Analytical and clinical validation of diagnostic tests for the detection of leucospermia in beef bulls.

The objectives of this study were to validate diagnostic tests to detect polymorphonuclear cells (PMNs) in bull semen, and to determine the prevalence of leucospermia in beef bulls with varying semen quality. We hypothesized that all tests have comparable diagnostic value, and that leucospermia is more prevalent in unsatisfactory breeders in association with poor semen quality. For the analytical validation, one ejaculate was obtained from five bulls. Aliquots of 50 × 106 purified sperm were incubated in triplicate with six concentrations of purified bovine PMNs: 1) no PMNs, 2) 0.25 × 106 PMN/ml, 3) 0.5 × 106 PMN/ml, 4) 2.5 × 106 PMN/ml, 5) 5 × 106 PMN/ml, 6) 10 × 106 PMN/ml. The PMNs were quantified using a hemacytometer, cytology, a leucocyte esterase dipstick test (LEDT), a peroxidase test, and CD45 immunolabeling. The number of leucocytes detected with the LEDT differed among treatments (P < 0.0001). The quantitative tests detected differences with the control treatment at a PMN concentration of ≥2.5 × 106 PMN/ml (P < 0.0001). Sperm motion parameters after 4 h of incubation at 38 °C were lower in samples with ≥5 × 106 PMN/ml (P < 0.05). For the clinical validation, semen samples from 305 beef bulls were evaluated. Unsatisfactory breeders (n = 83) had more CD45-positive cells (P = 0.016) and positive LEDT results (P = 0.008) than satisfactory breeders (n = 222). With CD45 immunostaining as the gold standard, the hemacytometer count had the highest clinical sensitivity (64.3 %) but the lowest specificity (73.3 %). A higher specificity was obtained with the peroxidase test (95.1 %) or semen cytology (98.8 %). In conclusion, the presence of ≥5 × 106 PMN/ml was associated with decreased semen quality in beef bulls. The hemacytometer count was the most sensitive bull-side test. But due to the low specificity, positive hemacytometer counts should be confirmed with the identification of peroxidase-positive cells or morphological identification of leucocytes on semen cytology. The CD45 immunostaining is the gold standard for the diagnosis of leucospermia in bulls.

Repeated injections of vitamin E and Se improves testicular morphology, testosterone and in vitro and in vivo sperm fertility in subfertile rabbits.

Subfertility is a multifactorial disorder that affects the rabbit production industry. However, subfertility may be treated by using a simple intervention such as vitamin supplementation. Vitamin E and selenium (Se) are potent antioxidants that protect the male reproductive system. The aim of this study is to determine the effects of vitamin E and Se on testicular size, semen quality and freezability, antioxidant activity, testosterone levels, and fertility in subfertile rabbits. Twenty-one New Zealand rabbits were classified as subfertile rabbits based on their semen characteristics and fertility records. The rabbits were randomly allocated into 3 equal groups (G1: control; G2: injected with Vit E 100 IU/head + Se 0.1 mg/kg b.w.; G3: injected with Vit E 200 IU/head + Se 0.2 mg/kg b.w. once weekly for 8 weeks).Once weekly for 8 W, blood samples were collected to measure serum testosterone level and total antioxidant capacity (TAC), and semen samples were collected by artificial vagina to assess the quality of fresh and frozen semen. At the 8th week of the study, 150 multiparous does were artificially inseminated with fresh semen to assess the fertility of rabbits after treatment; 50 does for each group. At the end of the study, rabbits were slaughtered to assess testicular morphometry. Fresh and post-thaw semen quality parameters were significantly (p < 0.05) higher in G3in comparison with G2and G1, respectively. Also, testosterone level was significantly (p < 0.05) increased at the 2nd week in G3in comparison with other groups. Conception and kindling rates were significantly (p < 0.05) higher in does which were inseminated with semen fromG3. In conclusion, injection of vitamin E and selenium at a higher dose (G3) improved the testicular morphology, quality of fresh and post-thaw semen, and most importantly, the fertility of subfertile rabbits.

