fertility, can be achieved if porcine semen is deposited deep in the uterus in front of the utero-tubal junction or directly into the oviduct. Only very few sperm are required for IVF using in vivo or in vitro matured oocytes. Transferring both gametes into the oviduct at the same time (gamete intrafallopian transfer – GIFT) can be used as an alternative to IVF. Even fewer sperm are required for intracytoplasmic sperm injection (ICSI) than for all other IVF methods. However, to date, these methods require laparoscopy or laparotomy for insemination, embryo or gamete transfer, which are not practicable as alternatives to castration.
In horses the preferred gender depends on the breed and range of use. Stallion sperm have a low sorting index and their sortability varies, not only among stallions but also among ejaculates. Additionally, the freezability of stallion sperm varies widely. Insemination with sex-sorted sperm has to be performed by hysteroscopy deep into the uterine horn, limiting the technology to high-value animals.
The sex-sorting process can cause sperm damage. The main sources of damage are incubation with the fluorescent stain and exposure to the UV laser, mechanical forces and electrical charge.
Future sorting methods may avoid the need to identify quantitative differences between X and Y chromosome-bearing sperm. This would require a specific marker related to only one sex. A promising system is based on gold nanoparticles, which can be functionalised with DNA probes. After internalisation of the probe into the sperm head, the Y chromosome-bearing sperm can be identified due to their strong plasmon resonance, which is more stable than fluorescent dyes. Non-invasive coupling of a specific DNA probe with the intact DNA double strand by triplex binding and accumulation of nanoparticles has been achieved, but to date internalisation of the gold nanoparticles requires further research. Another promising new method promotes the naturally occurring genomic variations by gene editing. It is not a question of if, only when these methods will be ready for the market and replace the existing sexing techniques.
This article is dedicated to Dr. Lawrence A. Johnson, who contributed most to the development and introduction of sperm sexing in farm animal reproduction. The authors of this paper are very thankful for his constant support and friendship. Larry Johnson celebrated his 80th birthday on July 9th, 2016. We also gratefully acknowledge all the students, technicians and scientists, who contributed in the laboratories of both authors, and who made the research on sperm sexing such an interesting part of our lives. We honour the personal friendships created during these projects, including that between the authors, which has encompassed some 25 years of collaboration.
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