Genomic and Phenotypic Udder Evaluation for Dairy Cattle Selection: A Review.

The traditional point of view regarding dairy cattle selection has been challenged by recent genomic studies indicating that livestock productivity prediction can be redefined based on the evaluation of genomic and phenotypic data. Several studies that included different genomic-derived traits only indicated that interactions among them or even with conventional phenotypic evaluation criteria require further elucidation. Unfortunately, certain genomic and phenotypic-derived traits have been shown to be secondary factors influencing dairy production. Thus, these factors, as well as evaluation criteria, need to be defined. Owing to the variety of genomic and phenotypic udder-derived traits which may affect the modern dairy cow functionality and conformation, a definition of currently important traits in the broad sense is indicated. This is essential for cattle productivity and dairy sustainability. The main objective of the present review is to elucidate the possible relationships among genomic and phenotypic udder evaluation characteristics to define the most relevant traits related to selection for function and conformation in dairy cattle. This review aims to examine the potential impact of various udder-related evaluation criteria on dairy cattle productivity and explore how to mitigate the adverse effects of compromised udder conformation and functionality. Specifically, we will consider the implications for udder health, welfare, longevity, and production-derived traits. Subsequently, we will address several concerns covering the application of genomic and phenotypic evaluation criteria with emphasis on udder-related traits in dairy cattle selection as well as its evolution from origins to the present and future prospects.

Bridging the Gap: Animal Models in Next-Generation Reproductive Technologies for Male Fertility Preservation.

This review aims to explore advanced reproductive technologies for male fertility preservation, underscoring the essential role that animal models have played in shaping these techniques through historical contexts and into modern applications. Rising infertility concerns have become more prevalent in human populations recently. The surge in male fertility issues has prompted advanced reproductive technologies, with animal models playing a pivotal role in their evolution. Historically, animal models have aided our understanding in the field, from early reproductive basic research to developing techniques like artificial insemination, multiple ovulation, and in vitro fertilization. The contemporary landscape of male fertility preservation encompasses techniques such as sperm cryopreservation, testicular sperm extraction, and intracytoplasmic sperm injection, among others. The relevance of animal models will undoubtedly bridge the gap between traditional methods and revolutionary next-generation reproductive techniques, fortifying our collective efforts in enhancing male fertility preservation strategies. While we possess extensive knowledge about spermatogenesis and its regulation, largely thanks to insights from animal models that paved the way for human infertility treatments, a pressing need remains to further understand specific infertility issues unique to humans. The primary aim of this review is to provide a comprehensive analysis of how animal models have influenced the development and refinement of advanced reproductive technologies for male fertility preservation, and to assess their future potential in bridging the gap between current practices and cutting-edge fertility techniques, particularly in addressing unique human male factor infertility.

A review of inbreeding depression in dairy cattle: current status, emerging control strategies, and future prospects.

Dairy cattle breeding has historically focused on relatively small numbers of elite bulls as sires of sons. In recent years, even if generation intervals were reduced and more diverse sires of sons could have been selected, genomic selection has not fundamentally changed the fact that a large number of individuals are being analyzed. However, a relatively small number of elite bulls are still siring those animals. Therefore inbreeding-derived negative consequences in the gene pool have brought concern. The detrimental effects of non-additive genetic changes such as inbreeding depression and dominance have been widely disseminated while seriously affecting bioeconomically important parameters because of an antagonistic relationship between dairy production and reproductive traits. Therefore, the estimation of benefits and limitations of inbreeding and variance of the selection response deserves to be evaluated and discussed to preserve genetic variability, a significant concern in the selection of individuals for reproduction and production. Short-term strategies for genetic merit improvement through modern breeding programs have severely lowered high-producing dairy cattle fertility potential. Since the current selection programs potentially increase long-term costs, genetic diversity has decreased globally as a consequence. Therefore, a greater understanding of the potential that selection programs have for supporting long-term genetic sustainability and genetic diversity among dairy cattle populations should be prioritized in managing farm profitability. The present review provides a broad approach to current inbreeding-derived problems, identifying critical points to be solved and possible alternative strategies to control selection against homozygous haplotypes while maintaining sustained selection pressure. Moreover, this manuscript explores future perspectives, emphasizing theoretical applications and critical points, and strategies to avoid the adverse effects of inbreeding in dairy cattle. Finally, this review provides an overview of challenges that will soon require multidisciplinary approaches to managing dairy cattle populations, intending to combine increases in productive trait phenotypes with improvements in reproductive, health, welfare, linear conformation, and adaptability traits into the foreseeable future.