Advantages of Curcuma longa in preventing male infertility caused by thioacetamide.

Background: Thioacetamide (TAA) is known to cause damage to various organs, including the testes, posing a significant health threat. On the other hand, Curcuma longa (Cl) has been recognized for its antioxidant properties, suggesting a potential protective role against TAA-induced toxicity in the testes. Aim: This study aims to investigate the effect of TAA on testicular function and structure while exploring the therapeutic and protective potential of C. longa versus TAA toxicity. Methods: Thirty-two male albino rats, with an age range of 11-12 weeks and a weight range of 180-200 g, were randomly allocated into four distinct groups. The control group received normal saline, while the Cl group ingested Cl orally at a dose of 500 mg/kg daily. The TAA group, received TAA through intraperitoneal injections at a dose of 200 mg/kg body weight three times per week. Lastly, the Cl with TAA group received Cl orally 2 hours before the TAA injections. After 8 weeks of treatment, we anesthetized the rats and saved blood samples for biochemical analysis. Results: The study revealed significant alterations in various biochemical parameters in the TAA-treated group, as compared with the control. Specifically, there was a significant increase in bilirubin, albumin, cholesterol, triglyceride, very low-density lipoprotein, white blood cells, low-density lipoprotein cholesterol, and platelets levels. Conversely, the Cl-treated group exhibited significant reductions in these parameters, along with notable increases in red blood cells, high-density lipoprotein cholesterol, and hemoglobin. Conclusion: C. longa demonstrates a protective effect on the testes against TAA-induced toxicity, potentially attributed to its antioxidant properties. This suggests a promising avenue for the use of Cl in mitigating the harmful effects of TAA on testicular function and structure.

Intratesticular versus intraperitoneal Busulfan administration: a comparative study on spermatogenesis suppression in quails and chickens.

Generation of transgenic birds can be achieved by temporal suppression of endogenous spermatogenesis in males prior to primordial germ cell implantation. One of many established methods to induce male sterility is the intraperitoneal injection of busulfan, an alkylating agent. Nevertheless, the use of busulfan injections, which may also affect hematopoietic stem cells, carries the risk of potential lethality in animals. Given their safety and non-toxic nature, it has been demonstrated that intratesticular busulfan injections in mammals are less effective than intraperitoneal injections. This study aimed to compare, for the first time, the sterility and toxicity effects of intraperitoneal vs. intratesticular busulfan injections in quail and chickens. Our experimental design involved a previously established single intraperitoneal busulfan injection of 40 mg/kg of body weight (BW). In quail, busulfan was then administered intratesticularly at 3 different concentrations (6, 12, and 20 mg/kg BW), while in chickens, the working concentration was 20 mg/kg BW. We found that a single intraperitoneal busulfan injection of 40 mg/kg of BW resulted in 100% mortality in the treated roosters. In quails, however, this concentration only caused a temporary suppression of fertility for a 15-d period. Moreover, we found that a higher dose of intratesticular injection of busulfan is required to suppress spermatogenesis in quail (20 mg/kg BW) compared to mammals (4 mg/kg BW). Following these findings, we further confirmed that intratesticular injection of 20 mg/kg BW busulfan into roosters did not affect their overall viability. However, it induced a temporary state of male sterility, consistent with the effects observed with intraperitoneal injections. Hence, our data demonstrate that quail and chicken respond differently to busulfan administration. Furthermore, the present study provides evidence that direct injection into the rooster testes causes less physiological stress than intraperitoneal injection.

Proteomic analysis of sperm from fertile stallions and subfertile stallions due to impaired acrosomal exocytosis.

