Predetermination of Sex in Production Animals Through the Use of Sperm Sexing, Gene Editing, and Spectroscopic Methods

Abstract 

Every year, staggering numbers of male chicks, calves, and piglets are killed in the U.S. The egg industry is responsible for the killing of 250 million male chicks every year [1]. Further, around 369,000 male dairy calves are slaughtered or sold for veal production annually [2]. Male piglets often undergo castration without sedation [9]. The animal welfare issues encompassing the culling and castration of unwanted male animals in food production can be addressed through a multitude of sex determination methods. Methods for sex determination in animals primarily address the issue of male animals being regarded as unwanted byproducts of food animal production. While additional methods in animal biotechnology used for predetermination of sex in livestock would help address these animal welfare concerns, application on a large scale across food industries must showcase feasibility and economic viability. Sperm sexing by flow cytometry, the CRISPR/Cas9 system, and spectroscopy methods in particular have the potential for widespread, efficient use in industry. 

Introduction 

The practice of culling male animals in food production is a controversial topic that has garnered attention and debate. Culling in the food animal production industry, primarily milk and egg production, refers to the deliberate removal or termination of male animals, often at a young age. 

Though controversial, one of the main reasons for culling (deliberately removing or terminating at a young age) male animals is the difference in productivity between males and females. In some cases, male animals do not possess the desirable traits that are sought after in terms of genetic potential, meat quality (swine), milk production, or egg-laying capacity. As a result, they are deemed less economically valuable than their female counterparts. Culling male animals allows producers to focus their resources and efforts on breeding and raising animals that are expected to yield higher returns. In swine, for example, males are either castrated without anesthetics or culled due to an unpleasant odor and taste in their meat known as “boar taint.” Boar taint is caused by the hormones androstenone, skatole, and indole during puberty, and is a significant hurdle for consumer acceptance [3]. Castration has been used to eliminate boar taint, but it causes concern for animal welfare as it is often performed without proper anesthesia or analgesics, leading to significant pain and distress for piglets. The welfare of livestock is based on freedom from hunger, thirst, discomfort, injury, disease, fear, and distress as well as the freedom to express normal behaviors [14]. In the context of piglet castration, this means freedom from discomfort and distress. These freedoms, encompassing the gold standard of animal welfare, have pushed some countries countries such as Norway to outlaw or enforce stricter regulations on castration without anesthesia or pain relief [4]. No equivalent federal laws currently exist in the United States. 

In recent years, there has been increasing interest in finding alternative solutions to address the ethical and welfare issues associated with the culling of male livestock, which are often considered unwanted byproducts. The hope is that advances in sexing methods will produce majority female offspring in food animals, which will reduce or eventually eliminate the need for sex-specific culling. This paper will specifically discuss methods of predetermination of sex through sperm sexing by flow cytometry, CRISPR/Cas9, and Raman spectroscopy. 

Sperm Sexing by Flow Cytometry 

Artificial insemination (AI) is a widely used reproductive technology in livestock production that fertilizes a female animal by introducing semen via an insemination rod into the reproductive tract. AI, introduced in 1930 and widely implemented in dairy cattle herds by 1938, revolutionized livestock breeding by allowing for the use of high-quality sires without the need for physical contact between male and female animals. This technique improves breeding efficiency and genetic selection [5]. This technique is commonly used in dairy cattle as it allows breeders to select sires with desirable traits for milk production as well as to produce greater proportions of female dairy calves through sexed semen. The rationale behind sexed semen, that is the separation of X-chromosome-bearing sperm (which produce female offspring) from Y-chromosome-bearing sperm (which produce male offspring), is that male dairy calves have limited economic value in traditional dairy farming systems as they do not produce milk. Thus, they are often sold and slaughtered for veal or sent to feedlots in the beef cattle industry [2]. Sexed semen allows farmers to allocate their resources (such as feed, housing, and healthcare) more efficiently to female calves that are directly contributing to milk production and herd improvement. In addition to AI, other advancements in reproductive technologies have further improved breeding efficiency and profitability. One such method is sperm sexing by flow cytometry, which was developed in the 1980s by Dr. Lawrence Johnson. This has become a valuable tool for commercial livestock operations utilizing AI [6]. 