Spermatogenesis in a vulnerable South American cervid, dwarf red brocket (Mazama rufina).

The brocket deer (Genus Mazama) is a highly diverse cervid group distributed from Mexico to Argentina, with a downward population trend. However, literature on the basic reproductive biology of the genus is scarce. This work aimed to study biometric, histological and stereological aspects of the testes of Dwarf Red Brocket (Mazama rufina). Testes from free-ranging adult brockets (n = 3) were retrieved from necropsies. Testes were histologically processed. From histological images, several stereological parameters were estimated, and seminiferous epithelium cycle morphology was described. Testes volumes were between 8.2 and 18.4 ml and weights from 8.3 to 19.4 g. Gonadosomatic index (% paired-testes weight to body weight) went from 0.17 to 0.64. The tubular cross-sectional diameter was 179.8 ± 2.8 µm. Estimated volume densities for parenchyma and interstitium were 78.8% and 21.2% respectively. There were (in millions/ml) 96.0 ± 13.1 germ cells and 37.7 ± 6.0 somatic cells. Specific cell densities were (all expressed in millions/ml) as follows: spermatogonia 13.1 ± 4.2; primary spermatocytes 43.1 ± 5.0; round spermatids 36.8 ± 8.0 (lower density near the caudal pole, p < 0.01); sustentacular (Sertoli) cells 16.8 ± 4.1 and interstitial endocrine (Leydig) cells 17.4 ± 3.4. Sertoli cell index (germ cells per Sertoli cell) was 6.72. Eight stages of the cycle were described, and frequencies estimated, resembling those of goats. M. rufina adult testis anatomy is similar to that of other cervids and domestic ruminants, with an apparently lower spermatogenic efficiency. This work is a first approximation to the physiology of the testis of M. rufina. Basic knowledge of the reproductive physiology of vulnerable species may allow biotechnological approaches for the restitution of animal populations.

Filgrastim (r-met-hu G-CSF) enhances the efficiency of spermatogenesis in prepubertal Bos indicus bulls.

This study aimed to test the effects of the drug r-met-hu-G-CSF (filgrastim) on spermatogenic efficiency in prepubertal Brahman bulls. Twelve intact, healthy prepubertal bulls were administered 0, 1 (LD = low dose) or 4 (HD = high dose) µg/Kg r-met-hu-G-CSF (daily for 4 days), and haematological analysis was performed. Bulls were castrated (D0 or D60). BW (body weight) and SC (scrotal circumference) were recorded. Testis weight and volume were taken at castration with samples for testis histology and stereology: germ cell types, spermatids count and DSP (daily sperm production per gram)/g of testicular parenchyma. Testicular weight, volume, BW, SC and gonadosomatic index (GSI) were NS (LD-HD; p > .05). At D0 (age 11 months), the most advanced germ cell types (maGCt) ranged from intermediate spermatogonia to pachytene spermatocytes. After 2 months, control animals had round spermatids as maGCt, LD animals 75% round spermatids and 25% elongated spermatids, and HD animals round spermatids. Spermatids/testis were higher in LD (1.23 ± 0.2 millions) than in controls (0.65 ± 0.1 millions, p < .05). Spermatogenic efficiency (DSP/g) was higher in LD (5.4 ± 0.4 million) than in controls (3.2 ± 0.2 million, p < .01). In conclusion, r-met-hu-G-CSF raises spermatogenic efficiency in prepubertal Brahman bulls.

Genomic Analysis, Progress and Future Perspectives in Dairy Cattle Selection: A Review.

