Proteomic Insights into Seminal Plasma and Spermatozoa Proteins of Small-Spotted Catsharks, Scyliorhinus canicula: Implications for Reproductive Conservation in Aquariums.

In the ex situ conservation of chondrichthyan species, successful reproduction in aquaria is essential. However, these species often exhibit reduced reproductive success under human care. A key aspect is that conventional sperm analyses do not provide insights into the functional competence of sperm. However, proteomics analysis enables a better understanding of male physiology, gaining relevance as a powerful tool for discovering protein biomarkers related to fertility. The present work aims to build the first proteome database for shark semen and to investigate the proteomic profiles of seminal plasma and spermatozoa from small-spotted catsharks (Scyliorhinus canicula) related to the underlying adaptations to both natural and aquarium environments, thereby identifying the reproductive impact in aquarium specimens. A total of 305 seminal plasma and 535 spermatozoa proteins were identified. Among these, 89 proteins (29.2% of the seminal plasma set) were common to both spermatozoa and seminal plasma. In the seminal plasma, only adenosylhomocysteinase protein showed differential abundance (DAP) between wild and aquarium animals. With respect to the spermatozoa proteins, a total of 107 DAPs were found between groups. Gene Ontology enrichment analysis highlighted the primary functional roles of these DAPs involved in oxidoreductase activity. Additionally, KEGG analysis indicated that these DAPs were primarily associated with metabolic pathways and carbon metabolism. In conclusion, we have successfully generated an initial proteome database for S. canicula seminal plasma and spermatozoa. Furthermore, we have identified protein variations, predominantly within spermatozoa, between aquarium and wild populations of S. canicula. These findings provide a foundation for future biomarker discovery in shark reproduction studies. However, additional research is required to determine whether these protein variations correlate with reproductive declines in captive sharks.

Magnetic-Assisted Control of Eggs and Embryos via Zona Pellucida-Linked Nanoparticles.

Eggs and embryo manipulation is an important biotechnological challenge to enable positioning, entrapment, and selection of reproductive cells to advance into a new era of nature-like assisted reproductive technologies. Oviductin (OVGP1) is an abundant protein in the oviduct that binds reversibly to the zona pellucida, an extracellular matrix that surrounds eggs and embryos. Here, the study reports a new method coupling OVGP1 to magnetic nanoparticles (NP) forming a complex (NPOv). NPOv specifically surrounds eggs and embryos in a reversible manner. Eggs/embryos bound to NPOv can be moved or retained when subjected to a magnetic force, and interestingly only mature-competent eggs are attracted. This procedure is compatible with normal development following gametes function, in vitro fertilization, embryo development and resulting in the birth of healthy offspring. The results provide in vitro proof-of-concept that eggs and embryos can be precisely guided in the absence of physical contact by the use of magnets.

Modulation of Caecal Microbiota and Metabolome Profile in Salmonella-Infected Broilers by Phage Therapy.

Bacteriophage therapy is considered one of the most promising tools to control zoonotic bacteria, such as Salmonella, in broiler production. Phages exhibit high specificity for their targeted bacterial hosts, causing minimal disruption to the niche microbiota. However, data on the gut environment's response to phage therapy in poultry are limited. This study investigated the influence of Salmonella phage on host physiology through caecal microbiota and metabolome modulation using high-throughput 16S rRNA gene sequencing and an untargeted metabolomics approach. We employed 24 caecum content samples and 24 blood serum samples from 4-, 5- and 6-week-old broilers from a previous study where Salmonella phages were administered via feed in Salmonella-infected broilers, which were individually weighed weekly. Phage therapy did not affect the alpha or beta diversity of the microbiota. Specifically, we observed changes in the relative abundance of 14 out of the 110 genera using the PLS-DA and Bayes approaches. On the other hand, we noted changes in the caecal metabolites (63 up-accumulated and 37 down-accumulated out of the 1113 caecal metabolites). Nevertheless, the minimal changes in blood serum suggest a non-significant physiological response. The application of Salmonella phages under production conditions modulates the caecal microbiome and metabolome profiles in broilers without impacting the host physiology in terms of growth performance.

Reproductive Performance of Female Rabbits Inseminated with Extenders Supplemented with GnRH Analogue Entrapped in Chitosan-Based Nanoparticles.

