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International Journal of Molecular Zoology (online), 2024, Vol.14, No.6 ISSN 1927-534X http://animalscipublisher.com/index.php/ijmz © 2024 AnimalSci Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved. Latest Content A Review of Canid Immunogenomics: How Domestication Shaped the Canine Immune System Jinya Li, Jing He, Mengyue Chen International Journal of Molecular Zoology, 2024, Vol. 14, No. 6, 297-304 Optimization of Reproductive Technologies in Water Buffalo: A Review of Current Practices Xiaoli Chen, Shiqiang Huang International Journal of Molecular Zoology, 2024, Vol. 14, No. 6, 305-314 Optimizing Goat Feed Formulation to Reduce Costs and Improve Production Efficiency Guoxiang Li, Liuhui Li, Chengjie Zhang International Journal of Molecular Zoology, 2024, Vol. 14, No. 6, 315-325 Evaluating the Effectiveness of Smart Sensors in Livestock Health Monitoring Haimei Wang International Journal of Molecular Zoology, 2024, Vol. 14, No. 6, 326-333 Feature Review on the Use of Genomic Selection in Chicken Breeding: Current Practices and Future Prospects Hongbo Liang, Jia Xuan International Journal of Molecular Zoology, 2024, Vol. 14, No. 6, 334-343
International Journal of Molecular Zoology, 2024, Vol.14, No.6, 297-304 http://animalscipublisher.com/index.php/ijmz 297 Review Article Open Access A Review of Canid Immunogenomics: How Domestication Shaped the Canine Immune System Jinya Li, Jing He, Mengyue Chen Animal Science Research Center, Cuixi Academy of Biotechnology, Zhuji, 311800, Zhejiang, China Corresponding author: mengyue.chen@cuixi.org International Journal of Molecular Zoology, 2024, Vol.14, No.6 doi: 10.5376/ijmz.2024.14.0026 Received: 03 Nov., 2024 Accepted: 05 Dec., 2024 Published: 16 Dec., 2024 Copyright © 2024 Li et al., This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Li J.Y., He J., and Chen M.Y., 2024, A review of canid immunogenomics: how domestication shaped the canine immune system, International Journal of Molecular Zoology, 14(6): 297-304 (doi: 10.5376/ijmz.2024.14.0026) Abstract This study reviews the genetic basis of immune function in dogs, with a focus on key immune genes such as major histocompatibility complexes (MHC) and toll like receptors (TLRs). The evolutionary changes of immune genes during domestication were examined, and the comparative analysis between domestic dogs and their wild relatives (such as wolves and coyotes) highlighted significant immune genomic variations caused by differences in pathogen exposure and selection pressure. Taking sled dogs as an example, the unique immune adaptation to extreme environments was demonstrated, revealing how selection pressure affects immune gene diversity and pathogen resistance. The development direction of canine immune genomics was reviewed, including emerging technologies, personalized health management, and protection of immune gene diversity in wild dogs. This study emphasizes the importance of immune genome research in advancing our understanding of the impact of dog health, evolutionary biology, and domestication on immune system function. Keywords Canid; Canine immunogenomics; Domestication; Immune system; Major histocompatibility complex (MHC) 1 Introduction Canid immunogenomics is a burgeoning field that explores the genetic underpinnings of the immune system in canids, including domestic dogs and their wild relatives such as wolves and foxes (vonHoldt et al., 2011). This area of study leverages high-throughput sequencing technologies to decode the complex genetic variations that contribute to immune responses and disease susceptibility in these species. Structural variations (SVs), copy number variations (CNVs), and epigenetic modifications are among the key genomic elements investigated to understand how they influence immune function (Koch et al., 2016; Wang et al., 2018). Domestication has profoundly impacted the canine immune system, primarily through selective breeding and adaptation to human environments. Studies have shown that domesticated dogs exhibit significant genomic differences from their wild counterparts, including variations in genes associated with immune responses. For instance, structural variations specific to dogs have been linked to genes involved in energy metabolism, neurological processes, and immune systems, suggesting that these changes were crucial for adapting to new diets and living conditions during domestication (Zhao, 2018). Additionally, methylation patterns and CNVs have been found to differ significantly between dogs and wolves, further highlighting the role of domestication in shaping the canine immune system (Ramírez et al., 2014; Serres-Armero et al., 2017). This study reviews the current research status of canine immunogenomics, including domesticated dogs and their wild relatives, providing a comparative perspective for better understanding the genetic and epigenetic mechanisms of immune response in canine animals. It focuses on the effects of domestication on the canine immune system, explores various genomic elements, including SVs, CNVs, and epigenetic modifications, and their roles in immune function. The aim of this study is to provide insights into the evolutionary dynamics of the canine immune system by synthesizing recent research findings and to identify areas for future research.