Genomics comprises a set of current and valuable technologies implemented as selection tools in dairy cattle commercial breeding programs. The intensive progeny testing for production and reproductive traits based on genomic breeding values (GEBVs) has been crucial to increasing dairy cattle productivity. The knowledge of key genes and haplotypes, including their regulation mechanisms, as markers for productivity traits, may improve the strategies on the present and future for dairy cattle selection. Genome-wide association studies (GWAS) such as quantitative trait loci (QTL), single nucleotide polymorphisms (SNPs), or single-step genomic best linear unbiased prediction (ssGBLUP) methods have already been included in global dairy programs for the estimation of marker-assisted selection-derived effects. The increase in genetic progress based on genomic predicting accuracy has also contributed to the understanding of genetic effects in dairy cattle offspring. However, the crossing within inbred-lines critically increased homozygosis with accumulated negative effects of inbreeding like a decline in reproductive performance. Thus, inaccurate-biased estimations based on empirical-conventional models of dairy production systems face an increased risk of providing suboptimal results derived from errors in the selection of candidates of high genetic merit-based just on low-heritability phenotypic traits. This extends the generation intervals and increases costs due to the significant reduction of genetic gains. The remarkable progress of genomic prediction increases the accurate selection of superior candidates. The scope of the present review is to summarize and discuss the advances and challenges of genomic tools for dairy cattle selection for optimizing breeding programs and controlling negative inbreeding depression effects on productivity and consequently, achieving economic-effective advances in food production efficiency. Particular attention is given to the potential genomic selection-derived results to facilitate precision management on modern dairy farms, including an overview of novel genome editing methodologies as perspectives toward the future.

Genomic Evaluation of Primiparous High-Producing Dairy Cows: Inbreeding Effects on Genotypic and Phenotypic Production-Reproductive Traits.

The main objective of this study was to analyze the effects of the inbreeding degree in high-producing primiparous dairy cows genotypically and phenotypically evaluated and its impacts on production and reproductive parameters. Eighty Holstein-Friesian primiparous cows (age: ~26 months; ~450 kg body weight) were previously genomically analyzed to determine the Inbreeding Index (II) and were divided into two groups: low inbreeding group (LI: <2.5; n = 40) and high inbreeding group (HI: ≥2.5 and ≤5.0; n = 40). Genomic determinations of production and reproductive parameters (14 in total), together with analyses of production (12) and reproductive (11) phenotypic parameters (23 in total) were carried out. Statistically significant differences were obtained between groups concerning the genomic parameters of Milk Production at 305 d and Protein Production at 305 d and the reproductive parameter Daughter Calving Ease, the first two being higher in cows of the HI group and the third lower in the LI group (p < 0.05). For the production phenotypic parameters, statistically significant differences were observed between both groups in the Total Fat, Total Protein, and Urea parameters, the first two being higher in the LI group (p < 0.05). Also, significant differences were observed in several reproductive phenotypic parameters, such as Number of Services per Conception, Calving to Conception Interval, Days Open Post Service, and Current Inter-Partum Period, all of which negatively influenced the HI group (p < 0.05). In addition, correlation analyses were performed between production and reproductive genomic parameters separately and in each consanguinity group. The results showed multiple positive and negative correlations between the production and reproductive parameters independently of the group analyzed, being these correlations more remarkable for the reproductive parameters in the LI group and the production parameters in the HI group (p < 0.05). In conclusion, the degree of inbreeding significantly influenced the results, affecting different genomic and phenotypic production and reproductive parameters in high-producing primiparous cows. The determination of the II in first-calf heifers is crucial to evaluate the negative effects associated with homozygosity avoiding an increase in inbreeding depression on production and reproductive traits.

Isolation, Culture, Cryopreservation, and Identification of Bovine, Murine, and Human Spermatogonial Stem Cells.

Spermatogonial stem cells (SSCs) are the germ cells at the basis of spermatogenesis in adult mammals. SSCs offer many biotechnological possibilities and are fundamental cells in the study of spermatogenesis (Aponte, World J Stem Cells 7:669-680, 2015). This chapter describes detailed procedures for SSC isolation, culture, cryopreservation, and characterization in bovine, murine, and human models.

Differential role of r-met-hu G-CSF on male reproductive function and development in prepubertal domestic mammals.