Rabbit is a reflexively ovulating species. Accordingly, in the practice of artificial insemination (AI) ovulation must be induced via exogenous GnRH (Gonadotropin-Releasing Hormone) administration, which may be performed intramuscularly, subcutaneously, or intravaginally. Unfortunately, the bioavailability of the GnRH analogue when added to the extender is lower due to the proteolytic activity in the seminal plasma and the poor permeability of the vaginal mucosa. The aim of the study was to refine the practice of AI practice in rabbits by replacing parenteral GnRH analogue administration (subcutaneous, intravenous, or intramuscular injection) with intravaginal application, while reducing its concentration in the diluent. Extenders containing the buserelin acetate in chitosan-dextran sulphate and chitosan-alginate nanoparticles were designed and 356 females were inseminated. Reproductive performance of females inseminated with the two experimental extenders, receiving 4 µg of buserelin acetate intravaginally per doe, was compared with that in the control group, the does of which were inseminated with the extender without the GnRH analogue and induced to ovulate with 1 µg of buserelin acetate administered intramuscularly. The entrapment efficiency of the chitosan-dextran sulphate complex was higher than that of chitosan-alginate. However, females inseminated with both systems showed similar reproductive performance. We conclude that both nanoencapsulation systems are an efficient way of intravaginal ovulation induction, allowing a reduction in the level of the GnRH analogue normally used in seminal doses from 15-25 µg to 4 µg.

Zoonotic Parasites in Playgrounds in Southern Spain: A One Health Approach.

Zoonotic parasitic diseases are considered a global threat to public health. In this sense, canines and felines may be infected by different cosmopolitan parasites, with playgrounds serving as an important focus of infection for humans, as well as domestic or wild animals. Knowledge of the epidemiological situation of parasites in animal reservoirs integrated into the environment, identifying the spread pathways, is a key element for an effective response to this threat. Thus, the aim of this study was to assess the frequency of intestinal parasites with zoonotic potential in 120 playgrounds in the Malaga province (Spain). Samples were processed and analysed following standard parasitological procedures. Some 36.7% of playgrounds were parasite-positive with one or more zoonotic parasites. The most common parasites recovered were nematodes (60.0%), followed by protozoan species (33.3%) and cestodes (6.7%). In the parasite-positive playgrounds, Toxocara spp. (17.0 ± 3.5%) and Giardia duodenalis (17.0 ± 3.4%) were the most predominant parasites. In addition, 34.1% of playgrounds were infected with multiple parasites. Our results show a high presence of parasitic forms with zoonotic potential in playgrounds in Malaga, Spain. Due to the close contact between pets and humans in playgrounds, the potential zoonotic risk may increase if prevention and control measures are not designed.

A 3D-Printed Large Holding Capacity Device for Minimum Volume Cooling Vitrification of Embryos in Prolific Livestock Species.

Although many devices have been developed to reduce sample volume, with an explosion of methods appearing in the literature over the last decade, commercially available devices with simultaneous vitrification of a larger number of embryos are scarce, with the apparent gap for their use in prolific livestock species. In this study, we investigated the effectiveness of a new three-dimensional (3D)-printed device that combines minimum volume cooling vitrification with simultaneous vitrification of a larger number of rabbit embryos. Late morulae/early blastocysts were vitrified with the open Cryoeyelet® device (n = 175; 25 embryos per device), the open Cryotop® device (n = 175; 10 embryos per device), and the traditional closed French mini-straw device (n = 125; 25 embryos per straw) and compared in terms of in vitro development and reproductive performance after transfer to adoptive mothers. Fresh embryos constituted the control group (n = 125). In experiment 1, there was no difference in the development rate to the blastocyst hatching stage between the CryoEyelet® and the other devices. In experiment 2, the CryoEyelet® device showed a higher implantation rate compared with the Cryotop® (6.3% unit of SD, p = 0.87) and French mini-straw® (16.8% unit of SD, p = 1.00) devices. In terms of offspring rate, the CryoEyelet® device was similar to the Cryotop® device but superior to the French straw device. Regarding embryonic and fetal losses, the CryoEyelet® showed lower embryonic losses compared to other vitrification devices. The analysis of bodyweight showed that all devices showed a similar outcomes-a higher birthweight but a lower body weight at puberty than those in the fresh transfer embryos group. In summary, the CryoEyelet® device can be used for the vitrification of many late morulae or early blastocyst stage rabbit embryos per device. Further studies should be performed to evaluate the CryoEyelet® device in other polytocous species for the simultaneous vitrification of a large number of embryos.

