Managing aggressive behaviour in bulls often involves a trade-off between handling ease and the anabolic benefits associated with endogenous testosterone production. This study compared the effects of GnRH-immunocastration and a phytogenic feed additive on reproductive parameters, testicular echotexture and behavioural activity in young Holstein bulls aged 12-15 months. Bulls were allocated to either an immunocastration group (IC, n = 6) or a phytogenic feed additive group (PFA, n = 7). The IC group received two subcutaneous injections of 1 mL Bopriva (400 μg GnRH-protein conjugate) at a three-week interval, whereas the PFA group received 50 g/day of a phytogenic feed additive throughout the 90-day experimental period. Evaluations included scrotal circumference (SC), serum testosterone concentrations, ultrasonographic testicular echotexture assessed by mean grey value (MGV) and behavioural observations. Repeated measurements obtained throughout the study were analysed using linear mixed models. Significant group × time interactions (p < 0.001) were observed for SC, testosterone concentrations and MGV. In the IC group, testosterone concentrations and SC progressively decreased beginning on Day 15, while MGV values declined from Day 30 onward. In contrast, the PFA group maintained relatively stable testosterone concentrations and ultrasonographic echotexture throughout the observation period. Qualitative behavioural observations indicated reduced mounting attempts and restlessness in both groups during the follow-up period. Immunocastration induced marked suppression of reproductive parameters and alterations in testicular echotexture in young bulls. Under the conditions of this study, supplementation with the phytogenic feed additive was associated with relatively stable reproductive parameters, while qualitative reductions in behavioural activity were also observed. Further studies including untreated control groups and quantitative behavioural scoring systems are warranted.

Cryopreservation of epididymal stallion spermatozoa is a valuable tool for fertility preservation, particularly after orchiectomy. However, the potential interactions between local anaesthesia and extender composition on post-thaw spermatozoal quality remain poorly understood. This study investigated the effects of intratesticular lidocaine injection and the efficacy of various extender (egg yolk [EY] vs. lecithin [LC]) and cryoprotectant (glycerol [GLY] vs. dimethyl sulfoxide [DMSO]) combinations on stallion epididymal spermatological parameters. Using a paired 2 × 2 × 2 factorial design, epididymal spermatozoa were collected from both lidocaine-injected (right) and non-injected (left) testes of eight stallions. Samples were cryopreserved in Tris-based extenders supplemented with EY + GLY, EY + DMSO, LC + GLY or LC + DMSO. Spermatozoal motility (fresh, post-equilibration and post-thaw), viability, plasma membrane and acrosome integrity (PMAI), mitochondrial reactive oxygen species (MROS), spermatozoa malondialdehyde (MDA) and glutathione (GSH) levels were evaluated. Intratesticular lidocaine administration had no significant effect on spermatozoal motility at any evaluation stage, and no differences were detected in any biochemical or spermatologic parameters assessed post-thaw (p > 0.05). Regarding cryopreservation media, a significant main effect of extender type was observed for spermatozoal viability (p < 0.05) and MROS levels (p < 0.001), with EY-based extenders providing superior protection compared to LC-based formulations. A significant Extender × Cryoprotectant interaction was detected for GSH levels (p < 0.05), with the EY + GLY combination yielding the highest antioxidant capacity. No significant differences were observed in MDA levels among treatment groups (p > 0.05). Intratesticular lidocaine administration during orchiectomy does not compromise epididymal spermatozoal quality after cryopreservation. Among the evaluated cryopreservation strategies, the combination of egg yolk and glycerol in a Tris-based extender provides the most effective protection against oxidative stress and membrane damage in stallion epididymal spermatozoa.