The understanding of mammalian spermatogenesis niche factors active during sexual development may be leveraged to impact reproduction in farm animals. The aim of this study was to evaluate the effects of r-met-hu/G-CSF (filgrastim) on prepubertal sexual development of guinea pigs (Cavia porcellus) and ram lambs (Ovis aries). Individuals of both species were administered r-met-hu/G-CSF daily for 4 days. During and after administration protocols, testicular function and development were assessed through hematological responses, hormonal profiles (gonadotropins, testosterone and cortisol) testicular morphometry and germ cell kinetics. As expected, r-met-hu/G-CSF acutely mobilized white-lineage blood cells in both species. LH was increased by r-met-hu/G-CSF in guinea pigs (P<0.01) but T remained unchanged. In ram lambs gonadotropins and T increased in dose-response fashion (P<0.01) while cortisol values were stable and similar in treated and control animals (P>0.05). In guinea pigs there were no differences in testicular weights and volumes 2-mo after r-met-hu/G-CSF application (P>0.05). However, ram lambs showed a dose-response effect regarding testis weight (P<0.05). 66.66% of ram lambs had initial testes not yet in meiosis or starting the first spermatogenic wave. After 60-days only 25% of control animals were pubertal while all treated animals (1140-µg) had reached puberty. We propose an integrated hypothesis that G-CSF can stimulate spermatogenesis through two possible ways. 1) r-met-hu/G-CSF may go through the brain blood barrier and once there it can stimulate GnRH-neurons to release GnRH with the subsequent release of gonadotrophins. 2) a local testicular effect through stimulation of steroidogenesis that enhances spermiogenesis via testosterone production and a direct stimulation over spermatogonial stem cells self-renewal. In conclusion, this study shows that r-met-hu/G-CSF differentially affects prepubertal sexual development in hystricomorpha and ovine species, a relevant fact to consider when designing methods to hasten sexual developmental in mammalian species.

Artificial Mitochondria Transfer: Current Challenges, Advances, and Future Applications.

The objective of this review is to outline existing artificial mitochondria transfer techniques and to describe the future steps necessary to develop new therapeutic applications in medicine. Inspired by the symbiotic origin of mitochondria and by the cell's capacity to transfer these organelles to damaged neighbors, many researchers have developed procedures to artificially transfer mitochondria from one cell to another. The techniques currently in use today range from simple coincubations of isolated mitochondria and recipient cells to the use of physical approaches to induce integration. These methods mimic natural mitochondria transfer. In order to use mitochondrial transfer in medicine, we must answer key questions about how to replicate aspects of natural transport processes to improve current artificial transfer methods. Another priority is to determine the optimum quantity and cell/tissue source of the mitochondria in order to induce cell reprogramming or tissue repair, in both in vitro and in vivo applications. Additionally, it is important that the field explores how artificial mitochondria transfer techniques can be used to treat different diseases and how to navigate the ethical issues in such procedures. Without a doubt, mitochondria are more than mere cell power plants, as we continue to discover their potential to be used in medicine.

Stemness in Cancer: Stem Cells, Cancer Stem Cells, and Their Microenvironment.

Stemness combines the ability of a cell to perpetuate its lineage, to give rise to differentiated cells, and to interact with its environment to maintain a balance between quiescence, proliferation, and regeneration. While adult Stem Cells display these properties when participating in tissue homeostasis, Cancer Stem Cells (CSCs) behave as their malignant equivalents. CSCs display stemness in various circumstances, including the sustaining of cancer progression, and the interaction with their environment in search for key survival factors. As a result, CSCs can recurrently persist after therapy. In order to understand how the concept of stemness applies to cancer, this review will explore properties shared between normal and malignant Stem Cells. First, we provide an overview of properties of normal adult Stem Cells. We thereafter elaborate on how these features operate in CSCs. We then review the organization of microenvironment components, which enables CSCs hosting. We subsequently discuss Mesenchymal Stem/Stromal Cells (MSCs), which, although their stemness properties are limited, represent essential components of the Stem Cell niche and tumor microenvironment. We next provide insights of the therapeutic strategies targeting Stem Cell properties in tumors and the use of state-of-the-art techniques in future research. Increasing our knowledge of the CSCs microenvironment is key to identifying new therapeutic solutions.

