Cryopreservation of ram spermatozoa is essential for genetic improvement and germplasm conservation but remains limited by cryo-induced post-thaw deterioration. Conventional egg-yolk-based (EY) extenders suffer from biological variability and biosecurity concerns, motivating the development of chemically defined alternatives. This in vitro study compared a commercial liposome-based extender (LB; OptiXcell®, IMV Technologies) with an EY extender for ram semen cryopreservation using a split-ejaculate design across two independent experiments (42 ejaculates; contrasting management and seasonal conditions). Post-thaw quality was assessed by computer-assisted sperm analysis (CASA; total and progressive motility, VAP, VCL, VSL), flow cytometry (plasma membrane integrity, acrosome integrity, mitochondrial membrane potential), and the hypo-osmotic swelling test (HOST). LB-cryopreserved semen showed higher post-thaw total motility (Experiment I: 34.8 ± 4.2% vs 25.1 ± 3.5%; Experiment II: 40.8 ± 4.1% vs 34.3 ± 4.1%) and progressive motility, with velocity parameters significantly improved. A greater proportion of spermatozoa with intact plasma membranes (Experiment I: 41.9 vs 27.4%; Experiment II: 51.5 vs 37.0%) and preserved acrosomes was detected in LB-treated samples. Mitochondrial membrane potential was higher with LB in Experiment II only (73.5 ± 4.2% vs 59.3 ± 4.2%), reflecting the context-dependent sensitivity of this endpoint. No difference was detected by HOST. Despite inter-individual variability, 94% of ejaculates exhibited higher plasma membrane integrity and two-thirds showed higher total motility with LB. These findings indicate that chemically defined liposome-based extenders improve multiple in vitro indicators of ram sperm function, whilst in vivo fertility trials remain necessary to confirm reproductive relevance.

Despite significant progress in porcine in vitro embryo production (IVP), major challenges persist, particularly regarding the biological quality and developmental competence of gametes and embryos produced in vitro. The lack of standardised and objective evaluation criteria continues to limit the efficiency, reproducibility, and translational value of porcine assisted reproductive technologies (ART). This review provides a comprehensive synthesis of current methodologies used to assess oocyte, sperm, and embryo quality in pigs, integrating insights from other mammalian models to highlight common principles and species-specific challenges. Oocyte competence remains a key determinant of IVP success, yet its assessment is hindered by variable maturation efficiency, polyspermy, and inconsistent handling procedures. Emerging approaches such as advanced imaging, metabolomics, and single-cell transcriptomics show potential for non-invasive and dynamic evaluation of oocyte quality, but their routine implementation in routine practice requires further validation and inter-laboratory standardisation. Similarly, improving sperm assessment requires moving beyond traditional parameters such as motility and morphology toward integrative frameworks that incorporate molecular, biochemical, and imaging-based markers of functionality. The strong link between gamete quality and fertilisation outcomes highlights the need for harmonised protocols and validated species-specific biomarkers to ensure reproducibility across laboratories. At the embryonic level, the development of multiparametric, data-driven frameworks combining morphological, metabolic, and molecular indicators represents a pivotal step toward objective embryo quality assessment. Although artificial intelligence, high-throughput omics, and machine learning hold great promise, their adoption will depend on rigorous validation, cost-effectiveness, and standardisation to minimise operator-dependent variability and guarantee reproducibility. Ultimately, advancing porcine IVP will depend on integrating biological insights with technological innovations through non-invasive tools and robust quality control systems adapted to the species unique physiological characteristics. This convergence offers a pathway toward more efficient, reproducible, and predictive ART in pigs, with broad implications for animal breeding, genetic preservation, and translational biomedical research.

The corpus luteum is the only structure producing progesterone during pregnancy in dogs. The aim of this study was to characterise morphological changes of corpora lutea in the bitch and assess their relationship with body weight, serum progesterone concentration, and multiple resorptions. We monitored 26 bitches weekly from ovulation confirmation to 35 days post-ovulation, measuring the corpora lutea diameter via ultrasound examination in combination with progesterone assays. The pregnancy rate was 80.7% (21/26), and all pregnancies were carried to term. Dogs were classified into small (5-15 kg), medium (16-39 kg), and large breeds (40-65 kg). Dog weight significantly influenced mean luteal diameter (P < 0.001), which ranged from a mean ± SD of 3.4 ± 0.5 mm for small dogs to 6.0 ± 0.7 mm for large dogs on the day of ovulation confirmation. From ovulation confirmation to peak, corpora lutea grew significantly (2.1 ± 1.2 mm; P < 0.001) and returned to their initial size by day 35. Surprisingly, one-third of maximum corpora lutea exceeded 1 cm before undergoing subsequent physiological diametric reduction. This growth in luteal diameter was positively correlated with serum progesterone concentration (P < 0.05). This study provides novel findings on canine corpus luteum characteristics, not previously described in literature, which could aid ovulation detection and differentiation between physiological and potentially pathological structures.