Examining the effects of Salmonella phage on the caecal microbiota and metabolome features in Salmonella-free broilers.

Bacteriophages selectively infect and kill their target bacterial host, being a promising approach to controlling zoonotic bacteria in poultry production. To ensure confidence in its use, fundamental questions of safety and toxicity monitoring of phage therapy should be raised. Due to its high specificity, a minimal impact on the gut ecology is expected; however, more in-depth research into key parameters that influence the success of phage interventions has been needed to reach a consensus on the impact of bacteriophage therapy in the gut. In this context, this study aimed to investigate the interaction of phages with animals; more specifically, we compared the caecum microbiome and metabolome after a Salmonella phage challenge in Salmonella-free broilers, evaluating the role of the phage administration route. To this end, we employed 45 caecum content samples from a previous study where Salmonella phages were administered via drinking water or feed for 24 h from 4, 5 to 6-weeks-old broilers. High-throughput 16S rRNA gene sequencing showed a high level of similarity (beta diversity) but revealed a significant change in alpha diversity between broilers with Salmonella-phage administered in the drinking water and control. Our results showed that the phages affected only a few genera of the microbiota's structure, regardless of the administration route. Among these, we found a significant increase in Streptococcus and Sellimonas in the drinking water and Lactobacillus, Anaeroplasma and Clostridia_vadinBB60_group in the feed. Nevertheless, the LC-HRMS-based metabolomics analyses revealed that despite few genera were significantly affected, a substantial number of metabolites, especially in the phage administered in the drinking water were significantly altered (64 and 14 in the drinking water and feed groups, respectively). Overall, our study shows that preventive therapy with bacteriophages minimally alters the caecal microbiota but significantly impacts their metabolites, regardless of the route of administration.

Gut-Brain Axis: Insights from Hippocampal Neurogenesis and Brain Tumor Development in a Mouse Model of Experimental Colitis Induced by Dextran Sodium Sulfate.

Chronic inflammatory bowel disorders (IBD) are idiopathic diseases associated with altered intestinal permeability, which in turn causes an exaggerated immune response to enteric antigens in a genetically susceptible host. A rise in psych cognitive disorders, such as anxiety and depression, has been observed in IBD patients. We here report investigations on a model of chemically induced experimental colitis by oral administration of sodium dextran sulfate (DSS) in C57BL/6 mice. We investigate, in vivo, the crosstalk between the intestine and the brain, evaluating the consequences of intestinal inflammation on neuroinflammation and hippocampal adult neurogenesis. By using different DSS administration strategies, we are able to induce acute or chronic colitis, simulating clinical characteristics observed in IBD patients. Body weight loss, colon shortening, alterations of the intestinal mucosa and fecal metabolic changes in amino acids-, lipid- and thiamine-related pathways are observed in colitis. The activation of inflammatory processes in the colon is confirmed by macrophage infiltration and increased expression of the proinflammatory cytokine and oxidative stress marker (Il-6 and iNOS). Interestingly, in the hippocampus of acutely DSS-treated mice, we report the upregulation of inflammatory-related genes (Il-6, Il-1ß, S-100, Tgf-ß and Smad-3), together with microgliosis. Chronic DSS treatment also resulted in neuroinflammation in the hippocampus, indicated by astrocyte activation. Evaluation of stage-specific neurogenesis markers reveals deficits in the dentate gyrus after acute and chronic DSS treatments, indicative of defective adult hippocampal neurogenesis. Finally, based on a possible causal relationship between gut-related inflammation and brain cancer, we investigate the impact of DSS-induced colitis on oncogenesis, using the Ptch1+/-/C57BL/6 mice, a well-established medulloblastoma (MB) mouse model, finding no differences in MB development between untreated and DSS-treated mice. In conclusion, in our experimental model, the intestinal inflammation associated with acute and chronic colitis markedly influences brain homeostasis, impairing hippocampal neurogenesis but not MB oncogenesis.