The determination of sperm oxidative stress provides significant information related to semen quality. Most studies investigate oxidative stress markers only at the level of seminal plasma, but this approach may provide an incomplete overview of the oxidative status of semen. The aim of the study is to determine the relationship between protein-normalized oxidative stress markers and spermatological parameters in ram semen fractions. A total of 38 ejaculates obtained from 24 rams were fractionated into seminal plasma, whole semen (non-centrifuged), and pellet at different concentrations. Total antioxidant status (TAS), total oxidant status (TOS), malondialdehyde (MDA), and oxidative stress index (OSI) were measured and normalized to total protein content using the Lowry method. Fraction-wise differences in oxidative stress parameters were analysed using linear mixed-effects models accounting for ram identity, while relationships between spermatological and oxidative parameters were evaluated using Spearman correlation analysis. Principal component analysis (PCA) and hierarchical clustering were performed as exploratory multivariate visualization tools. Motility and viability were positively correlated (ρ = 0.856, p < 0.001), while both parameters were negatively correlated with abnormal sperm rate (p < 0.05). In addition, TOS and MDA levels were significantly lower in seminal plasma than in sperm pellet fractions, supporting the presence of fraction-dependent oxidative profiles (p < 0.05). Exploratory PCA suggested that overall multivariate variation was mainly associated with spermatological parameters, whereas oxidative markers had a more limited contribution. These findings provide insight into the relationship between oxidative status and different semen fractions. Additionally, fraction-specific oxidative profiling may provide complementary information beyond seminal plasma measurements alone.

South American camelids (SAC), including llamas (Lama glama) and alpacas (Vicugna pacos), exhibit distinctive reproductive features, such as induced ovulation and highly viscous seminal plasma (SP), that complicate conventional semen collection and objective sperm evaluation. Consequently, spermatozoa recovered from the cauda epididymis or through surgical vas deferens deviation (VDD) have become valuable alternatives for research and germplasm preservation. These sperm sources also provide a unique opportunity to investigate sperm physiology in the absence of SP. This review critically compares the functional characteristics of ejaculated, epididymal and VDD-derived spermatozoa and examines how methodological variables, including collection procedures, post-mortem interval, tissue handling and surgical protocols, contribute to variability among studies. Current evidence indicates that many functional differences observed among sperm sources, particularly in motility and plasma membrane functionality, are largely associated with the presence or absence of SP rather than intrinsic differences in sperm quality. These findings support a reinterpretation of SP as a physiological regulator of sperm quiescence, membrane stability and functional competence rather than merely a technical obstacle. We also review advanced analytical approaches, including flow cytometry and imaging flow cytometry, highlighting their potential to improve the evaluation of camelid sperm physiology and cryobiology, while summarizing current evidence on the developmental competence of sperm from different anatomical sources for in vitro embryo production. Overall, this review provides an updated synthesis of the literature, identifies major methodological limitations and highlights priorities for future research to advance reproductive biotechnologies in SAC.

Gestational success in ruminants depends on the maintenance of a dynamic maternal-foetal microenvironment, resulting from complex interactions among the conceptus, extraembryonic membranes, maternal tissues, and gestational fluids. This study performed a comparative and longitudinal proteomic characterization of allantoic and amniotic fluids throughout pregnancy in sheep, aiming to elucidate the molecular mechanisms involved in maternal-foetal communication and embryonic development. Samples of allantoic and amniotic fluids aseptically collected from extraembryonic compartments after uterine opening following animal slaughter and allocated into three gestational groups according to foetal development: early gestation (0-50 days; n = 6), mid gestation (50-100 days; n = 3) and late gestation (100-150 days; n = 5). After centrifugation and protein quantification using the Bradford method, standardized aliquots were subjected to in-solution protein digestion and analysed by nano-LC-ESI-Q-TOF mass spectrometry. A total of 201 proteins were identified in the allantoic fluid and 159 proteins in the amniotic fluid, with five proteins showing significant differences across gestational stages. In the allantoic fluid, differentially abundant proteins included alpha-2-macroglobulin, gelsolin, basement membrane-specific heparan sulphate proteoglycan core protein and alpha-globin chain. In the amniotic fluid, changes were observed in peroxiredoxin-2, mucin-5 AC, fetuin-B, cystatin domain-containing protein and globin domain-containing protein. Multivariate analyses revealed progressive remodelling of the foetal extraembryonic microenvironment, associated with proteolytic control, tissue remodelling, redox homeostasis, epithelial protection and metabolic processes. Collectively, these findings reinforce the active role of foetal extraembryonic fluids in gestational physiology and highlight their potential as sources of biomarkers associated with reproductive success in sheep.

