Maria Pilar Viudes de Castro

Artificial insemination represents a fundamental tool in contemporary rabbit production but continues to depend largely on the administration of exogenous GnRH analogues to trigger ovulation, raising concerns related to animal welfare and long-term sustainability. This study combined a comparative histological analysis of the distal vaginal tract in nulliparous and multiparous New Zealand White does with an on-farm evaluation of two customized 3D-printed insemination cannulas tailored to each reproductive status. A thicker cannula (maximum diameter 10 mm) was used in multiparous rabbits and a thinner cannula (maximum diameter 7 mm) in nulliparous rabbits. Histological and immunohistochemical analyses characterized the distribution of subepithelial mechanoreceptors (Pacinian, Ruffini, and Meissner corpuscles), epithelial receptors (Merkel cells), and paravaginal nerve ganglia along the first 6 cm of the vagina. Subepithelial mechanoreceptors showed the highest densities within the first two centimeters of the vaginal tract, Merkel cells increased in the third and fourth centimeters, and nerve ganglia were predominantly located at a depth of five to six centimeters. Among subepithelial mechanoreceptors, only Pacinian corpuscles were significantly more abundant in nulliparous than in multiparous females, whereas epithelial Merkel cell abundance did not differ between groups. Multiparous does showed larger paravaginal ganglia but lower neuronal density. In total, 289 inseminations were performed in receptive does using the two cannula designs, allowing the functional evaluation of these anatomically adapted devices under commercial farm conditions. Double physical stimulation resulted in fertility rates comparable to those obtained with intramuscular GnRH administration both in nulliparous does (0.93 ± 0.05 vs. 0.79 ± 0.08) and multiparous does (0.76 ± 0.05 vs. 0.85 ± 0.06), without compromising total or live litter size. The inseminations were performed by two independent operators, and no significant differences were observed between groups, confirming the reproducibility of the insemination procedure. Consequently, these findings demonstrate that appropriately designed cannulas and stimulation protocols can reliably induce ovulation in the absence of hormonal treatments, supporting the development of a sustainable artificial insemination system that maintains reproductive efficiency while enhancing animal welfare in rabbit production systems.

This study evaluated the effectiveness of a 3D-printed cannula in inducing ovulation in female rabbits without exogenous GnRH analogues. A total of 325 inseminations were performed across different physiological stages: nulliparous, multiparous lactating, and multiparous non-lactating does. Two insemination methods were compared: a physical stimulation technique using the 3D-printed cannula and a control method involving hormonal induction with buserelin acetate. Females were either synchronized with eCG or left untreated. Delivery rates and litter sizes were recorded to assess fertility outcomes. Results indicated that the insemination procedure had a noticeable effect on delivery rate, with the control group showing greater rates (79%) compared to the cannula group (65%). In conclusion, these findings suggest that physical stimulation via the cannula can induce ovulation without exogenous hormones. This hormone-free approach offers a promising and welfare-friendly alternative for rabbit artificial insemination, aligning with efforts to reduce hormone use in livestock production. Further optimization of cannula design and technique may improve results, particularly in less responsive groups, supporting sustainable and ethical reproductive management.

Sperm cryopreservation is an excellent way to store genetic materials for a long period. Semen cryopreservation is associated with structural and functional damage of the spermatozoa caused by cold shock, osmotic stress, intracellular ice crystal formation, and the production of reactive oxygen species, which reduce their functional lifespan and fertilization capacity. Here, we describe the main rabbit sperm cryopreservation protocol.