Chitosans (CS) have been of great interest due to their properties and numerous applications. However, CS have poor solubility in neutral and basic media, which limits their use in these conditions. In contrast, chitooligosaccharides (COS) have better solubility in water and lower viscosity in aqueous solutions whilst maintaining interesting biological properties. CS and COS, unlike other sugars, are not single polymers with a defined structure but are groups of molecules with modifiable structural parameters, allowing the adaptation and optimization of their properties. The great versatility of CS and COS makes these molecules very attractive for different applications, such as cryopreservation. Here, we investigated the effect of the degree of polymerization (DP), degree of N-acetylation (DA) and concentration of a series of synthesized CS and COS, water-soluble at physiological pH, on their cytotoxicity in an L929 fibroblast cell culture. Our results demonstrated that CS and COS showed no sign of toxicity regarding cell viability at low concentrations (≤10 mg/mL), independently of their DP and DA, whereas a compromising effect on cell viability was observed at a high concentration (100 mg/mL).

Docosahexaenoic acid (DHA) is an n-3 polyunsaturated fatty acid (PUFA) that improves fertility by increasing membrane fluidity. Moreover, embryos produced by donor females supplied with n-3 PUFA did not show any difference in terms of the lipid profile after 7 days of culture. The present study aimed to investigate the effects of DHA (20 and 100 μM) coupled with carnosine (5 mg/mL), an antioxidant, during oocyte maturation and embryo development on the developmental and cryosurvival rates and the number of pluripotent cells. Free fatty acid receptor-4 (FFAR4), which is able to bind DHA, was visualised by immunostaining. The addition of DHA in the in vitro development (IVD) medium decreased the percentage of pluripotent SOX2 positive cells compared with the control (8.4% vs. 10.9%) without affecting the number of cells (196.7 vs. 191.6 cells) or the developmental (20.9% vs. 23.9% blastocysts rate on D7) and cryosurvival rates (86.3% vs 86.2%). Such a decrease in pluripotent cells, relevant to the differentiation of the first lineage within the inner cell mass, represents an improvement in the embryo quality. On the contrary, embryos without any pluripotent SOX2-positive cells would not be able to achieve gestation. Future studies should follow up these results by carrying out embryo transfers to assess the beneficial effects of DHA supplementation.

Embryo lipid profile is affected by in vitro culture conditions that lead to an increase in lipids. Efforts have been made to optimize embryo lipid composition as it is associated with their quality. The objective of this study was to evaluate whether the diet supplementation of donor cows (n-3 or n-6 polyunsaturated fatty acids), or the slow freezing protocols (ethylene glycol sucrose vs. glycerol-trehalose), or the physiological stage of the donor (nulliparous heifers vs. primiparous lactating cows) may impact the bovine embryo lipid profile. Lipid extracts of 97 embryos were individually analyzed by liquid chromatography-high resolution mass spectrometry, highlighting 246 lipids, including 85% being overabundant in cow embryos compared to heifer embryos. Among 105 differential lipids, 72 were overabundant after ethylene glycol sucrose protocol, including a single glycerophosphate PA(32:1) representing 27.3% of the significantly modulated lipids, suggesting that it is degraded when glycerol-trehalose protocol is used. No lipids were different according to the n-3 or n-6 supplementation of the donor cows. In conclusion, the embryonic lipid profile was mainly affected by the physiological stage of the donors and the slow freezing protocols. The overabundance of lipids in lactating cow embryos and the resulting lower quality of these embryos are consistent with the lower pregnancy rate observed in cows compared to heifers. Unlike glycerol-trehalose protocol, ethylene glycol sucrose freezing allowed to preserve glycerophospholipids, potentially improving the slow freezing of in vitro-produced embryos. Further studies are required to modulate embryo quality and freezability by modulating the lipidome and by integrating all stages of embryonic production.