International Journal of Molecular Zoology, 2024, Vol.14, No.6, 297-304 http://animalscipublisher.com/index.php/ijmz 298 2 Genetic Basis of the Canine Immune System 2.1 Overview of the canine immune system The canine immune system, like that of other vertebrates, is composed of innate and adaptive components that work together to protect the host from pathogens (Chen, 2024). The innate immune system provides the first line of defense through pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs), which detect pathogen-associated molecular patterns (PAMPs) and initiate immediate immune responses (Vaure and Liu, 2014; Vijay, 2018). The adaptive immune system, on the other hand, involves the major histocompatibility complex (MHC) molecules that present antigens to T cells, facilitating a more specific and long-lasting immune response (Migalska et al., 2019). 2.2 Major histocompatibility complex (MHC) in canines The MHC genes are crucial for the adaptive immune response in canines, encoding proteins that present foreign antigens to T cells. These genes exhibit high polymorphism, which is believed to be maintained by pathogen-mediated selection (Bartocillo et al., 2021). In canines, MHC class I molecules, such as DLA-88*50801, have been structurally characterized to reveal diverse peptide-binding modes, which are essential for recognizing a wide array of pathogens (Xiao et al., 2016). Studies on raccoon dogs, a non-model canid species, have shown that MHC class I genes are subject to positive selection and balancing selection, indicating their evolutionary adaptation to pathogen pressures. 2.3 Toll-like receptors (TLRs) and pathogen recognition TLRs are a family of PRRs that play a pivotal role in the innate immune system by recognizing PAMPs and initiating immune responses (Figure 1) (Fitzgerald and Kagan, 2020). In canines, TLRs such as TLR2, TLR4, and TLR5 are involved in recognizing bacterial components and other pathogens (Quéméré et al., 2015). These receptors are highly polymorphic, which allows for a broad recognition spectrum and adaptability to various pathogens (Minias et al., 2021). The expression and functionality of TLR4, for instance, vary across different species, including dogs, which has implications for vaccine development and therapeutic interventions. The evolutionary dynamics of TLRs in canines suggest that these receptors are under continuous selection pressure to maintain their diversity and functionality in pathogen recognition (Quéméré et al., 2021). 3 Impact of Domestication on Immune Genes 3.1 Evolutionary changes in immune genes during domestication Domestication has significantly influenced the evolution of immune genes in canids. Structural variations (SVs) in the genome, such as insertions, deletions, and translocations, have been identified as key factors in the domestication process. These SVs are particularly enriched in genes associated with immune systems, indicating that immune function has been a critical area of adaptation during domestication (Wang et al., 2018). For instance, the insertion of a new copy of the AKR1B1 gene in dogs, which is highly expressed in the small intestine and liver, suggests an enhanced ability for de novo fatty acid synthesis and antioxidant activity, likely in response to dietary changes during the agricultural revolution. 3.2 Adaptation to human-influenced environments The adaptation of canids to human-influenced environments has also shaped their immune systems. The shift from wild habitats to human-dominated landscapes exposed domestic dogs to a new range of pathogens, necessitating changes in their immune responses. This is evident in the increased expression of immune-related genes and the presence of structural variations that enhance immune function. Additionally, the European roe deer, which has expanded into agricultural landscapes, shows that exposure to new pathogens can drive the evolution of immune genes, such as toll-like receptors (TLRs), which continue to evolve dynamically in response to pathogen-mediated positive selection (Quéméré et al., 2015). 3.3 Genetic bottlenecks and immune gene diversity Domestication has also led to genetic bottlenecks, which have had a profound impact on immune gene diversity. Small population sizes during domestication and strong artificial selection for specific traits have increased the
International Journal of Molecular Zoology, 2024, Vol.14, No.6, 297-304 http://animalscipublisher.com/index.php/ijmz 299 number of deleterious genetic variants in domestic dogs compared to their wild counterparts, such as gray wolves (Marsden et al., 2015). This is reflected in the higher ratio of amino acid-changing heterozygosity to silent heterozygosity in dogs, indicating a higher genetic load. The bottlenecks associated with domestication and breed formation have reduced the efficiency of natural selection, leading to an accumulation of deleterious variants in regions of the genome implicated in selective sweeps. This highlights the importance of maintaining large population sizes to prevent the accumulation of deleterious variants and preserve immune gene diversity. Figure 1 Multiple TLR family members can detect PAMPs on individual microorganisms (Adopted from Fitzgerald and Kagan, 2020) 4 Comparative Immunogenomics of Wild and Domestic Canids 4.1 Immune gene diversity in wild canids Wild canids, such as wolves, coyotes, and foxes, exhibit significant immune gene diversity, which is crucial for their survival in diverse and often challenging environments. For instance, studies have shown that wild canids like the gray wolf (Canis lupus) and the red fox (Vulpes vulpes) possess a wide array of immune genes that enable them to respond effectively to various pathogens (Vinkler et al., 2023). The genetic diversity in these species is shaped by both historical and contemporary evolutionary forces, including genetic drift and pathogen-mediated selection. For example, toll-like receptors (TLRs) in wild canids continue to evolve dynamically, reflecting ongoing adaptation to pathogen pressures.