Alcohol-based solutions for bovine testicular tissue fixation.

Tissue fixation, a central element in histotechnology, is currently performed with chemical compounds potentially harmful for human health and the environment. Therefore, alternative fixatives are being developed, including alcohol-based solutions. We evaluated several ethanol-based mixtures with additives to study fixative penetration rate, tissue volume changes, and morphologic effects in the bovine testis. Fixatives used were Bouin solution, 4% formaldehyde (F4), 70% ethanol (E70), E70 with 1.5% glycerol (E70G), E70 with 5% acetic acid (E70A), E70 with 1.5% glycerol and 5% acetic acid (E70AG), and E70 with 1.5% glycerol, 5% acetic acid, and 1% dimethyl sulfoxide (DMSO; E70AGD). Five-millimeter bovine testicular tissue cubes could be completely penetrated by ethanol-based fixatives and Bouin solution in 2-3 h, whereas F4 required 21 h. Bouin solution produced general tissue shrinkage, whereas the other fixatives (alcohol-based and F4) caused tissue volume expansion. Although Bouin solution is an excellent fixative for testicular tissue, ethanol-based fixatives showed good penetration rates, low tissue shrinkage, and preserved sufficient morphology to allow identification of the stages of the seminiferous epithelium cycle, therefore representing a valid alternative for histotechnology laboratories. Common additives such as acetic acid, glycerol, and DMSO offered marginal benefits for the process of fixation; E70AG showed the best preservation of morphology with excellent nuclear detail, close to that of Bouin solution.

Tumor de células granulares del esófago: reporte de un caso / Esophageal granular cell tumor: Case report

Introducción: el tumor de células granulares del esófago (TCG) es una neoplasia rara y su diagnóstico preciso se basa en el examen histopatológico. Con el incremento de la endoscopia como medida de tamizaje se ha visto un leve aumento en la incidencia, por lo que debe tenerse en cuenta como diagnóstico diferencial en el momento de abordar una lesión subepitelial. Metodología: presentación de un caso clínico con TCG cuya endoscopia de vías digestivas altas (EVDA) muestra una lesión subepitelial en el tercio distal del esófago, y que debido a las características histopatológicas, clínicas y ecosonográficas se decide seguimiento y manejo expectante. Conclusiones: es importante el conocimiento de las características, comportamientos y estrategias de manejo del TCG, puesmuchos son asintomáticos y estables en el seguimiento, por lo que no necesitan tratamientos agresivos. Por el riesgo de malignidad, es importante su control riguroso.

Introduction: Esophageal granular cell tumors (GCTs) are rare. Their precise diagnosis is based on histopathological examination of the specimen. However, owing to the use of endoscopy as a screening tool the incidence of these lesions has presently mildly increased and must be considered as a differential diagnosis of subepithelial lesions. Methodology: A case is presented of a GCT as a subepithelial lesion in the distal part of the esophagus found by esophagogastroduodenoscopy (EGD). Conservative management and follow-up was decided due to the histopathological, clinical and ultrasound features of the lesion. Conclusions: Knowledge regarding GCTs´i characteristics, behavior and management is important for many are asymptomatic and remain clinically stable during follow-up, requiring no aggressive treatment. A rigorous follow-up is recommended due to its malignant potential.

Distinction of Ecuadorian varieties of fermented cocoa beans using Raman spectroscopy.

Cocoa (Theobroma cacao) is a crop of economic importance. In Ecuador, there are two predominant cocoa varieties: National and CCN-51. The National variety is the most demanded, since its cocoa beans are used to produce the finest chocolates. Raman measurements of fermented, dried and unpeeled cocoa beans were performed using a handheld spectrometer. Samples of the National and CCN-51 varieties were collected from different provinces and studied in this work. For each sample, 25 cocoa beans were considered and each bean was measured at 4 different spots. The most important Raman features of the spectra were assigned and discussed. The spectroscopic data were processed using chemometrics, resulting in a distinction of varieties with 91.8% of total accuracy. Differences in the average Raman spectra of cocoa beans from different sites but within the same variety can be attributed to environmental factors affecting the cocoa beans during the fermentation and drying processes.