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Currently, in vitro embryo production (IVP) is successfully commercially applied in cattle. However, the high sensitivity of embryos to cryopreservation in comparison to in vivo (IVD) embryos slows the dissemination of this biotechnology. Reduced cryotolerance is frequently associated with lipid accumulation in the cytoplasm mainly due to in vitro culture conditions. The objective of this study was to evaluate the lipid composition of biopsied and sexed embryos, produced either in vivo or in vitro from the same Holstein heifers before and after a slow freezing protocol. Lipid extracts were analysed by liquid chromatography-high resolution mass spectrometry, which enabled the detection of 496 features. Our results highlighted a lipid enrichment of IVP embryos in triglycerides and oxidised glycerophospholipids and a reduced abundance in glycerophospholipids. The slow freezing process affected the lipid profiles of IVP and IVD embryos similarly. Lysophosphatidylcholine content was reduced when embryos were frozen/thawed. In conclusion, the embryonic lipid profile is impacted by IVP and slow freezing protocols but not by sex. Lysophosphatidylcholine seemed highly sensitive to cryopreservation and might contribute to explain the lower quality of frozen embryos. Further studies are required to improve embryo freezability by modulating the lipidome.

Currently, invitro embryo production (IVP) is successfully applied commercially in cattle. However, the high sensitivity of embryos to cryopreservation compared with invivo-derived (IVD) embryos still impairs the dissemination of this biotechnology. Reduced cryotolerance is frequently associated with lipid accumulation in the cytoplasm mainly due to invitro culture conditions. The objective of this study was to evaluate the lipid content of fresh and frozen sexed bovine grade 1 IVP or IVD embryos. The same 8 Holstein heifers were used in a Latin square design for both IVP and IVD embryo production. Zygotes were cultured in synthetic oviductal fluid (SOF) supplemented with 1% oestrus cow serum. The same bull was used for IVP and IVD. All expanded Day 7 blastocysts (n=40 IVP and 40 IVD) were biopsied and sexed. Half of the embryos (n=20 in each group) was slow frozen (1.5M ethylene glycol, 0.1m sucrose) and thawed before lipid extraction. Remaining embryos underwent lipid extraction in the fresh state. Briefly, the liposoluble fraction of the embryos was extracted according to the Bligh and Dyer method using chloroform and methanol. Liquid chromatography–high-resolution mass spectrometry (LC-HRMS) analysis was performed and operated in positive ionization mode. Lipids with variance intensities greater than 30% in quality control samples were removed as well as those identified as background noise. Partial least square discriminant analysis (PLS-DA) was used to show the relationship between variance in the data and difference among embryo origin (IVP vs. IVD), state before extraction (fresh vs. frozen), and sex of the embryos (male vs. female). The differentially lipid species groups were identified using Wilcoxon test, and considered significantly different when P<0.05. LC-HRMS analysis allowed us to identify 75 lipids. PLS-DA showed that embryo origin (IVP vs. IVD) and state before extraction (fresh vs. frozen) can be determined by LC-HRMS profiles by group in PLS-DA plot, despite slight overlaps. Sex of the embryos did not allow us to differentiate the lipid profile. However, 15 lipids varied significantly between male and female IVD, predominantly triglycerides (TG), whereas no lipid varied between the sexes in the IVP homologues. Moreover, 26 lipids varied significantly between IVP and IVD fresh embryos with enrichment of IVP embryos in TG, phosphatidyl choline, cholesteryl ester, and less diglyceride and lysophospholipid (LP) compared with IVD embryos. The comparison of the lipid profiles before and after freezing for IVP embryos showed that only 7 lipids varied significantly between fresh and frozen states with a decrease in LP for the frozen embryos. For the invivo counterparts, 13 lipids varied significantly, including the same LP as those identified for IVP embryos in the same way. Our results showed that the embryonic lipid profile is mainly affected by IVP and slow freezing protocols and, to a lesser extent, by sex. Further studies are needed to improve IVP protocols and optimize the cryotolerance of IVP embryos in cattle.