Background/Objectives: The use of antibiotics in livestock contributes to antimicrobial resistance, highlighting the need for alternative solutions. Among these, chelating agents, like ethylenediaminetetraacetic acid (EDTA) and Chitosan, have shown potential in reducing bacterial contamination in seminal doses used in artificial insemination (AI), while preserving sperm quality. The objective of this study was to evaluate the potential use of EDTA and Chitosan as alternatives to antibiotics for the liquid storage of rabbit seminal AI doses. Methods: EDTA (20 mM) and Chitosan (0.05%) were tested both individually and in combination, by adding them to the semen extender, and their effects were compared with extenders containing antibiotics or none. The extenders were evaluated for microbial resistance and their ability to maintain sperm quality in vitro during refrigeration at 16 ± 1 °C for 72 h. To assess antimicrobial efficacy, Enterococcus faecalis was used. Seminal doses stored for 24 h were used for insemination under commercial conditions, and fertility rate and total kits born were evaluated. Results: Adding 0.05% Chitosan to the extender resulted in sperm parameters and bacterial load comparable to those achieved with antibiotics during refrigerated storage, yielding similar fertility rate and total kits born outcomes 24 h post-storage. In contrast, the use EDTA alone or in combination with Chitosan was less effective at controlling Enterococcus faecalis than the antibiotic extenders, which also resulted in a reduction of sperm total motility over storage period (0–72 h) and negatively impacted fertility rate and total kits born. Conclusions: Chitosan’s protective effect on sperm function, combined with its antimicrobial activity, makes it a promising alternative antimicrobial agent for the liquid storage of rabbit seminal AI doses.

Gonadotropin‐releasing hormone (GnRH) ‐supplemented extenders have emerged as a welfare‐orientated method to induce ovulation in the artificial insemination (AI) of rabbits. The main factor that limits the bioavailability of GnRH analogue on intravaginal administration is the proteolytic activity of enzymes present in rabbit seminal plasma. The use of GnRH analogues with higher biological potency would allow us to decrease their concentration in the seminal dose without compromising effectiveness. The current study was designed to assess the efficacy of various GnRH analogues concerning their ability to induce ovulation in rabbit AI. The base solution used for experimental extenders contained an aminopeptidase inhibitor. Four experimental groups were used, females from the Control group were induced to ovulate with an intramuscular administration of 1 μg of buserelin, while in the other three groups females received an intravaginal administration of 3.5 μg of buserelin (BUS), deslorelin (DES) or fertirelin (FER) within the seminal dose. Results showed that the ovulation frequency was similar in all groups studied. A concentration of 3.5 μg of the different GnRH analogues tested in this study showed similar potency in inducing ovulation in non‐lactating females, yielding comparable results in terms of pregnancy rate at birth and prolificacy.

Rabbits have played a significant role in both livestock production and the advancement of reproductive scientific research. Their unique biological traits, including induced ovulation and a reproductive process that closely mirrors that of humans, have been pivotal in their use as a model. Moreover, their body size is perfectly aligned with the 3Rs principles: Replacement, Reduction, and Refinement. Consequently, techniques for gamete collection and embryo recovery, followed by their use in artificial insemination or embryo transfer, are characterized by being minimally invasive. However, refining in vitro fertilization and embryo culture techniques continues to present challenges. The incorporation of cutting‐edge genomic editing tools, such as CRISPR/Cas9, has reestablished rabbits as essential models in genetic and biomedical research, driving scientific progress. This review aims to describe the most effective reproductive biotechnologies for both male and female rabbits and how these methodologies are in line with the 3Rs principles—Replacement, Reduction, and Refinement—highlighting their significance in conducting ethical research.

Artificial insemination (AI) has become a popular technique in rabbit farms worldwide. This report discusses the progress made on semen extenders used in rabbit AI, setting out the latest innovations. Fresh and frozen semen have different requirements, so the extender composition will vary depending on the type of semen used. We discuss the endocrine supplementation of extenders for ovulation induction, the use of active molecules as an alternative to conventional antibiotics and the extenders developed for rabbit sperm cryopreservation.

The development of reproductive technologies in this species is contributing decisively to the development of rabbit farming and the preservation of genetic resources. Obtaining embryos is an essential step to both genetic diffusion and the preservation of genetic resources from genetic erosion or natural disasters. In rabbits, it is common for embryos to be recovered post-mortem after ovarian hyperstimulation with gonadotrophins, although the quality and number of embryos are variable, affecting the embryo viability and offspring rate by the donor. In vivo embryo production within a conservation programme aims not only to obtain a large number of embryos, but also that they come from a greater number of male and female origins, in order to ensure an adequate representation of the original population. This is why both the quality and quantity of embryos obtained per donor rabbit and the rate of donors with offspring after embryo cryopreservation must be considered, as well as the response of the embryos to the chemical, physical and physiological stress to which they are subjected in the rederivation process and its postnatal repercussions on those that survive. Rederived rabbits from cryopreserved and transferred embryos showed phenotypic growth changes, which calls into question the neutrality of the technique and its usefulness in those cases in which a control population is required.

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.