Sperm sexing by flow cytometry utilizes a flow cytometer to sort X- and Y-bearing sperm. This allows for the selection of either X or Y spermatozoa that will produce the desired sex, which is typically female (X) in the case of production mammals. Sperm sexing by flow cytometry is a relatively recent method of predetermination of sex, yet it is one of the most widely used techniques/methods due to its ability to improve breeding efficiency and profitability [6]. According to Xie et al., this method uses a flow cytometer to analyze the DNA content X- and Y- bearing sperm to sort sperm cells, where the content in Y-bearing sperm is 3-4% less than X [10]. Sperm cells containing the chromosome for the desired sex can then be sorted out and used in artificial insemination. 

The process of sperm sexing by flow cytometry involves the use of a laser beam that passes through sperm cells as they are suspended in fluid. During this process, the sperm cells’ DNA content is analyzed by measuring the amount of fluorescence emitted by DNA-specific dyes that have been added to the fluid. Based on the differences in fluorescence intensity between X- and Y-bearing sperm cells, the flow cytometer sorts the sperm cells according to their sex chromosome content [7]. The sorted sperm cells are then collected, concentrated, and packaged for artificial insemination, most commonly in dairy cattle. While sexed semen is generally more expensive than conventional semen, the potential benefits in terms of increased milk production, genetic improvement, and reduced management challenges associated with male calves can outweigh the initial costs. However, the issue with sperm sexing lies in the fact that sorting reduces the fertility of the sperm, in which cattle pregnancy rates with sexed sperm are about 80% of those with nonsexed sperm [6].  Because the process of flow cytometry can cause stress for sperm cells and slightly reduce fertility, this may result in a decrease in pregnancy rates for cows. Additionally, sperm sexing, which is usually done in the dairy cattle industry, is not practical in other animals such as pigs and laboratory mice, warranting further research into alternative methods such as CRISPR/Cas9 and other gene editing tools [9]. 

CRISPR/Cas9 for the Predetermination of Sex at the Genetic Level 

CRISPR/Cas9 technology involves a complex molecular mechanism used to modify genomes, which can also be used for the predetermination of sex in offspring. It is a tool that has shown promise in application for livestock, particularly in species that exhibit issues with sexing by flow cytometry (e.g swine and mice). CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) works by utilizing a specific system of enzymes, known as the CRISPR-associated (Cas) system, to cut and modify targeted DNA sequences. The CRISPR/Cas9 system consists of two main components: a guide RNA molecule and a Cas9 enzyme. The guide RNA is designed to match a specific target DNA sequence, and when it binds to this sequence, it directs the Cas enzyme to cut the DNA at that location. Once the DNA is cut, the cell's natural repair mechanisms take over, either repairing the cut with the original DNA sequence or incorporating a new piece of DNA introduced by scientists [8]. In livestock, CRISPR/Cas9 can be used to introduce a genetic modification that causes the animal to only produce offspring of a particular sex [13]. 

The use of a genetic modification for sex selection was explored in the 2021 article “CRISPR/Cas9 effectors facilitate generation of single-sex litters and sex-specific phenotypes”, in which Douglas et al. developed a CRISPR/Cas9 system in laboratory mice models in order to produce the more preferable sex, particularly as it applies to animal agriculture [13]. Their findings indicated potential for implementation in other vertebrates as well. Topoisomerase (Top1) is a gene which aids in DNA replication and repair across mammalian species. The CRISPR/Cas9 strategy produces single-sex litters in mice by targeting and inactivating Top1 in a synthetic lethal system, which causes cell death. Essentially, researchers determined that using CRISPR/Cas9 to disrupt this gene would cause early embryonic death of the unwanted sex. This would in turn allow for efficient allocation of resources to the desired sex as there will be a reduction of about fifty-percent in mean litter size [13]. The results of this study showed potential for the application of using gene editing to disrupt Top1 to livestock. 