The aim of this study was to evaluate the effect of anethole addition to the ovine oocyte in vitro maturation (IVM) medium on viability and chromatin configuration, intracellular production of reactive oxygen species (ROS), enzymatic activity of superoxide dismutase (SOD) and catalase (CAT), as well as the levels of reduced thiols in IVM medium, in addition to its antioxidant potential (DPPH and ABTS). Cumulus-oocyte complexes (COCs) were randomly matured in IVM medium in the absence (control group, CTRL) or presence of anethole at concentrations of 30 (AN30), 300 (AN300), and 2000 μg/mL (AN2000). COCs were matured in groups under mineral oil at 39°C in a humidified atmosphere containing 5% CO2 in air for 24 h. At the end of IVM, part of the COCs was used to assess viability and chromatin configuration using Hoechst 33,342. The remaining COCs were used to assess intracellular ROS levels using the fluorescent probe H2DCFDA. Furthermore, the IVM medium was collected to evaluate antioxidant status (SOD, CAT, and reduced thiols). Finally, the antioxidant potential of different anethole concentrations was evaluated using DPPH and ABTS assays. It was observed that AN300 promoted higher meiotic resumption rates compared to the other treatments (p < 0.05) and a lower rate of oocyte degeneration compared to AN30 (p < 0.05). However, regarding the proportion of oocytes at MII, it was observed that only AN2000 was lower than AN300 (p < 0.05). Moreover, AN30 resulted in lower ROS levels compared to AN300 and AN2000 (p < 0.05). SOD activity was lower in AN2000 compared to the other treatments (p < 0.05). AN2000 also showed the highest antioxidant capacity in the DPPH and ABTS assays among the anethole concentrations, although it was lower than the control group. In conclusion, anethole supplementation during IVM positively affected oocyte meiotic progression in sheep.

Testicular ultrasonography is increasingly used in ram breeding soundness evaluations, yet methodological standardization of quantitative grayscale image analysis remains limited. The primary objective of this study was to determine the effects of region-of-interest (ROI) placement and imaging plane on testicular parenchymal numerical pixel values (NPVs) under a fixed ultrasonographic acquisition protocol, in Güney Karaman rams. A secondary objective was to compare NPV among age groups and between two predefined reproductive-period sampling points, August and March. Twenty-nine healthy, fertility-proven rams were assigned to three age groups: 1.5 (n = 8), 3.1 (n = 12), and 4.6 years (n = 9). B-mode ultrasonography was performed with a 7.5-MHz linear transducer during the breeding (August) and non-breeding season (March). From each testis, 10 standardized regions of interest were analysed (six longitudinal and four transversal, with five above and five below the mediastinum testis). ImageJ analysis yielded 1160 spot measurements. A linear mixed model with random intercepts for animal and testis ID accounted for the hierarchical repeated-measures structure. NPVs increased with age, were higher in August than in March (71.65 ± 1.75 vs. 60.89 ± 1.75, p < 0.001), and were markedly higher below than above the mediastinum testis (89.50 ± 1.75 vs. 43.03 ± 1.75, p < 0.001). Transversal images yielded higher NPVs than longitudinal images (68.61 ± 1.77 vs. 63.93 ± 1.74, p < 0.001). Random-effects analysis showed that 31%, 7%, and 62% of total variance were attributable to between-animal, between-testis, and spot-level variation, respectively. These findings demonstrate that ram testicular echogenicity is influenced by biological and methodological factors, highlighting the need for standardized multi-site sampling protocols.