Rapid Oxford Nanopore Technologies MinION Sequencing Workflow for Campylobacter jejuni Identification in Broilers on Site­A Proof-of-Concept Study

Campylobacter is recognised as one of the most important foodborne bacteria, with a worldwide health and socioeconomic impact. This bacterium is one of the most important zoonotic players in poultry, where efficient and fast detection methods are required. Current official culture methods for Campylobacter enumeration in poultry usually include >44 h of culture and >72 h for identification, thus requiring at least five working shifts (ISO/TS 10272-2:2017). Here, we have assembled a portable sequencing kit composed of the Bento Lab and the MinION and developed a workflow for on-site farm use that is able to detect and report the presence of Campylobacter from caecal samples in less than five hours from sampling time, as well as the relationship of Campylobacter with other caecal microbes. Beyond that, our workflow may offer a cost-effective and practical method of microbiologically monitoring poultry at the farm. These results would demonstrate the possibility of carrying out rapid on-site screening to monitor the health status of the poultry farm/flock during the production chain.

Sildenafil Citrate Enhances Renal Organogenesis Following Metanephroi Allotransplantation into Non-Immunosuppressed Hosts.

In order to harness the potential of metanephroi allotransplantation to the generation of a functional kidney graft on demand, we must achieve further growth post-transplantation. Sildenafil citrate (SC) is widely known as a useful inductor of angiogenesis, offering renoprotective properties due to its anti-inflammatory, antifibrotic, and antiapoptotic effects. Here, we performed a laparoscopic metanephroi allotransplantation after embedding sildenafil citrate into the retroperitoneal fat of non-immunosuppressed adult rabbit hosts. Histology and histomorphometry were used to examine the morphofunctional changes in new kidneys 21 days post-transplantation. Immunofluorescence of E-cadherin and renin and erythropoietin gene expression were used to assess the tubule integrity and endocrine functionality. After the metanephroi were embedded in a 10 µM SC solution, the new kidneys' weights become increased significantly. The E-cadherin expression together with the renin and erythropoietin gene expression revealed its functionality, while histological mature glomeruli and hydronephrosis proved the new kidneys' excretory function. Thus, we have described a procedure through the use of SC that improves the outcomes after a metanephroi transplantation. This study gives hope to a pathway that could offer a handsome opportunity to overcome the kidney shortage.

Comparative Study of Semen Parameters and Hormone Profile in Small-Spotted Catshark (Scyliorhinus canicula): Aquarium-Housed vs. Wild-Captured.

Several chondrichthyan species are threatened, and we must increase our knowledge of their reproductive biology in order to establish assisted reproductive protocols for ex situ or in situ endangered species. The small-spotted catshark (Scyliorhinus canicula) is one of the most abundant shark species of the Mediterranean coast and is easy to maintain in aquaria; therefore, it is considered an ideal reproductive model. This study aimed to compare S. canicula male reproductive function in aquarium-housed (n = 7) and wild-captured animals, recently dead (n = 17). Aquarium-housed animals had lower semen volume (p = 0.005) and total sperm number (p = 0.006) than wild-captured animals, but similar sperm concentrations. In terms of sperm parameters, aquarium-housed sharks showed higher total sperm motility (p = 0.004), but no differences were observed regarding sperm viability, mitochondrial membrane potential, or membrane integrity. A morphometric study pointed to a significantly longer head (p = 0.005) and acrosome (p = 0.001) in wild-captured animals. The results of the spermatozoa morphological study of S. canicula were consistent with previous results obtained in other chondrichthyan species. With regard to sex hormones, testosterone levels were significantly lower in aquarium-housed animals (p ≤ 0.001), while similar levels of 17ß-estradiol and progesterone were found. In short, the present study provides evidence of good in vitro semen quality in S. canicula housed in an aquarium, underlining their excellent potential for application in reproductive technologies for this and other chondrichthyan species.

Early Embryo Exposure to Assisted Reproductive Manipulation Induced Subtle Changes in Liver Epigenetics with No Apparent Negative Health Consequences in Rabbit.

Embryo manipulation is a requisite step in assisted reproductive technology (ART). Therefore, it is of great necessity to appraise the safety of ART and investigate the long-term effect, including lipid metabolism, on ART-conceived offspring. Augmenting our ART rabbit model to investigate lipid metabolic outcomes in offspring longitudinally, we detected variations in hepatic DNA methylation ART offspring in the F3 generation for embryonic exposure (multiple ovulation, vitrification and embryo transfer). Through adult liver metabolomics and proteomics, we identified changes mainly related to lipid metabolism (e.g., polyunsaturated fatty acids, steroids, steroid hormone). We also found that DNA methylation analysis was linked to changes in lipid metabolism and apoptosis genes. Nevertheless, these differences did not apparently alter the general health status. Thus, our findings suggest that ART is likely to be a player in embryo epigenetic events related to hepatic homeostasis alteration in adulthood.