Premature udder development constitutes an alarm signal in pregnant mares. The objective of this clinical case report is to highlight the importance of transabdominal ultrasound examination of the fetus in these cases and to report a unique case of prenatal diagnosis of obstructive uropathy based on the observation of severe fetal hydronephrosis and megacystitis in utero. A 4-year-old French chaser primiparous mare was referred for evaluation of premature udder development during the ninth month of pregnancy. The mare had clinical signs within normal limits, a developed and sensitive udder with secretions, and no vulvar discharge. Transrectal examination revealed the presence of an immobile fetus. Combined uteroplacental thickness was within normal limits. Transabdominal ultrasound revealed a single live fetus in posterior presentation with several abdominal abnormalities. Unilateral hydronephrosis and megacystitis lead to a hypothetical diagnosis of fetal multiple urinary tract malformation with outflow obstruction. Treatment was discontinued and the mare was monitored. Abortion occurred spontaneously a week later. Postmortem examination revealed a ruptured bladder of abnormally large dimensions and a severely distended left kidney without parenchyma (filled with free urine) and lack of permeability in the left ureter and urethra. Postmortem diagnosis was consistent with our prenatal ultrasonographic diagnosis. Even though described during human pregnancy with various etiologies and severity, prenatal diagnosis of fetal hydronephrosis and megacystitis has not been reported in equine veterinary medicine before. These malformations need to be characterized more precisely in the future. This case highlights the importance of transabdominal ultrasonography to detect equine fetal abnormalities.

Controlling the supercooling state during ice crystallization process is important for differential scanning calorimetry (DSC) analysis. In cryopreservation solutions, the control of the nucleation temperature (T-n) is necessary as it allows the simulation of slow-freezing procedures. Herein, the effect of Snomax (R) on T-n value in aqueous solutions containing dimethyl-sulfoxide (Me2SO) (H2O + 10 %(v/v)Me2SO) was studied. The effect of Me2SO, a cryoprotectant, on nucleation control was investigated by evaluating lambda(het), which describes the influence of a molecule on the nucleation behaviour of an ice-nucleating agent (INA). Note that lambda(het) is specific to the molecule added in solution and to the maximum melting temperature of the solution. The knowledge of lambda(het) helps evaluate the ranges of the supercooling magnitude at which the INA in solution can trigger nucleation. This study shows how Snomax can be used in DSC experiments using the H2O + 10 %(v/v)Me2SO solution when ice nucleation must be controlled.

Induced pluripotent stem cells (iPSCs) are obtained by genetically reprogramming adult somatic cells via the overexpression of specific pluripotent genes. The resulting cells possess the same differentiation properties as blastocyst-stage embryonic stem cells (ESCs) and can be used to produce new individuals by embryonic complementation, nuclear transfer cloning, or in vitro fertilization after differentiation into male or female gametes. Therefore, iPSCs are highly valuable for preserving biodiversity and, together with somatic cells, can enlarge the pool of reproductive samples for cryobanking. In this study, we subjected rabbit iPSCs (rbiPSCs) and rabbit ear tissues to several cryopreservation conditions with the aim of defining safe and non-toxic slow-freezing protocols. We compared a commercial synthetic medium (STEM ALPHA.CRYO3) with a biological medium based on fetal bovine serum (FBS) together with low (0–5%) and high (10%) concentrations of dimethyl sulfoxide (DMSO). Our data demonstrated the efficacy of a CRYO3-based medium containing 4% DMSO for the cryopreservation of skin tissues and rbiPSCs. Specifically, this medium provided similar or even better biological results than the commonly used freezing medium composed of FBS and 10% DMSO. The results of this study therefore represent an encouraging first step towards the use of iPSCs for species preservation.

Optimizing thawing conditions are required to maximize recovery of semen capacities after cryopreservation. This study aimed to assess the effect of thawing procedures on motility recovery and maintenance of semen capacities over time. A fractional factorial design approach was used to reduce the number of repetitions and simultaneously analyze the different interactions. Thirty canine frozen semen samples were thawed under different thawing conditions. Motility and velocity parameters were recorded using a computer-assisted semen analyzer up to 6 hours after thawing. Ten quadratic models were found to be significant, with the most significant effects observed on the velocity parameters. Second, this study allowed us to evaluate the effect of the proAKAP4 marker on frozen/thawed semen, with regard to motility recovery. ProAKAP4 is the precursor of A-kinase anchor protein 4. It has been studied in several species as a marker to assess sperm quality. In our study, the expression of proAKAP4 was determined using flow cytometry. No correlation was found between post-thaw motility parameters and proAKAP4 levels. However, the effects of thawing temperature, incubation time, and straw size were significant and similar to those observed for velocity parameters.