International Journal of Molecular Zoology, 2024, Vol.14, No.6, 297-304 http://animalscipublisher.com/index.php/ijmz 300 4.2 Immunogenomic variations in domestic breeds Domestic dog breeds exhibit distinct immunogenomic variations compared to their wild counterparts. The domestication process has led to structural variations (SVs) in the genome, which have significant implications for immune system function. For example, domestic dogs have been found to possess specific SVs, such as insertions and deletions, that are enriched in genes related to immune responses (Wang et al., 2018). Additionally, copy number variations (CNVs) in domestic dogs show significant differences from those in wild canids, with certain CNVs being associated with immune response genes (Serres-Armero et al., 2017). Despite the population bottlenecks during domestication, domestic dogs maintain a similar proportion of CNV loci as wild canids, suggesting selective pressures favoring these variations. 4.3 Host-pathogen co-evolution in wild vs. domestic canids The co-evolution of hosts and pathogens has led to distinct immunogenomic landscapes in wild and domestic canids (Canuti et al., 2022). In wild canids, the continuous exposure to a wide range of pathogens drives the evolution of diverse immune genes, enabling these animals to adapt to new and emerging infectious diseases (Quéméré et al., 2015). For instance, the red fox has shown susceptibility to SARS-CoV-2, highlighting the ongoing interaction between wild canids and novel pathogens (Porter et al., 2022). In contrast, domestic dogs have undergone significant genomic changes due to human-mediated selection, which has influenced their immune system. The domestication process has introduced new immune challenges, such as those related to close contact with humans and other domestic animals, leading to unique immunogenomic adaptations. 5 Case Study: Immune Adaptations in Sled Dogs 5.1 Unique immune gene profiles in sled dog breeds Sled dogs, such as the Alaskan malamute and Siberian husky, exhibit unique immune gene profiles that have evolved to support their demanding lifestyles in harsh environments. These breeds have been subject to both natural and artificial selection pressures, leading to distinct genetic adaptations. For instance, structural variations (SVs) in the dog genome, including insertions, deletions, and translocations, have been linked to immune system functions (Figure 2) (Wang et al., 2018). These SVs are enriched in genes associated with energy metabolism and immune responses, which are crucial for sled dogs that endure extreme cold and physical exertion (Zhao, 2018). Figure 2 Structural variation in the dog genome (Adopted from Wang et al., 2018) Image caption: (a) Circle diagram showing SVs detected by the dog-dhole alignment (yellow) and the dog-wolf alignment (black). (b) SVs in the dog genome by identified by multiz alignment. Each ring from the inner ring outwards represents translocations, insertions, deletions, repeats and inversions, respectively (Adopted from Wang et al., 2018)
International Journal of Molecular Zoology, 2024, Vol.14, No.6, 297-304 http://animalscipublisher.com/index.php/ijmz 301 5.2 Pathogen resistance in sled dogs Sled dogs have also developed enhanced resistance to various pathogens, a trait that is vital for their survival in environments where they are exposed to a wide range of infectious agents. Studies on other canid species, such as the raccoon dog, have shown that major histocompatibility complex (MHC) genes play a significant role in pathogen resistance through high allelic diversity and positive selection (Bartocillo et al., 2021). Similar mechanisms are likely at play in sled dogs, where pathogen-driven selection has shaped their immune gene repertoire. Additionally, the identification of positively selected genes linked to immunity in other dog populations, such as African dogs, suggests that sled dogs may possess unique genetic adaptations that confer resistance to specific pathogens (Liu et al., 2018). 5.3 Lessons from sled dogs for understanding canine immunogenomics The study of sled dogs provides valuable insights into canine immunogenomics and the broader implications of domestication on immune system evolution. The genetic adaptations observed in sled dogs highlight the role of structural variations and positive selection in shaping immune responses. These findings underscore the importance of considering both natural and artificial selection pressures in the study of domesticated species (Pilot et al., 2016; Serres-Armero et al., 2021). Furthermore, the unique immune profiles of sled dogs can inform research on other canid species and contribute to our understanding of the genetic basis of disease resistance and environmental adaptation in domestic dogs (Wilbe et al., 2010; Vinkler et al., 2023). 6 Future Directions in Canine Immunogenomics 6.1 Emerging technologies for immunogenomic research The field of canine immunogenomics is rapidly evolving with the advent of new technologies. High-quality draft genomes of various canid species, such as the gray wolf and dhole, have provided insights into structural variations (SVs) that are crucial for understanding phenotypic evolution, disease susceptibility, and environmental adaptations in dogs (Wang et al., 2018). Whole genome re-sequencing and the development of fine-scale genomic maps of segmental duplications (SDs) have enabled the identification of copy number variations (CNVs) that play significant roles in sensory perception, immune response, and metabolic processes. Additionally, the Dog10K Consortium aims to sequence 10 000 canid genomes, which will capture the genetic diversity underlying phenotypic and geographical variability, further advancing our understanding of canine immunogenomics (Ostrander et al., 2019). 