Thoroughbred stallions that carry a double-homozygous genotype A/A-A/A for SNPs rs397316122 and rs69101140 in exon 5 of the FKBP6 gene (chr13; EquCab3.0) are uniquely subfertile due to impaired acrosomal exocytosis (IAE). In this study, the sperm proteome in frozen/thawed semen from subfertile Thoroughbred stallions was studied and compared to that of frozen/thawed sperm from fertile Thoroughbred stallions. A total of 2,220 proteins was identified, of which 140 proteins were found to be differentially abundant in sperm from the subfertile stallions compared to that of fertile stallions (83 less and 57 more abundant). Proteins of differential abundance in sperm from the subfertile stallions were mainly overrepresented in the "metabolism" and the "metabolism of lipids" pathways. One of these proteins, arylsulfatase F (ARSF), was studied by immunofluorescence. A lower proportion of sperm displaying ARSF signal at the acrosome region was observed in sperm from subfertile Thoroughbred stallions. In addition, heterologous zona pellucida binding assays revealed that sperm from subfertile Thoroughbred stallions bound at a lower proportion to zonae pellucidae than sperm from fertile Thoroughbred stallions. In conclusion, a group of differential abundance proteins, including some of acrosome origin, were identified in sperm from subfertile stallions with acrosome dysfunction.

Genome-wide sequence divergence of satellite DNA could underlie meiotic failure in male hybrids of bighead catfish and North African catfish (Clarias, Clariidae).

Hybrid sterility, a hallmark of postzygotic isolation, arises from parental genome divergence disrupting meiosis. While chromosomal incompatibility is often implicated, the underlying mechanisms remain unclear. This study investigated meiotic behavior and genome-wide divergence in bighead catfish (C. macrocephalus), North African catfish (C. gariepinus), and their sterile male hybrids (important in aquaculture). Repetitive DNA analysis using bioinformatics and cytogenetics revealed significant divergence in satellite DNA (satDNA) families between parental species. Notably, one hybrid exhibited successful meiosis and spermatozoa production, suggesting potential variation in sterility expression. Our findings suggest that genome-wide satDNA divergence, rather than chromosome number differences, likely contributes to meiotic failure and male sterility in these catfish hybrids.

mRNA-Seq of testis and liver tissues reveals a testis-specific gene and alternative splicing associated with hybrid male sterility in dzo.

Alternative splicing (AS) plays an important role in the co-transcription and post-transcriptional regulation of gene expression during mammalian spermatogenesis. The dzo is the male F1 offspring of an interspecific hybrid between a domestic bull (Bos taurus ♂) and a yak (Bos grunniens ♀) which exhibits male sterility. This study aimed to identify the testis-specific genes and AS associated with hybrid male sterility in dzo. The iDEP90 program and rMATS software were used to identify the differentially expressed genes (DEG) and differential alternative splicing genes (DSG) based on RNA-seq data from the liver (n = 9) and testis (n = 6) tissues of domestic cattle, yak, and dzo. Splicing factors (SF) were obtained from the AmiGO2 and the NCBI databases, and Pearson correlation analysis was performed on the differentially expressed SFs and DSGs. We focused on the testis-specific DEGs and DSGs between dzo and cattle and yak. Among the top 3,000 genes with the most significant variations between these 15 samples, a large number of genes showed testis-specific expression involved with spermatogenesis. Cluster analysis showed that the expression levels of these testis-specific genes were dysregulated during mitosis with a burst downregulation during the pachynema spermatocyte stage. The occurrence of AS events in the testis was about 2.5 fold greater than in the liver, with exon skipping being the major AS event (81.89% to 82.73%). A total of 74 DSGs were specifically expressed in the testis and were significantly enriched during meiosis I, synapsis, and in the piRNA biosynthesis pathways. Notably, STAG3 and DDX4 were of the exon skipping type, and DMC1 was a mutually exclusive exon. A total of 36 SFs were significantly different in dzo testis, compared with cattle and yak. DDX4, SUGP1, and EFTUD2 were potential SFs leading to abnormal AS of testis-specific genes in dzo. These results show that AS of testis-specific genes can affect synapsis and the piRNA biosynthetic processes in dzo, which may be important factors associated with hybrid male sterility in dzo.