In a similar study surrounding the predetermination of sex in pigs, preliminary studies using CRISPR/Cas9 were conducted to produce all female mice. This technology could be applied to swine genetics in order to provide a new solution for boar taint instead of unethical castration. Under the scientific supervision of Dr. Björn Petersen, Stefanie Kurtz discovered that alteration of the SRY gene (sex-determining region located on the Y chromosome) in swine causes a “sex reversal” for males: “Structurally, the SRY encodes for testis-determining transcription factor (TDF)...[which] serves as the main factor triggering the sex determination cascade” [9]. This sex determining region of the Y chromosome is crucial for the development of testes and gonads; disruption of the SRY using CRISPR/Cas9 will essentially lead to a female phenotype in an animal that is otherwise genetically male (XY), where typical male reproductive organs fail to develop. A different study carried out by Ning Wu et al. demonstrated this concept with mice: “the Sry knockdown male gonads showed female-like gonad with poorly organized [testes] cords” [15]. This indicates that, theoretically, by knocking out the SRY gene in the Y chromosome responsible for male gender development in pigs prior to embryo transfer using genome editors, entire litters of pigs could be born phenotypically female. However, there are other male and female sex determination genes that have also been identified, which make it unclear if SRY is the only sex-determining gene in mammals, particularly swine [10]. 

Predetermination of Sex in Poultry In Ovo via Raman Spectroscopy 

Applying sperm sexing by flow cytometry and CRISPR/Cas9 to poultry, namely layer hens, gives rise to an array of issues that are not associated with sex determination in mammals. Sperm-sexing, which is mostly used in cattle, cannot be used in poultry as the sex of the offspring is determined by the female chromosome (W) and not the male chromosome (Z). As such, the genotypes of females and males are ZW and ZZ respectively [11]. This difference in chromosomes also leads to difficulty in the application of CRISPR/Cas9, as avian male chromosomes (Z) are larger than mammals (Y) and thus have more genes. Further, the sex of the developing chicks can be influenced by environmental factors such as temperature [11]. Therefore, there is a need for alternative methods of predetermination of sex in poultry. 

Researchers are currently investigating alternative methods to predetermining sex of egg-laying chickens in order to avoid the common practice of culling male chicks early in life. In ovo sex determination technology would allow for identification of sex within the egg, allowing for producers to separate male- and female-bearing eggs before hatching. This requires that the technology be widely-practicable in industry as well as rapid, cost-efficient, and precise. Negative side effects that could impact the health of the bird need to be limited as well. Furthermore, sex determination should be performed prior to the roughly seven-day point in incubation at which a chick is able to feel and respond to pain [11]. 

Taking these factors into account, Raman spectroscopy has the potential to be a suitable technology for this purpose. Raman spectroscopy (discovered in 1928) is a type of vibrational spectroscopy that uses monochromatic light to analyze the spectrum of scattered light after it interacts with a germinal or blood cell. It is useful for in ovo sex identification based on the spectral signature of blood or germinal cells of female and male birds. Its ability to retain information in as little as 80 hours after the start of incubation makes it a particularly appealing system [11]. First, a shell window (essentially a hole in the egg) is made for vessel sampling using a CO2 laser. By using a near-infrared excitation wavelength, live cells are not damaged, and a diode laser emitting a wavelength of 785 nm is used to excite Raman scattering. The laser is precisely focused on the samples as precision is necessary for high accuracy. According to Galli et al., recorded spectra of male and female embryos show distinguishable differences: “Spectra of male embryos exhibit stronger phosphodiester linkage stretching vibrations of nucleic acids and C–C stretching modes, respectively. Spectra of female embryos show slightly stronger amide III and CHx deformation modes” [12]. The recorded Raman signal is affected by a strong background fluorescence signal originating from hemoglobin, and the median fluorescence signal is stronger for male embryos due to a higher hematocrit, or proportion of red blood cells in the blood. Using Raman spectroscopy allows breeders to separate eggs containing the unwanted sex and dispose of them prior to the seven-day point in which a chick is able to detect and respond to pain. 

Benefits & Drawbacks 

While these methods hold promise for producing primarily female offspring in livestock, they each have their own distinct advantages and limitations. Sperm sexing by flow cytometry is widely utilized in the industry due to its cost efficiency, practicality, and established track record. However, its drawback lies in its accuracy, which typically ranges from 85% to 95%, in addition to slightly decreased semen quality [6]. The process involves evaluating and sorting each individual sperm cell, which can be time-consuming and restricts the quantity of sperm that can be accurately analyzed [6]. Consequently, this lower accuracy rate has caused industry farmers to be hesitant to invest in sexed semen. Moreover, the centrifugation step for sperm concentration prior to packaging can result in a small loss of sperm viability [6]. 