6.2 Integrating immunogenomics with canine health management Integrating immunogenomic data with canine health management can lead to improved disease prevention and treatment strategies. For instance, the identification of the dog erythrocyte antigen (DEA) 1 blood group in both domestic and non-domestic canids has implications for blood transfusion practices, ensuring compatibility and reducing the risk of transfusion reactions (Charpentier et al., 2020). Moreover, understanding the genetic basis of immune responses, such as the role of natural killer (NK) cells in cancer immunotherapy, can inform the development of targeted treatments for dogs with naturally occurring cancers (Gingrich et al., 2018). The creation of a canine PD-L1 antibody and a caninized PD-L1 mouse model exemplifies how immunogenomic research can translate into effective immunotherapies for both canine and human cancers (Oh et al., 2023). 6.3 Conservation and genetic management of wild canid populations Conservation efforts for wild canid populations can benefit significantly from immunogenomic research. The study of structural variations and CNVs in wild canids, such as gray wolves, can reveal genetic adaptations that are essential for their survival and inform conservation strategies (Serres-Armerong et al., 2017). Additionally, the identification of novel genetic variants, such as the distinct lineage of canine distemper virus (CDV) circulating among domestic dogs in India, highlights the importance of monitoring and managing disease outbreaks in wild canid populations to prevent cross-species transmission and ensure their long-term viability (Bhatt et al., 2019). Integrating immunogenomic data with conservation practices can help maintain genetic diversity and resilience in wild canid populations.
International Journal of Molecular Zoology, 2024, Vol.14, No.6, 297-304 http://animalscipublisher.com/index.php/ijmz 302 7 Concluding Remarks The review of canid immunogenomics has highlighted several key findings regarding how domestication has shaped the canine immune system. Comparative studies of natural killer (NK) cells in dogs have shown that canine NK cells exhibit distinct transcriptional profiles under various conditions and are more similar to human NK cells than to those of mice, providing valuable insights for translational NK studies. Research on zoonotic intestinal helminths has revealed that these parasites modulate the canine immune system by altering T cell responses and preventing dendritic cell maturation, which helps in understanding the immune evasion strategies of these parasites. The study of the canine transmissible venereal tumor (CTVT) has uncovered specific genomic aberrations that enable its long-term persistence and adaptation, shedding light on the mechanisms of clonal transmissibility and immune evasion. Additionally, the evolution of MHC class I genes in raccoon dogs has demonstrated the role of pathogen-driven positive selection and long-term balancing selection in maintaining allelic diversity, which is crucial for immunological fitness. The findings from these studies have several implications for future research. The similarity between canine and human NK cells suggests that dogs could serve as a valuable model for studying human NK cell biology and developing NK cell-based immunotherapies. The immunomodulatory effects of zoonotic helminths on the canine immune system highlight the need for further investigation into parasite-host interactions and the development of novel therapeutic strategies to manage parasitic infections. The insights gained from the study of CTVT can inform research on other transmissible cancers and contribute to the development of targeted therapies that can disrupt the mechanisms of immune evasion and clonal propagation. The extensive allelic diversity of MHC class I genes in raccoon dogs underscores the importance of studying non-model canid species to understand the evolutionary pressures shaping immune gene diversity and to identify potential targets for enhancing disease resistance. The evolution of the canine immune system has been profoundly influenced by domestication, pathogen interactions, and genetic diversity. Domestication has likely led to selective pressures that have shaped immune responses to better suit the environments and lifestyles of domestic dogs. The interaction with various pathogens, including viruses, bacteria, and parasites, has driven the evolution of immune genes, such as MHC class I, to enhance pathogen recognition and immune response. The study of canid immunogenomics not only provides insights into the adaptive mechanisms of the canine immune system but also offers valuable models for understanding human immunology and developing novel therapeutic approaches. Future research should continue to explore the genetic and environmental factors