The interspecific hybrid offspring of a domestic bull (Bos taurus) and a yak (Bos grunniens) display heterosis in meat and milk production. The hybrid offspring are particularly adaptable to the harsh environments of the Qinghai-Tibet Plateau. However, the male F1 to F3 offspring of this interspecies hybrid are infertile, and spermatogenesis is arrested at meiosis preventing the prolonged utilization of the benefits of heterosis. This study aimed to identify the testis-specific genes and alternative splicing (AS) associated with hybrid male sterility using RNA-Seq data from the liver and testis tissues of domestic cattle, yaks, and their F1 offspring (dzo). The expression of the testis-specific genes became disordered during mitosis and meiosis in dzo. Their testis-specific genes with AS events were enriched during synapsis and in the piRNA biosynthetic processes. In addition, we identified the potential splicing factors associated with abnormal testis-specific AS gene expression in dzo. These results reveal the important role of AS in the meiotic arrest of dzo.

Target silencing of porcine SPAG6 and PPP1CC by shRNA attenuated sperm motility.

The quality of sperm significantly influences the reproductive efficiency of pig herds. High-quality sperm is necessary for efficient fertilization and to maximize the litter numbers in commercial pig farming. However, the understanding of genes regulating porcine sperm motility and viability is limited. In this study, we validated porcine sperm/Sertoli-specific promoters through the luciferase reporter system and identified vital genes for sperm quality via loss-of-function means. Further, the shRNAs driven by the ACE and SP-10 promoters were used to knockdown the SPAG6 and PPP1CC genes which were provisionally important for sperm quality. We assessed the effects of SPAG6 and PPP1CC knockdown on sperm motility by using the sperm quality analyzer and flow cytometry. The results showed that the ACE promoter is active in both porcine Sertoli cells and sperms, whereas the SP-10 promoter is operating exclusively in sperm cells. Targeted interference with SPAG6 and PPP1CC expression in sperm cells decreases the motility and increases apoptosis rates in porcine sperms. These findings not only offer new genetic tools for targeting male germ cells but also highlight the crucial roles of SPAG6 and PPP1CC in porcine sperm function.

Protamine 2 and phospholipase C zeta 1 are possible biomarkers for the diagnosis of male subfertility in frozen-thawed stallion semen.

Subfertility is one of the main issues in horse breeding and the study of mRNAs in sperm might help in elucidating the reasons that lead to this diagnosis. The present study aims at assessing the differences in the expression of 10 potential candidate genes in stallions of different fertility. Frozen-thawed semen of 29 stallions was included. Each sample was classified into two groups according to pregnancy rates (PR) achieved with this semen: "good fertility" (GF; n = 17; PR ≥ 30 %) or "poor fertility" (PF; n = 12; PR <20 %). All stallions underwent a breeding soundness examination (BSE) before semen production and were only included into the semen cryopreservation program when raw semen characteristics at BSE met minimal requirements. Semen was cryopreserved following European Union regulations and all stallions met the respective health requirements. Each sample was assessed for concentration (NucleoCounter SP-100), motility (CASA), membrane functionality (SYBR-14/PI), mitochondrial membrane potential (JC-1), morphology (SpermacStain), acrosome integrity (SpermacStain), membrane integrity (HOS test) and chromatin integrity (Aniline blue). Sperm RNAs were extracted using the Direct-zol RNA Miniprep Kit (Zymo Research) and RT-qPCR was performed for each target gene. ACTB and RPL32 were included as reference genes (RGs) for normalization. For each variable of each group, mean, standard deviation and SEM were calculated. The difference in gene expression levels between the GF and PF group were analyzed using the Mann-Whitney U test and Spearman's rank correlation. Significant results were considered with p < 0.05. Sperm quality parameters did not differ significantly between the two groups except for concentration, that was significantly higher in GF (p = 0.043). In GF a positive correlation was identified for PRM1/PRM2 with r = +0.6, while PRM1/ACR (r = -0.495), PRM2/ZPBP (r = -0.645) and CRISP3/ACR (r = -0.551) were inversely correlated. In PF direct correlations were registered for PRM1/PRM2 (r = +0.629), PRM1/PRM3 (r = +0.657), PRM2/SPA17 (r = +0.685), SPA17/PLCZ1 (r = +0.786) and PRM3/ACR (r = +0.627). In the total sample (GF + PF), positive correlations were detected for PRM1/PRM2 (r = +0.625), PRM1/PRM3 (r = +0.368); PRM2/SPA17 (r = +0.465), SPA17/PLCZ1 (r = +0.637) and PLCZ1/ZAN (r = +0.587). Only two of the genes considered were differentially expressed in the 2 groups: PRM2 and PLCZ1, that were significantly (p < 0.05) overexpressed in the GF group. Stallions frozen-thawed semen with higher expression levels of PRM2 and PLCZ1 are more likely to belong to animals with a good pregnancy rate. Further studies are needed to investigate the role of sperm transcripts in male subfertility in stallions.