A review of cleaning and disinfection guidelines and recommendations following an outbreak of classical scrapie.

Classical scrapie is a prion disease of small ruminants, the infectious agent of which has been shown to be extremely persistent in the environment. Cleaning and disinfection (C&D) after a scrapie outbreak is currently recommended by many governments' veterinary advisors and implemented in most farms affected. Yet, the effectiveness of these procedures remains unclear. The aim of this study was to review existing literature and guidelines regarding farm C&D protocols following classical scrapie outbreaks and assess their effectiveness and the challenges that translation of policy and legislative requirements present at a practical level. A review of the literature was conducted to identify the on-farm C&D protocols used following outbreaks of scrapie, assess those materials with high risk for persistence of the scrapie agent on farms, and review the existing evidence of the effectiveness of recommended C&D protocols. An expert workshop was also organised in Great Britain (GB) to assess: the decision-making process used when implementing C&D protocols on GB farms, the experts' perceptions on the effectiveness of these protocols and changes needed, and their views on potential recommendations for policy and research. Outputs of the literature review revealed that the current recommended protocol for C&D [1 h treatment with sodium hypochlorite containing 20,000 ppm free chlorine or 2 M sodium hydroxide (NaOH)] is based on laboratory experiments. Only four field farm experiments have been conducted, indicating a lack of data on effectiveness of C&D protocols on farms by the re-occurrence of scrapie infection post re-stocking. Recommendations related to the control of outdoor environment, which are difficult and expensive to implement, vary between countries. The expert workshop concluded that there are no practical, cost-effective C&D alternatives to be considered at this time, with control therefore based on C&D only in combination with additional time restrictions on re-stocking and replacement with non-susceptible livestock or more genetically resistant types, where available. Participants agreed that C&D should still be completed on scrapie affected farms, as it is considered to be "good disease practice" and likely to reduce the levels of the prion protein. Participants felt that any additional protocols developed should not be "too prescriptive" (should not be written down in specific policies) because of significant variation in farm types, farm equipment and installations. Under this scenario, control of classical scrapie on farms should be designed with a level of C&D in combination with re-stocking temporal ban and replacement with livestock of limited susceptibility.

Antibacterial Activity of Some Molecules Added to Rabbit Semen Extender as Alternative to Antibiotics.

Although great attention is paid to hygiene during semen collection and processing, bacteria are commonly found in the semen of healthy fertile males of different species. As the storage of extended semen might facilitate bacterial growth, extenders are commonly supplemented with antibiotics. This study aimed to evaluate the antibacterial activity of ethylenediaminetetraacetic acid (EDTA), bestatin and chitosan-based nanoparticles added to rabbit semen extender and their effect on reproductive performance under field conditions. Four different extenders were tested, supplemented with antibiotics (TCG+AB), with EDTA and bestatin (EB), with EDTA, bestatin and chitosan-based nanoparticles (QEB) or without antibiotics (TCG-AB). Extended semen was cooled at 15 °C for three days. Cooled samples were examined for bacterial growth and semen quality every 24 h for 3 days. The enterobacteria count increased considerably during storage at 72 h in semen extended with TCG+AB and TCG-AB, while extenders EB and QEB showed a bacteriostatic effect over time. After 24, 48 and 72 h, quality characteristics were retained in all groups, with no significant motility differences, either in acrosome integrity, membrane functionality or the viability of spermatozoa. Additionally, bacterial concentration present in fresh semen did not affect reproductive performance. In conclusion, EDTA and bestatin exerted a potent bacteriostatic effect over time and could be used as an alternative to conventional antibiotics in rabbit semen extenders.

Development of Decellularized Oviductal Hydrogels as a Support for Rabbit Embryo Culture.