that influence immune function in canids, with the goal of improving health outcomes for both dogs and humans. Acknowledgments We would like to thank Professor Meng for his/her invaluable guidance, insightful suggestions, and continuous support throughout the development of this study. Conflict of Interest Disclosure The authors affirm that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest. References Bartocillo A., Nishita Y., Abramov A., and Masuda R., 2021, Evolution of MHC class I genes in Japanese and Russian raccoon dogs, Nyctereutes procyonoides (Carnivora: Canidae), Mammal Research, 66: 371-383. https://doi.org/10.1007/s13364-021-00561-y Bhatt M., Rajak K., Chakravarti S., Yadav A., Kumar A., Gupta V., Chander V., Mathesh K., Chandramohan S., Sharma A., Mahendran K., Sankar M., Muthuchelvan D., Gandham R., Baig M., Singh R., and Singh R., 2019, Phylogenetic analysis of haemagglutinin gene deciphering a new genetically distinct lineage of canine distemper virus circulating among domestic dogs in India, Transboundary and Emerging Diseases, 66(3): 1252-1267. https://doi.org/10.1111/tbed.13142
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International Journal of Molecular Zoology, 2024, Vol.14, No.6, 305-314 http://animalscipublisher.com/index.php/ijmz 305 Review Article Open Access Optimization of Reproductive Technologies in Water Buffalo: A Review of Current Practices Xiaoli Chen, Shiqiang Huang Tropical Animal Resources Research Center, Hainan Institute of Tropical Agricultural Resources, Sanya, 572000, Hainan, China Corresponding author: shiqiang.huang@hitar.org International Journal of Molecular Zoology, 2024, Vol.14, No.6 doi: 10.5376/ijmz.2024.14.0027 Received: 05 Nov., 2024 Accepted: 07 Dec., 2024 Published: 18 Dec., 2024 Copyright © 2024 Huang and Chen, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Chen X.L., and Huang S.Q., 2024, Optimization of reproductive technologies in water buffalo: a review of current practices, International Journal of Molecular Zoology, 14(6): 305-314 (doi: 10.5376/ijmz.2024.14.0027) Abstract This study provides a comprehensive overview of the biological and physiological basis of water buffalo reproduction, including reproductive anatomy, seasonal breeding, and hormonal regulation. It critically examines current reproductive technologies such as artificial insemination, estrus synchronization, in vitro fertilization, embryo transfer, and sex-sorted semen, while also exploring emerging technologies like genome editing, precision breeding, and reproductive biomarkers. A detailed case study highlights the application of embryo transfer technology for genetic improvement, emphasizing practical outcomes and lessons learned. Key factors influencing the adoption of these technologies, including socio-economic, infrastructural, and regulatory considerations, are discussed, alongside challenges such as biological constraints and technological barriers. Finally, the study proposes strategies for improving reproductive efficiency through training, infrastructure development, and policy support. This study underscores the importance of optimizing reproductive technologies to achieve sustainable water buffalo production and suggests future research directions to further enhance these efforts. Keywords Water buffalo; Reproductive technologies; Artificial insemination; Genome editing; Embryo transfer 1 Introduction Water buffaloes play a crucial role in global agriculture, particularly in regions such as South Asia and the Mediterranean, where they are a primary source of milk, meat, and draught power. They are well-adapted to harsh environments and can thrive on low-quality forage, making them invaluable in ecologically disadvantaged agricultural systems. In countries like Nepal, buffaloes contribute significantly to milk and meat production, underscoring their economic importance (Devkota et al., 2022). However, despite their adaptability, the reproductive efficiency of water buffaloes is often compromised due to various biological and management challenges (Bm, 2019). Reproductive efficiency is a critical factor in water buffalo production, as it directly impacts the economic viability of buffalo farming. Challenges such as delayed puberty, silent estrus, and prolonged postpartum intervals lead to suboptimal fertility rates, causing significant economic losses for farmers (Singhal et al., 2021). The reproductive performance of buffaloes is further affected by environmental factors like climatic stress and poor nutrition, which can delay puberty and extend postpartum anoestrus (Bm, 2019). Effective reproductive management strategies, including assisted reproductive technologies (ART) and hormonal protocols, are essential to enhance fertility and improve overall herd productivity (Warriach et al., 2015; Coman et al., 2024). This study attempts to explore the significance of reproductive efficiency in water buffalo production, discuss the challenges faced in optimizing reproductive technologies, and provide an overview of the effectiveness of various reproductive technologies and management strategies. By synthesizing recent developments and identifying areas for improvement, this review seeks to offer insights that can enhance the reproductive performance of water buffaloes and support the sustainability of buffalo farming globally.