Spermatogonial stem cells: Current biotechnological advances in reproduction and regenerative medicine.

Spermatogonial stem cells (SSCs) are the germ stem cells of the seminiferous epithelium in the testis. Through the process of spermatogenesis, they produce sperm while concomitantly keeping their cellular pool constant through self-renewal. SSC biology offers important applications for animal reproduction and overcoming human disease through regenerative therapies. To this end, several techniques involving SSCs have been developed and will be covered in this article. SSCs convey genetic information to the next generation, a property that can be exploited for gene targeting. Additionally, SSCs can be induced to become embryonic stem cell-like pluripotent cells in vitro. Updates on SSC transplantation techniques with related applications, such as fertility restoration and preservation of endangered species, are also covered on this article. SSC suspensions can be transplanted to the testis of an animal and this has given the basis for SSC functional assays. This procedure has proven technically demanding in large animals and men. In parallel, testis tissue xenografting, another transplantation technique, was developed and resulted in sperm production in testis explants grafted into ectopical locations in foreign species. Since SSC culture holds a pivotal role in SSC biotechnologies, current advances are overviewed. Finally, spermatogenesis in vitro, already demonstrated in mice, offers great promises to cope with reproductive issues in the farm animal industry and human clinical applications.

Morphometric evaluation of the spermatogonial stem cell distribution and niche in vertebrates.

Morphometry is a classical quantitative method often used in biology to provide a data basis for functional interpretations/interactions of a particular organ or system. Herein we took advantage of this valuable approach to evaluate the spermatogonial stem cell niche using the horse testis and immunocytochemical localization of GFRA1 [glial cell line-derived neurotrophic factor receptor produced by Sertoli cells)] as an example. Using the NIH ImageJ free software, we describe in detail all the necessary steps to investigate this specific and crucial microenvironment. Based on several recently published papers from our research group, this approach has proved to be fast, simple, and adaptable to a wide range of species and has the potential to be easily reproducible in different laboratories.

Biotechnological approaches to the treatment of aspermatogenic men.

Aspermatogenesis is a severe impairment of spermatogenesis in which germ cells are completely lacking or present in an immature form, which results in sterility in approximately 25% of patients. Because assisted reproduction techniques require mature germ cells, biotechnology is a valuable tool for rescuing fertility while maintaining biological fatherhood. However, this process involves, for instance, the differentiation of preexisting immature germ cells or the production/derivation of sperm from somatic cells. This review critically addresses four potential techniques: sperm derivation in vitro, germ stem cell transplantation, xenologous systems, and haploidization. Sperm derivation in vitro is already feasible in fish and mammals through organ culture or 3D systems, and it is very useful in conditions of germ cell arrest or in type II Sertoli-cell-only syndrome. Patients afflicted by type I Sertoli-cell-only syndrome could also benefit from gamete derivation from induced pluripotent stem cells of somatic origin, and human haploid-like cells have already been obtained by using this novel methodology. In the absence of alternative strategies to generate sperm in vitro, in germ cells transplantation fertility is restored by placing donor cells in the recipient germ-cell-free seminiferous epithelium, which has proven effective in conditions of spermatogonial arrest. Grafting also provides an approach for ex-vivo generation of mature sperm, particularly using prepubertal testis tissue. Although less feasible, haploidization is an option for creating gametes based on biological cloning technology. In conclusion, the aforementioned promising techniques remain largely experimental and still require extensive research, which should address, among other concerns, ethical and biosafety issues, such as gamete epigenetic status, ploidy, and chromatin integrity.