The oviducts (fallopian tubes in mammals) function as the site of fertilization and provide necessary support for early embryonic development, mainly via embryonic exposure to the tubal microenvironment. The main objective of this study was to create an oviduct-specific extracellular matrix (oviECM) hydrogel rich in bioactive components that mimics the native environment, thus optimizing the developmental trajectories of cultured embryos. Rabbit oviducts were decellularized through SDS treatment and enzymatic digestion, and the acellular tissue was converted into oviductal pre-gel extracellular matrix (ECM) solutions. Incubation of these solutions at 37 °C resulted in stable hydrogels with a fibrous structure based on scanning electron microscopy. Histological staining, DNA quantification and colorimetric assays confirmed that the decellularized tissue and hydrogels contained no cellular or nuclear components but retained important components of the ECM, e.g. hyaluronic acid, glycoproteins and collagens. To evaluate the ability of oviECM hydrogels to maintain early embryonic development, two-cell rabbit embryos were cultured on oviECM-coated surfaces and compared to those cultured with standard techniques. Embryo development was similar in both conditions, with 95.9% and 98% of the embryos reaching the late morula/early blastocyst stage by 48 h under standard culture and oviECM conditions, respectively. Metabolomic analysis of culture media in the presence or absence of embryos, however, revealed that the oviECM coating may include signalling molecules and release compounds beneficial to embryo metabolism.

Effect of Embryo Vitrification on the Steroid Biosynthesis of Liver Tissue in Rabbit Offspring.

Preimplantation embryo manipulations during standard assisted reproductive technologies (ART) have significant repercussions on offspring. However, few studies to date have investigated the potential long-term outcomes associated with the vitrification procedure. Here, we performed an experiment to unravel the particular effects related to stress induced by embryo transfer and vitrification techniques on offspring phenotype from the foetal period through to prepuberal age, using a rabbit model. In addition, the focus was extended to the liver function at prepuberal age. We showed that, compared to naturally conceived animals (NC), offspring derived after embryo exposure to the transfer procedure (FT) or cryopreservation-transfer procedure (VT) exhibited variation in growth and body weight from foetal life to prepuberal age. Strikingly, we found a nonlinear relationship between FT and VT stressors, most of which were already present in the FT animals. Furthermore, we displayed evidence of variation in liver function at prepuberal age, most of which occurred in both FT and VT animals. The present major novel finding includes a significant alteration of the steroid biosynthesis profile. In summary, here we provide that embryonic manipulation during the vitrification process is linked with embryo phenotypic adaptation detected from foetal life to prepuberal age and suggests that this phenotypic variation may be associated, to a great extent, with the effect of embryo transfer.

Roles of host genetics and sperm microbiota in reproductive success in healthy rabbit.

Although the effects of sperm microbiota and sperm quality have been described previously, recent studies provide evidence that female genital modifications triggered by seminal components could be of significant importance to identify some disturbances associated with fertility. So, sperm microbiota could play a key role in sperm quality, contributing to fertilisation. To understand how sperm microbiota diversity is influenced by the host genetics, the symbiotic bacteria in four inbred lines raised in the same animal facility and their effects on sperm quality and fertility were analysed. Forty healthy rabbits from four selected Spanish commercial lines were used in this research (three based on litter performance, designated A, V and LP, and one selected for daily body weight gain, called R). Significant variations in the seminal concentration, morphology and some motion parameters were found among inbred lines, but sperm motility and viability were similar among inbred lines. After mating, inbred lines selected for litter size had the same fertility rate, significantly higher than inbred line selected for body weight (82 ± 3.3%, 79 ± 3.5% and 89 ± 4.5% versus 61 ± 3.7%, for the A, V and LP vs R lines, respectively, p < 0.05). Bacteria belonging to Proteobacteria, Firmicutes, Fusobacteria and Bacteroidetes were identified in sperm microbiota. At genus level, the bacterial community composition in the sperm microbiota was influenced by host genetics. A total of 35, 16, 34, and 51 genera were accurately detected in the A, V, LP, and R lines, respectively. Moreover, Enhydrobacter, Ferruginibacter, Myroides Paracoccus, Rheinheimera, Tepidiphilus, Tetradesmus obliquus and Thauera genera were present only in the inbred lines selected for litter size. Moreover, the discriminant analysis revealed Lysinibacillus and Flavobacterium genera as potential biomarkers for fertility. Thus, these two genera may play a key role in fertility. Our results demonstrated the existence of a rabbit inbred line-specific variation in bacterial occurrence in sperm microbiota. Moreover, fertility differentials among inbred lines that were not predicted by routine semen analysis could be partly explained by the symbiotic state of the semen microbiota.

Impact of embryo technologies on secondary sex ratio in rabbit.