International Journal of Molecular Zoology, 2024, Vol.14, No.6, 305-314 http://animalscipublisher.com/index.php/ijmz 306 2 Biological and Physiological Basis of Reproduction in Water Buffalo 2.1 Reproductive anatomy and physiology Water buffaloes (Bubalus bubalis) are multipurpose livestock known for their milk, meat, and draught power. However, they exhibit low fecundity characterized by delayed puberty, less pronounced estrus signs, and long postpartum anestrus periods (Qg et al., 2020). The reproductive anatomy of buffaloes is similar to that of other bovines, but their physiological characteristics, such as ovarian cyclicity and estrus expression, are often influenced by environmental and management factors (Bm, 2019). The ovarian function during the estrous cycle is crucial for the application of assisted reproductive technologies (ART), which aim to synchronize follicular development and ovulation (Baruselli et al., 2018). 2.2 Seasonal breeding and its implications Buffaloes are seasonal breeders, with reproductive activity peaking during periods of decreasing day length, typically from late summer to early autumn. This seasonality is influenced by both exogenous factors like photoperiod and climate, and endogenous factors such as hormonal regulation (D’Occhio et al., 2020). Seasonal breeding patterns can lead to challenges in maintaining consistent milk production and reproductive efficiency throughout the year. In regions like Nepal, buffaloes show active breeding from July to December, with low reproductive activity from April to June and January to March (Devkota et al., 2022). This seasonality necessitates management strategies to mitigate its impact, such as hormonal treatments to induce estrus and ovulation during the non-breeding season. 2.3 Hormonal regulation of reproduction The hormonal regulation of reproduction in buffaloes involves complex interactions between endogenous hormones and external factors. Melatonin, produced by the pineal gland, plays a significant role in regulating reproductive seasonality by influencing gonadotropin secretion and gonadal function (D’Occhio et al., 2020). The presence of melatonin receptors, such as MT1 and MT2, and their genetic polymorphisms have been associated with variations in reproductive activity (Gunwant et al., 2018). Hormonal protocols, including melatonin implantation and estrus synchronization, have been developed to enhance reproductive performance, particularly during the non-breeding season. These protocols aim to control follicular and luteal functions, allowing for timed artificial insemination and improved conception rates (Baruselli et al., 2018). In summary, the reproductive efficiency of water buffaloes is intricately linked to their biological and physiological characteristics, seasonal breeding patterns, and hormonal regulation. Understanding these factors is essential for optimizing reproductive technologies and improving productivity in buffalo herds. 3 Current Reproductive Technologies in Water Buffalo 3.1 Artificial insemination (AI) Artificial insemination (AI) is a pivotal technology for genetic improvement and controlling the breeding period in water buffalo. However, AI in buffalo is more challenging than in cattle due to irregular estrous cycles, subdued estrous behavior, and reproductive seasonality, which can lead to higher rates of anestrus and embryonic mortality outside the breeding season. Recent advancements in AI protocols focus on controlling the luteal phase with prostaglandins and progesterone, and managing follicle development and ovulation using hormones like GnRH, hCG, eCG, and estradiol. These protocols facilitate fixed-timed AI, eliminating the need for estrous detection and improving reproductive efficiency (Neglia et al., 2020; Coman et al., 2024). 3.2 Estrus synchronization techniques Estrus synchronization (ES) is often used alongside AI to enhance reproductive efficiency in buffalo. The difficulty in detecting estrus in buffaloes can lead to suboptimal timing of AI, reducing reproductive success. Various ES protocols, such as those involving prostaglandin and GnRH, have been developed to improve pregnancy rates. Studies have shown that administering ovulatory hormones like GnRH or hCG at the time of AI can significantly enhance pregnancy rates, making these protocols both effective and cost-efficient for field application (Atabay et al., 2020; Ahmad and Arshad, 2020; Du et al., 2021).