Establishing the normal range of sperm DNA fragmentation index (% DFI) for rhesus macaques.

The Sperm Chromatin Structure Assay (SCSA) is a robust test with high repeatability and precision. It is a clinically accepted assay that defines risk for infertility in men by measuring the degree of DNA fragmentation (% DFI) in sperm. The objective of this study was to adapt and validate the SCSA for rhesus macaques (Macaca mulatta) and establish a range for % DFI in fertile males. Sperm samples from two different males were used to produce a % DFI validation curve before establishing a range using additional samples from n = 11 males. Sperm labeled with acridine orange were analyzed by flow cytometry to measure green fluorescence (native or intact DNA) and red fluorescence (fragmented DNA). Data were exported to FlowJo software to determine the % DFI for each sample. DNA fragmentation ranged from 0.1 to 2.4% DFI, with a mean ± SD = 1.1 ± 0.7% DFI (validation curve optimized to R2 > 0.95). In conclusion, we were able to successfully validate the SCSA in our institution and establish the first normal range for sperm DNA fragmentation in rhesus macaques. Our study provides a quantitative baseline for future evaluations to assess macaque fertility through the SCSA test.

Abnormal centriolar biomarker ratios correlate with unexplained bull artificial insemination subfertility: a pilot study.

The mechanisms underlying male infertility are poorly understood. Most mammalian spermatozoa have two centrioles: the typical barrel-shaped proximal centriole (PC) and the atypical fan-like distal centriole (DC) connected to the axoneme (Ax). These structures are essential for fertility. However, the relationship between centriole quality and subfertility (reduced fertility) is not well established. Here, we tested the hypothesis that assessing sperm centriole quality can identify cattle subfertility. By comparing sperm from 25 fertile and 6 subfertile bulls, all with normal semen analyses, we found that unexplained subfertility and lower sire conception rates (pregnancy rate from artificial insemination in cattle) correlate with abnormal centriolar biomarker distribution. Fluorescence-based Ratiometric Analysis of Sperm Centrioles (FRAC) found only four fertile bulls (4/25, 16%) had positive FRAC tests (having one or more mean FRAC ratios outside of the distribution range in a group's high-quality sperm population), whereas all of the subfertile bulls (6/6, 100%) had positive FRAC tests (P = 0.00008). The most sensitive biomarker was acetylated tubulin, which had a novel labeling pattern between the DC and Ax. These data suggest that FRAC and acetylated tubulin labeling can identify bull subfertility that remains undetected by current methods and may provide insight into a novel mechanism of subfertility.

Evaluation of domestic animal sperm head morphology via flow cytometric DNA labelling and pulse shape analysis using bull and stallion spermatozoa as model species.