Increasing evidence indicates that assisted reproductive technologies (ARTs) disturb skewed sex-ratio and induce sex-dimorphic postnatal effects. Undoubtedly, the combination of multiple ovulation and embryo transfer (MOET) together with the use of vitrification technique (MOVET) is currently being used in breeding programs. However, since the first case of sex skewing reported in 1991, the accumulative and long-term transmission of skewed sex-ratio to future generations has not been thoroughly evaluated. Here we test as MOVET program induce a skewed sex ratio, and we consider skewed sex ratio transmission to future generations. To this end, we first evaluated the F1 generation, demonstrating that a MOVET program causes a severe imbalance skewed secondary sex ratio (SSR) towards male by 12%. This imbalanced persist after a second MOVET program (F2 generation), with an accumulative skewed SSR towards male by 25%. Finally, using a crossbred generation derived from crossing F1 males derived from a MOVET program with naturally-conceived (NC) females, we show that the imbalance skewed SRR persist. Bodyweight comparison between MOVET animals and NC counterparts revealed significant changes at birth, weaning and adulthood. However, there was a significant interaction between F2 MOVET animals and sex, demonstrating an apparent accumulative sex-dimorphic effect. At adulthood, MOVET derived males presented a lower body weight. In conclusion, we show that the MOVET program causes a direct, accumulative and long-term transmission of skewed SSR.

Metabolomic Analysis Reveals Changes in Preimplantation Embryos Following Fresh or Vitrified Transfer.

Although assisted reproduction technologies (ARTs) are recognised as safe, and most of the offspring seem apparently healthy, there is clear evidence that ARTs are associated with changes in the embryo's developmental trajectory, which incur physiological consequences during the prenatal and postnatal stages of life. The present study aimed to address the influence of early (day-3 embryos) embryo transfer and cryopreservation on embryo survival, size, and metabolome at the preimplantation stage (day-6 embryos). To this end, fresh-transferred (FT) and vitrified-transferred (VT) embryos were compared using naturally-conceived (NC) embryos as a control reference. The results show that as in vitro manipulation was increased (NC < FT < VT), both embryo survival rate (0.91 ± 0.02, 0.78 ± 0.05 and 0.63 ± 0.05, for NC, FT, and VT groups, respectively) and embryo size (3.21 ± 0.49 mm, 2.15 ± 0.51 mm, 1.76 ± 0.46 mm of diameter for NC, FT, and VT groups, respectively) were significantly decreased. Moreover, an unbiased metabolomics analysis showed overall down-accumulation in 40 metabolites among the three experimental groups, with embryo transfer and embryo cryopreservation procedures both exerting a cumulative effect. In this regard, targeted metabolomics findings revealed a significant reduction in some metabolites involved in metabolic pathways, such as the Krebs cycle, amino acids, unsaturated fatty acids, and arachidonic acid metabolisms. Altogether, these findings highlight a synergistic effect between the embryo transfer and vitrification procedures in preimplantation embryos. However, the ex vivo manipulation during embryo transfer seemed to be the major trigger of the embryonic changes, as the deviations added by the vitrification process were relatively smaller.

Rederivation by Cryopreservation of a Paternal Line of Rabbits Suggests Exhaustion of Selection for Post-Weaning Daily Weight Gain after 37 Generations.

Rabbit selection programmes have mainly been evaluated using unselected or divergently selected populations, or populations rederived from cryopreserved embryos after a reduced number of generations. Nevertheless, unselected and divergent populations do not avoid genetic drift, while rederived animals seem to influence phenotypic traits such as birth and adult weights or prolificacy. The study aimed to evaluate the effect of a long-term selection for post-weaning average daily weight gain (ADG) over 37 generations with two rederived populations. Specifically, two coetaneous populations were derived from vitrified embryos with 18 generational intervals (R19 and R37), reducing or avoiding genetic drift and environmental and cryopreservation effects. After two generations of both rederived populations (R21 vs. R39 generations), all evaluated traits showed some progress as a result of the selection, the response being 0.113 g/day by generation. This response does not seem to affect the estimated Gompertz growth curve parameters in terms of the day, the weight at the inflexion point or the adult weight. Moreover, a sexual dimorphism favouring females was observed in this paternal line. Results demonstrated that the selection programme had improved ADG without variations in adult body weight but, after 37 generations of selection, this trait seems exhausted. Given the reduction in the cumulative reproductive performance and as a consequence in the selection pressure, or possibly/perhaps due to an unexpected effect, rederivation could be the cause of this weak selection response observed from generation 18 onwards.