International Journal of Molecular Zoology, 2024, Vol.14, No.6, 305-314 http://animalscipublisher.com/index.php/ijmz 307 3.3 In vitro fertilization (IVF) and embryo transfer (ET) In vitro fertilization (IVF) and embryo transfer (ET) are advanced reproductive technologies that allow for the genetic improvement of buffalo herds. These techniques involve the collection of oocytes, fertilization in vitro, and the transfer of embryos to recipient females. The combination of IVF with sexed semen can further enhance genetic gains by allowing for the selection of offspring sex. Although these technologies offer significant potential, their application in buffalo is still limited by factors such as the efficiency of oocyte retrieval and embryo development (Pellegrino et al., 2016; Baruselli et al., 2018). 3.4 Use of sex-sorted semen The use of sex-sorted semen in AI and IVF is a promising approach to control the sex ratio of offspring in buffalo breeding. This technology involves sorting sperm to favor either X- or Y-chromosome-bearing sperm, allowing for the production of predominantly female or male calves. Studies have demonstrated the feasibility of using sexed semen in buffalo, achieving high pregnancy rates and sex accuracy. However, the success of this technology can vary based on factors such as season, technician skill, and the genetic background of the buffalo (Lu et al., 2015; Chebel and Cunha, 2020). In summary, the optimization of reproductive technologies in water buffalo involves a combination of AI, estrus synchronization, IVF, and the use of sex-sorted semen. These technologies, when effectively applied, can significantly enhance reproductive efficiency and genetic improvement in buffalo herds. However, challenges such as estrous detection, seasonality, and the efficiency of advanced reproductive techniques need to be addressed to fully realize their potential. 4 Emerging Technologies in Water Buffalo Reproduction 4.1 Genome editing and its potential in reproductive enhancement Genome editing, particularly using CRISPR/Cas9 technology, has shown significant promise in enhancing reproductive capabilities in water buffalo. This technology allows for precise genetic modifications, such as the integration of specific genes into the Y chromosome, which can be used for sex control in pre-implantation embryos. The successful generation of transgenic cloned buffalo embryos demonstrates the potential of genome editing to improve reproductive outcomes by enabling the selection of desired traits at the embryonic stage (Figure 1) (Zhao et al., 2020; Singh et al., 2020). Figure 1 Evaluation of sgRNA efficiency and knock-in strategies in buffalo fetal fibroblast cells (Adapted from Zhao et al., 2020) Image caption: (A) The RGS reporter system was used to determine the efficiencies of sgRNAs; the cleavage activities were evaluated by FACS and are presented as the ratio of GFP+/RFP+ cells to all transfected cells; (B) Histograms show the relative cleavage activity of different sgRNAs; (C) Schematic overview of the strategy used to target the Actb locus in buffalo fetal fibroblast cells; (D) Knock-in efficiencies were evaluated by FACS and are presented as the ratio of mCherry+ cells to all transfected cells. **P < 0.01, ns, no significant difference (Adopted from Zhao et al., 2020)
International Journal of Molecular Zoology, 2024, Vol.14, No.6, 305-314 http://animalscipublisher.com/index.php/ijmz 308 4.2 Precision breeding using genomic selection Precision breeding through genomic selection is an emerging approach that leverages detailed genomic data to enhance breeding programs. This method allows for the selection of genetically superior animals, thereby accelerating genetic gain. The integration of genomic selection with assisted reproductive technologies, such as in vitro embryo production, can significantly improve the efficiency of breeding programs by ensuring that only the best genetic material is propagated (Singh et al., 2020; Currin et al., 2021). 4.3 Advances in reproductive biomarkers for fertility prediction Recent advances in the identification of reproductive biomarkers have improved the ability to predict fertility in water buffalo. Proteomic profiling of spermatozoa has identified specific proteins associated with high fertility, which can be used as biomarkers to assess the fertilizing potential of semen before artificial insemination. This approach helps mitigate economic losses due to failed pregnancies by ensuring that only semen with high fertilizing potential is used (Karanwal et al., 2023; Andrei et al., 2024). In summary, the integration of genome editing, precision breeding, and the use of reproductive biomarkers represents a significant advancement in optimizing reproductive technologies in water buffalo. These emerging technologies hold the potential to enhance genetic gain, improve fertility rates, and increase the overall efficiency of breeding programs (Warriach et al., 2015; Zicarelli, 2019). Figure 2 Representation of differentially abundant proteins (DAPs) in high and low fertile spermatozoa (Adopted from Karanwal et al., 2023) Image caption: (A) Heat map showing differentially abundant proteins (DAPs) among the replicates of HF and LF groups. Intense blue colour represents high abundance of proteins while white colour represents low abundance of proteins. (B) Volcano plot of all DAPs identified in the proteomics data determine by log fold change vs. –log10 p-value. Red points: DAPs that were significantly high abundant in high fertile bull (fold change >2; p < 0.05). Blue points: DAPs that were significantly low abundant in high fertile bull (fold change <0.5; p < 0.05). Grey points: DAPs that showed neutral abundance. Volcano plot showing the significantly abundant proteins determine by log fold change (log fold) vs. -log10 p-value. (C) PCA plot representing the level of variances between the replicates of HF and LF samples (Adopted from Karanwal et al., 2023)