The aim of the present study was to test a rapid, robust flow cytometric technique for the detection of sperm head abnormalities of domestic bulls and stallions. The so-called PulSA approach detects the pulse profiles of propidium-iodide labelled spermatozoa. In the first experiment, species-specific threshold values were established on sperm samples that were tested for sperm head abnormalities with a classic visual morphology analysis. In the second experiment, serial mixtures of bull and stallion spermatozoa mimicking different percentages of sperm head abnormalities were analysed. Non-metric multidimensional scaling showed a clear separation between the normal and mixed samples. The PulSA approach may be a useful tool in identifying sub- or infertile breeding males as well as in studying the evolutionary aspects of sperm morphology and morphometry.

Role of oxidative stress in male infertility.

Abstract: Infertility affects millions of couples worldwide. Oxidative stress (OS) causes peroxidation of lipids and damage to spermatozoa, thus, reducing the quality of seminal parameters. In addition, the differences in the levels of antioxidants and reactive oxygen species (ROS) caused by intrinsic and extrinsic variables linked to lifestyle, diet, genetics, and OS also contribute to male infertility. High levels of ROS result in sperm damage of sperm parameters due to lipid peroxidation and oxidation of proteins. Other significant causes of ROS include changes in sex hormone levels, sperm DNA damage, including mutations, and immature spermatozoa. Treating the root causes of OS, by changing one's lifestyle, as well as antioxidant therapy, may be helpful strategies to fight OS-related infertility. However, the determination of male infertility induced by OS is currently a challenge in the field of reproductive health research. This review intends to describe the role of oxidative stress on male infertility and the current understanding of its management. Lay summary: The inability to conceive affects many couples globally. Oxidative stress refers to imbalances between different oxygen species which can lead to male fertility problems by damaging sperm and semen. Oxidative stress may be caused by several factors, including diets high in fats, sugars and processed foods, lifestyle (including smoking, alcohol consumption and having a sedentary lifestyle), and genetics. Treatment that focuses on the root cause may help combat male infertility. However, there is currently no consensus on the best way to treat male fertility problems, particularly those associated with oxidative stress. This paper describes the role of oxidative stress on male infertility and discusses the current techniques employed in treating male fertility issues.

Stiffness estimated by strain elastography reflects canine testicular spermatogenesis and histology.

Ultrasound elastography was proposed for the evaluation of testicular focal lesions, but no studies verified the agreement between the whole histological architecture of the testis and the stiffness measured by elastography. The present study explored the use of strain elastography in the evaluation of testis with normal or abnormal spermatogenesis, classified based on epididymal sperm attributes, and the consistency between elastographic parameters and the testicular histological feature. Strain elastography was performed during the routine andrological examination in 22 dogs presented for elective orchiectomy. Epididymal sperm attributes and testicular histology were analyzed after orchiectomy. Based on the epididymal sperm characteristics, testes were classified according to normal or abnormal spermatogenesis, and strain elastographic attributes were compared between groups. Possible correlations between strain elastography and histological features were also explored. Consistent with the literature in humans, testes with abnormal spermatogenesis were stiffer (mean strain elastographic index 3.6 ± 0.6) compared with normal testes (mean strain elastographic index 1.9 ± 0.2; P < 0.01). The strain elastographic index was negatively correlated with the area occupied by seminiferous tubules (Pearson's rho = -0.716; P = 0.0003), the mean diameter (Pearson's rho = -0.742; P = 0.0002), and thickness of the seminiferous tubule (Pearson's rho = -0.728; P = 0.0002). Surprisingly, no correlations were found between the area occupied by connective tissue in histological sections and elastographic attributes, suggesting that the increased stiffness was not related to the increased amount of connective tissue. This study demonstrated that strain elastography could be used to support the andrological examination, but measurements should be acquired in specific regions to be reliable.

An Update on Male Canine Infertility.