Animal-derived products are widely used in sperm cryopreservation for their cryoprotective properties. These components, however, must be replaced because of sanitary risks. STEMALPHA.CRYO3 (Ref. 5617, Stem Alpha), called CRYO3, is a chemically defined preservation medium currently used for freezing human tissue and adult stem cells. The aim of this study was to evaluate the effects of a CRYO3-based medium and of two cooling rates on invitro parameters and invivo fertility of ram sperm. Six rams (Blanche du Massif Central) were subjected to sperm collection four times using an artificial vagina. Sperm were split and frozen in three media: an egg yolk and milk-based medium (positive control), a CRYO3-based medium (tested medium), and a medium without additives (negative control). The two cooling rates were related to the distance between the straws and the surface of liquid nitrogen during the freezing process (5 and 20cm). Sperm membrane integrity (propidium iodide/SYBR-14), acrosome integrity (fluorescein isothiocyanate-peanut agglutinin/propidium iodide; FITC-PNA/PI), and mitochondrial membrane potential (JC-1) were assessed using flow cytometry, whereas functional membrane integrity was assessed using a hypo-osmotic swelling test and motion characteristics were evaluated using computer-assisted sperm analysis. Pregnancy rate, parturition rate, and prolificacy were evaluated after performing laparoscopic inseminations (n=75 ewes). Moreover, we characterised the freezing media thermodynamically using a differential scanning calorimeter. Statistical analyses were performed using R software. Invitro parameters were assessed using a mixed model including the time and the medium as fixed effects and the ram as a random effect. Pregnancy and parturition rates, following a binomial distribution, and prolificacy, assumed to follow a Poisson distribution, were analysed using generalised linear models, including the medium as a fixed effect and the ram as a random effect. Differences with P<0.05 were considered statistically significant. The cooling rates had no significant effect except on the wobble motion parameter. The positive control medium showed significantly higher results than the CRYO3-based medium and the negative control medium for all invitro parameters except for straightness motion parameter. Conversely, field trials showed no significant difference between the media for pregnancy rate (71, 64, and 74%), parturition rate (68, 61, and 74%) and prolificacy (2.0, 2.1, and 1.7), for the positive control, CRYO3-based medium, and the negative control, respectively. This study showed that the product, CRYO3, cannot replace egg yolk and milk in freezing extenders. Moreover, we showed that laparoscopic inseminations allowed a 74% parturition rate due to an easy and inexpensive medium comprising only a Tris buffer and glycerol. Although it could not be used on a large scale, this medium remains an option for international transport or long-term storage of genetic diversity.

Background Although data are still needed, early neutering of cats appears to be as safe as neutering at the traditional age of six months or older and beneficial to the individual and the population. The aim of this observational study was to obtain an overview of veterinarians’ opinions and practices about feline early neutering (ie, until the age of four months). Methods In this retrospective work, a web survey was distributed to French practitioners. Results A total of 609 veterinarians returned the survey. Majority of the veterinarians (56 per cent) reported never performing early neutering, 42 per cent reported performing it inconsistently, and 2 per cent reported consistently performing it. When carried out, it was mainly on kittens from commercial breeding, following breeders’ request, and performed at the age of three months. An overwhelming majority (93 per cent) of veterinarians performing early neutering reported no incidents. Veterinarians who did not practise early neutering neutered mostly (81 per cent) kittens from four to six months of age. Conclusion In conclusion, early neutering is still not a common practice among French veterinarians. Opinions differed as to its advantages and disadvantages, although reported incidents were scarce. Veterinarians who do not perform early neutering reported a lack of interest in this practice rather than reluctance.

The use of dimethyl sulfoxide (Me2SO) as a cryoprotectant agent (CPA) is controversial. Indeed, this cryoprotectant agent (CPA) is cytotoxic and potentially mutagenic. Therefore, other cryoprotectants must be used to reduce the proportion of Me2SO in slow-freezing solutions. In this chapter, we propose to present the first evaluation of new non-penetrating cryoprotectants: the chitooligosaccharides (COS). These molecules are chitosan oligomers, which are biocompatible, antioxidant, and bacteriostatic. We first review the use of saccharides through cryopreservation processes. We question the possibility to reduce penetrating CPA during slow-freezing procedures. We propose to use COS as extracellular CPA to reduce the use of Me2SO. We question the biocompatibility of COS on mouse embryos through the analysis of the cells’ development. Next, we evaluate these molecules in slow-freezing solutions with a reduced quantity of Me2SO. Our experimental approach is a physical method often used to characterize slow-freezing solutions. Differential scanning calorimetry (DSC) allows to evaluate the crystallization and melting processes, the amount of crystallized water, and the equilibrium temperature and consequently to evaluate the impact of different cryoprotectants. This study gives a better understanding on how slow-freezing protocols could be improved with extracellular CPA.