Biotechnological approaches to the treatment of aspermatogenic men

Aspermatogenesis is a severe impairment of spermatogenesis in which germ cells are completely lacking or present in an immature form, which results in sterility in approximately 25% of patients. Because assisted reproduction techniques require mature germ cells, biotechnology is a valuable tool for rescuing fertility while maintaining biological fatherhood. However, this process involves, for instance, the differentiation of preexisting immature germ cells or the production/derivation of sperm from somatic cells. This review critically addresses four potential techniques: sperm derivation in vitro, germ stem cell transplantation, xenologous systems, and haploidization. Sperm derivation in vitro is already feasible in fish and mammals through organ culture or 3D systems, and it is very useful in conditions of germ cell arrest or in type II Sertoli-cell-only syndrome. Patients afflicted by type I Sertoli-cell-only syndrome could also benefit from gamete derivation from induced pluripotent stem cells of somatic origin, and human haploid-like cells have already been obtained by using this novel methodology. In the absence of alternative strategies to generate sperm in vitro, in germ cells transplantation fertility is restored by placing donor cells in the recipient germ-cell-free seminiferous epithelium, which has proven effective in conditions of spermatogonial arrest. Grafting also provides an approach for ex-vivo generation of mature sperm, particularly using prepubertal testis tissue. Although less feasible, haploidization is an option for creating gametes based on biological cloning technology. In conclusion, the aforementioned promising techniques remain largely experimental and still require extensive research, which should address, among other concerns, ethical and biosafety issues, such as gamete epigenetic status, ploidy, and chromatin integrity.

Propagation of bovine spermatogonial stem cells in vitro.

The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. A specialized medium and several growth factors were tested to study the in vitro behavior of bovine type A spermatogonia, a cell population that includes the SSCs and can be specifically stained for the lectin Dolichos biflorus agglutinin. During short-term culture (2 weeks), colonies appeared, the morphology of which varied with the specific growth factor(s) added. Whenever the stem cell medium was used, round structures reminiscent of sectioned seminiferous tubules appeared in the core of the colonies. Remarkably, these round structures always contained type A spermatogonia. When leukemia inhibitory factor (LIF), epidermal growth factor (EGF), or fibroblast growth factor 2 (FGF2) were added, specific effects on the numbers and arrangement of somatic cells were observed. However, the number of type A spermatogonia was significantly higher in cultures to which glial cell line-derived neurotrophic factor (GDNF) was added and highest when GDNF, LIF, EGF, and FGF2 were all present. The latter suggests that a proper stimulation of the somatic cells is necessary for optimal stimulation of the germ cells in culture. Somatic cells present in the colonies included Sertoli cells, peritubular myoid cells, and a few Leydig cells. A transplantation experiment, using nude mice, showed the presence of SSCs among the cultured cells and in addition strongly suggested a more than 10 000-fold increase in the number of SSCs after 30 days of culture. These results demonstrate that bovine SSC self-renew in our specialized bovine culture system and that this system can be used for the propagation of these cells.

Basic features of bovine spermatogonial culture and effects of glial cell line-derived neurotrophic factor.

Spermatogonial stem cells (SSC) are a small self-renewing subpopulation of type A spermatogonia, which for the rest are composed of differentiating cells with a very similar morphology. We studied the development of primary co-cultures of prepubertal bovine Sertoli cells and A spermatogonia and the effect of glial cell line-derived neurotropic factor (GDNF) on the numbers and types of spermatogonia, the formation of spermatogonial colonies and the capacity of the cultured SSC to colonize a recipient mouse testis. During the first week of culture many, probably differentiating, A spermatogonia entered apoptosis while others formed pairs and chains of A spermatogonia. After 1 week colonies started to appear that increased in size with time. Numbers of single (A(s)) and paired (A(pr)) spermatogonia were significantly higher in GDNF treated cultures at Days 15 and 25 (P < 0.01 and 0.05, respectively), and the ratio of A(s) to A(pr) and spermatogonial chains (A(al)) was also higher indicating enhanced self-renewal of the SSC. Furthermore, spermatogonial outgrowths in the periphery of the colonies showed a significantly higher number of A spermatogonia with a more primitive morphology under the influence of GDNF (P < 0.05). Spermatogonial stem cell transplantation experiments revealed a 2-fold increase in stem cell activity in GDNF treated spermatogonial cultures (P < 0.01). We conclude that GDNF rather than inducing proliferation, enhances self-renewal and increases survival rates of SSC in the bovine spermatogonial culture system.