Infertility in the dog is a common reason for presentation of stud dogs for assessment with veterinarians. This article aims to discuss and outline some of the tests that can be done to try to ascertain the underlying cause of abnormalities found in a semen assessment. Topics discussed are semen alkaline phosphatase measurement, retrograde ejaculation assessment, ultrasound of the male reproductive tract, semen culture, human chorionic gonadotropin response testing, dietary assessment for phytoestrogens, environmental impacts on spermatogenesis, testicular biopsy, supplements to improve semen quality and quantity, and when to expect an improvement in semen quality after starting treatment.

Comparative proteomic analysis identifies differentially expressed proteins associated with meiotic arrest in cattle-yak hybrids.

Cattle-yak, the interspecific hybrid between yak and taurine cattle, exhibits male-specific sterility. Massive loss of spermatogenic cells, especially spermatocytes, results in azoospermia in these animals. Currently, the mechanisms underlying meiosis block and defects in spermatocyte development remain elusive. The present study was designed to investigate the differences in the protein composition of spermatocytes isolated from 12-month-old yak and cattle-yak testes. Histological analysis confirmed that spermatocytes were the most advanced germ cells in the testes of yak and cattle-yak at this developmental stage. Comparative proteomic analysis identified a total of 452 differentially abundant proteins (DAPs) in the fluorescence-activated cell sorting (FACS) isolated spermatocytes from cattle-yak and yak. A total of 291 proteins were only present in yak spermatocytes. Gene Ontology analysis revealed that the downregulated DAPs were mostly enriched in the cellular response to DNA damage stimulus and double-strand breaks (DSBs) repair via break-induced replication, while the proteins specific for yak were related to cell division and cycle, spermatogenesis, and negative regulation of the extrinsic apoptotic signaling pathway. Ultimately, these DAPs were related to the critical process for spermatocyte meiotic events, including DSBs, homologous recombination, synapsis, crossover formation, and germ cell apoptosis. The database composed of proteins associated with spermatogenesis, including KPNA2, HTATSF1, TRIP12, STIP1, LZTFL1, LARP7, MTCH2, STK31, ROMO1, CDK5AP2, DNMT1, RBM44, and CHRAC1, is the focus of further research on male hybrid sterility. In total, these results provide insight into the molecular mechanisms underlying failed meiotic processes and male infertility in cattle-yak.

Transcriptome analysis reveals dysregulated gene expression networks in Sertoli cells of cattle-yak hybrids.

Cattle-yak, the hybrid offspring of yak and taurine cattle, exhibits male sterility with normal female fertility. Spermatogenesis is arrested in adult cattle-yak, and apoptosis is elevated in spermatogenic cells. Currently, the mechanisms underlying these defects remain elusive. Sertoli cells are the only somatic cells that directly interact with spermatogenic cells in the seminiferous tubules and play essential roles in spermatogenesis. The present study was designed to investigate gene expression signatures and potential roles of Sertoli cells in hybrid sterility in cattle-yak. Immunohistochemical analysis showed that the 5 mC and 5hmC signals in Sertoli cells of cattle-yaks were significantly different from those of age-matched yaks (P < 0.05). Transcriptome profiling of isolated Sertoli cells identified 402 differentially expressed genes (DEGs) between cattle-yaks and yaks. Notably, niche factor glial cell derived neurotrophic factor (GDNF) was upregulated, and genes involved in retinoic acid (RA) biogenesis were changed in Sertoli cells of cattle-yak, suggesting possible impairments of spermatogonial fate decisions. Further studies showed that the numbers of proliferative gonocytes and undifferentiated spermatogonia in cattle-yak were significantly higher than those in yak (P < 0.01). Exogenous GDNF significantly promoted the proliferation of UCHL1-positive spermatogonia in yaks. Therefore, we concluded that altered GDNF expression and RA signaling impacted the fate decisions of undifferentiated spermatogonia in cattle-yak. Together, these findings highlight the role of Sertoli cells and their derived factors in hybrid sterility.

Single-cell transcriptome analysis and in vitro differentiation of testicular cells reveal novel insights into male sterility of the interspecific hybrid cattle-yak.