On the other hand, the CRISPR/Cas9 method of sex predetermination in livestock faces limitations regarding the role of the SRY gene in pigs' male phenotype development, which is yet to be fully understood compared to mice. The SRY gene was shown to play a major role in male sex determination in mice and that “a knockout of the SRY gene in mice [...] displayed suppressed testis development in the fetal gonadal ridges resulting in a female phenotype” [9]. The female phenotype would encompass both internal and external female genitalia. However, CRISPR/Cas9 poses some challenges when it comes to targeting male sex determining regions on the larger avian male Z chromosome (as opposed to the smaller mammalian Y male chromosome) that have more genes. Nevertheless, the CRISPR/Cas9 method boasts high accuracy, with a 100% efficacy rate in sex ratio selection of mammalian species studied [13]. 

In the case of sexing poultry using Raman spectroscopy, manually opening the shell can potentially disrupt embryonic function and structure. However, the technology and lasers used have been designed to be noninvasive to living cells and contactless to reduce chances of contamination, so developmental issues and reduced hatch rates may be less of a concern [12]. Furthermore, the application of this technology on a practical scale would require further research to develop automated methods. 

Economic viability is another crucial aspect to consider. Although the CRISPR/Cas9 technology initially incurs costs for designing guide RNAs, optimizing gene-editing protocols, and establishing the necessary laboratory infrastructure, in the long run it can be a more cost-effective and flexible approach compared to sperm sexing. Conversely, sperm sexing by flow cytometry requires specialized equipment, reagents, and technical expertise, which can be more expensive. However, the per-sample processing cost may be lower compared to the setup costs of the CRISPR/Cas9 system. Assessing the cost-effectiveness of these techniques depends on specific research or breeding program requirements, the number of animals involved, species used, and long-term goals. 

Long-term goals of a poultry operation also influence the economic feasibility of the use of Raman spectroscopy on a large scale. Raman spectroscopy equipment can be relatively expensive, and the initial investment may include the cost of the spectrometer (upwards of $12,000), laser source, and other accessories [16]. However, the long term benefit that potentially comes with allocating resources (i.e heat, energy, labor) to only eggs carrying female offspring and discarding those with males may help offset these costs. 

Conclusion 

The predetermination of sex in food animal production is a field that continues to evolve. Traditional techniques like sperm sexing by flow cytometry have proven cost-effective and practical for large-scale operations but may face limitations in accuracy and potential impacts on fertility. On the other hand, emerging techntaintologies such as CRISPR/Cas9 show promising efficiency in sex predetermination but introduce additional complexities and potential costs. While methods like Raman spectroscopy address challenges in sex predetermination for poultry, their lack of practicality in industrial systems (i.e lack of automated techniques) underscores the need for future research and development. Ultimately, the choice of sex predetermination methods in livestock breeding requires a careful evaluation of factors such as accuracy, economic feasibility, practical implementation, and potential long-term effects, with ongoing research contributing to the refinement of techniques for efficient, sustainable, and ethical livestock production systems.

About the Author: Anthony Magana

Anthony Magana is a third year Biotechnology major interested in pursuing a career in veterinary medicine. As a pre-veterinary student, he is interested in the role that biotechnology plays in animal health, welfare, and medicine and decided to apply this interest to this literature review originally written for his UWP 049 class. Overall, Anthony is passionate about the overlap between human and animal health and medicine. He is involved in campus organizations such as Mercer Clinic for Pets of the Homeless, Pre-Vet Students Supporting Diversity, and the biotechnology club.

Author's Note

The purpose of this paper is to investigate a variety of methods for the predetermination of sex in livestock animals. The methods discussed (CRISPR/Cas9 gene editing, sperm sorting, and Raman spectroscopy) help address the ethical issues that arise in regards to the slaughter of thousands of young male livestock animals in the egg, dairy, and meat industries. As a biotechnology major with an interest in animal biotechnology and veterinary medicine, I enjoy researching solutions to ethical concerns such as these and pondering the future research that this topic could stimulate. I hope that at the end of reading this paper, readers will also be prompted to investigate creative solutions to issues in agriculture in order to create a more sustainable and ethical system. 

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