International Journal of Molecular Zoology, 2024, Vol.14, No.6, 305-314 http://animalscipublisher.com/index.php/ijmz 309 5 Case Study: Application of Embryo Transfer Technology in Improving Genetic Traits 5.1 Background and objectives of the case study The primary objective of this case study is to explore the application of embryo transfer technology in water buffaloes to enhance genetic traits, particularly for increased milk and meat production. The Philippine Carabao Center has been at the forefront of this initiative, aiming to produce genetically superior water buffaloes through advanced reproductive biotechnologies. This involves the use of in vitro embryo production and transfer techniques to improve the genetic pool of water buffaloes in the Philippines (Duran et al., 2017). 5.2 Methods employed in the field The methods employed include the collection of ovaries from slaughtered river buffaloes, followed by in vitro maturation and fertilization of oocytes. The embryos are then cryopreserved using vitrification and transported for non-surgical transfer into recipient buffaloes. The study also explored the use of swamp buffaloes as surrogate mothers and the potential for twinning by transferring embryos in pairs (Duran et al., 2017). Additionally, ovum pick-up (OPU) combined with in vitro embryo production (IVEP) has been utilized to exploit the genetics of high-yield buffaloes, addressing challenges such as low in vivo embryo recovery (Baruselli et al., 2018; Baruselli et al., 2020). 5.3 Results and outcomes The results from the studies conducted demonstrated varying success rates. In one study, a 16.36% pregnancy rate and a 10.91% calving rate were achieved with river buffalo recipients. When swamp buffaloes were used as surrogates, a 12.5% pregnancy rate and a 10% calving rate were observed. The study on twinning showed a 23.1% calving rate with a 3.8% twinning rate when embryos were transferred in pairs (Duran et al., 2017). These outcomes highlight the potential of embryo transfer technology in improving genetic traits in water buffaloes, although efficiency varies based on the methods and conditions applied. 5.4 Lessons learned and recommendations The case study underscores the importance of optimizing embryo transfer techniques to improve genetic traits in water buffaloes. Key lessons include the need for precise synchronization of follicular development and ovulation, as well as the importance of selecting appropriate surrogate mothers to enhance pregnancy and calving rates (Baruselli et al., 2018; Baruselli et al., 2020). It is recommended to further refine in vitro embryo production techniques and explore the use of genomic-assisted selection to accelerate genetic gains. Additionally, addressing factors such as environmental conditions, donor selection, and the use of advanced technologies like sex-sorted sperm cells could further improve the efficiency and outcomes of embryo transfer programs in water buffaloes (Gaddis et al., 2017; Currin et al., 2021). 6 Factors Influencing Adoption of Reproductive Technologies 6.1 Socio-economic considerations The adoption of reproductive technologies in water buffalo is significantly influenced by socio-economic factors. Buffaloes are crucial for the economic sustenance of many farmers, particularly in developing countries, due to their contributions to milk, meat, and draught power (Bm, 2019; Nanda et al., 2019). However, the economic impact of reproductive failures can be substantial, affecting the profitability of buffalo farming systems. For instance, longer calving intervals negatively impact profitability, emphasizing the need for efficient reproductive management (Nava-Trujillo et al., 2020). The cost of implementing advanced reproductive technologies can be prohibitive for smallholder farmers, who often lack the financial resources to invest in such innovations. Therefore, socio-economic constraints play a critical role in the decision-making process regarding the adoption of these technologies. 6.2 Infrastructure and technical expertise requirements The successful implementation of reproductive technologies in buffaloes requires adequate infrastructure and technical expertise. Many buffaloes are managed in areas with restricted access, which can limit the application of procedures like ovum pick-up and in vitro embryo production (Konrad et al., 2017). The availability of skilled
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