BACKGROUND: Interspecific hybridization plays vital roles in enriching animal diversity, while male hybrid sterility (MHS) of the offspring commonly suffered from spermatogenic arrest constitutes the postzygotic reproductive isolation. Cattle-yak, the hybrid offspring of cattle (Bos taurus) and yak (Bos grunniens) can serve as an ideal MHS animal model. Although meiotic arrest was found to contribute to MHS of cattle-yak, yet the cellular characteristics and developmental potentials of male germline cell in pubertal cattle-yak remain to be systematically investigated. RESULTS: Single-cell RNA-seq analysis of germline and niche cell types in pubertal testis of cattle-yak and yak indicated that dynamic gene expression of developmental germ cells was terminated at late primary spermatocyte (meiotic arrest) and abnormal components of niche cell in pubertal cattle-yak. Further in vitro proliferation and differentially expressed gene (DEG) analysis of specific type of cells revealed that undifferentiated spermatogonia of cattle-yak exhibited defects in viability and proliferation/differentiation potentials. CONCLUSION: Comparative scRNA-seq and in vitro proliferation analysis of testicular cells indicated that not only meiotic arrest contributed to MHS of cattle-yak. Spermatogenic arrest of cattle-yak may originate from the differentiation stage of undifferentiated spermatogonia and niche cells of cattle-yak may provide an adverse microenvironment for spermatogenesis.

A review of the use of antioxidants in bovine sperm preparation protocols.

Reactive oxygen species (ROS) and oxidative stress (OS), the imbalance between the production of free radicals and the cellular antioxidant defenses, are discussed in relation to their role in bovine sperm physiology. Oxidative stress has been associated to male infertility and low fertility rates in Assisted Reproductive Techniques (ART). Antioxidant supplementation is an interesting approach to overcome OS-related infertility and assisted reproduction drawbacks. Several studies have been conducted to identify the potential sources of ROS in a typical ART setting and the impact of antioxidant supplementation on semen quality and pregnancy outcome. Procedures such as freezing and thawing, centrifugation and incubation are thought to produce significant amounts of ROS with a negative impact on sperm quality parameters and reproductive competence. Given the important role of ROS in sperm function, the addition of antioxidants in sperm media to prevent OS and to improve the reproductive outcome requires attention. Currently, there is limited evidence to support the ameliorative effect of antioxidant supplementation on fertilization and embryo development in farm animals. This review summarizes the different types and concentrations of antioxidants used in sperm preparation media of bovine species and their effectiveness in neutralizing excessive ROS production while preserving physiological sperm function.

Localization and expression of SLX4 in the testis of sterile male cattle-yak.

Cattle-yak, the hybrid offspring of yak (Bos grunniens) and cattle (Bos taurus), serves as a unique model to dissect the molecular mechanisms underlying reproductive isolation. While female cattle-yaks are fertile, the males are completely sterile due to spermatogenic arrest at the meiosis stage and massive germ cell apoptosis. Interestingly, meiotic defects are partially rescued in the testes of backcrossed offspring. The genetic basis of meiotic defects in male cattle-yak remains unclear. Structure-specific endonuclease subunit (SLX4) participates in meiotic double-strand break (DSB) formation in mice, and its deletion results in defects in spermatogenesis. In the present study, we examined the expression patterns of SLX4 in the testes of yak, cattle-yak, and backcrossed offspring to investigate its potential roles in hybrid sterility. The results showed that the relative abundances of SLX4 mRNA and protein were significantly reduced in the testis of cattle-yak. The results of immunohistochemistry revealed that SLX4 was predominately expressed in spermatogonia and spermatocytes. Chromosome spreading experiments showed that SLX4 was significantly decreased in the pachytene spermatocytes of cattle-yak compared with yak and backcrossed offspring. These findings suggest that SLX4 expression was dysregulated in the testis of cattle-yak, potentially resulting in the failure of crossover formation and collapses of meiosis in hybrid males.