The amount and source of nutrition are important factors that influence an ewe's reproductive ability, but the effects of diet on the reproductive performance of its offspring are unclear. This study aimed to investigate the impact of maternal nutrition (level of nutrition and source) on the reproductive performance of ewes and their female lamb offspring. Seventy-two Dorper × Santa Inês ewes were allocated in a completely randomized block design, in five treatments: CTL (n = 14) with 100% of requirement, and the energy source was corn; RES (n = 14) with 90% of predicted requirement, and the energy source was corn; SUPP (n = 15) with 110% of requirement, and the energy source was corn; CR (n = 15) treatment SUPP plus chromium propionate; and FAT (n = 14) treatment SUPP plus calcium salts of palm oil. Non-parametric data were evaluated using the Fisher exact test, and parametric data were evaluated using the Tukey test, both at a 5% significance level. There was no difference in pregnancy rate (p = 0.1944) or prolificacy (p = 0.5729) among the treatments; however, the CR diet resulted in a faster return to estrus after lambing (p < 0.001). The RES diet decreased lamb survival (p < 0.005) and reduced offspring reproductive potential. Using chromium positively affected the reproductive parameters of female offspring, whereas FAT adversely affected them. In conclusion, the source and level of dietary energy influence fetal programming in ewes. The restriction negatively affected the reproductive performance of the offspring, and supplementation with chromium propionate improved it.

Evaluating the reproductive microbiome in small ruminant livestock, such as goats and sheep, is crucial for improving breeding performance, promoting animal health, and optimizing pregnancy outcomes. A healthy microbiome in the reproductive tract inhibits infections, regulates immune activity, and reduces the risk of microbial imbalances that negatively affect pregnancy. Understanding this microbiome also supports better disease control, informs advanced animal husbandry practices for goats and sheep, and contributes to global food security. This study aimed to characterize the genital microbiome of three local Portuguese goat breeds-Preta de Montesinho, Serrana, and Serpentina-using 16S rRNA metabarcoding. Samples were collected from 39 goats and, for comparison, seven sheep from a local breed-Serra da Estrela. Microbiome differences were obtained when integrating breed and sex factors in goats. The predominant genera in both vaginal and preputial samples included Fusobacterium, Aerococcus, Corynebacterium, Sediminibacterium, Campylobacter, and Ralstonia. Significant compositional differences were observed across breed, sex, and species as indicated by Bray-Curtis dissimilarity. By characterizing the caprine reproductive microbiota through metabarcoding, this study fills an important knowledge gap and establishes a baseline for future research on disease-associated changes in the male and female urogenital tract microbiota of small ruminants.

Reproductive seasonality in subtropical goats is considered weak, yet seasonal differences in ovarian dynamics remain poorly characterized. This study compared ovarian follicular and luteal dynamics during breeding (September-November) and non-breeding (May-July) seasons in six multiparous Beetal goats monitored for three consecutive estrous cycles per season, (i.e., 18 cycles per season) and maintained under semi-intensive conditions. Ovarian activity in each goat was monitored daily for three consecutive cycles using transrectal ultrasonography, and progesterone concentrations were analysed to evaluate follicular wave patterns, ovulation and luteal function. The distribution of cycle types did not differ significantly between seasons (p > 0.05); however, normal cycles occurred more frequently during the breeding season as a non-significant trend (p = 0.08). Inter-ovulatory intervals were longer, and multi-wave patterns were more frequent during the non-breeding season (p < 0.05). Preovulatory follicle size (p = 0.66) and growth rate (p = 0.18) were unaffected by season, indicating conserved follicular characteristics. Corpus luteum diameter was influenced by cycle type but not season, whereas progesterone concentrations showed a significant season-by-stage interaction (p = 0.04), indicating that seasonal differences were not uniform across the luteal cycle. Double ovulations occurred only during the breeding season. These findings indicate weak but measurable seasonal modulation of ovarian function in subtropical goats, providing insights for improving reproductive management strategies.

Cooled boar sperm is susceptible to oxidative stress, therefore, the addition of antioxidants to the extender has been a recurring alternative. The objective of this research was to evaluate the effect of silydianin (SILYD) on the preservation of cooled boar semen. Fifteen ejaculates from five boar were extended and supplemented with SILYD at 10, 20, 40 and 60 μM. Then, the samples were cooled for five days at 16°C. Every 24 h, sperm motility and kinematic were assessed with a computer-assisted sperm analysis (CASA) system. At 0 and 96 h, reactive oxygen species (ROS) and total antioxidant capacity (TAC) were evaluated by fluorescence microplate reader, and functionality membrane integrity (FMI) was assessed by hypoosmotic swelling test (HOST). Sperm viability (SV), lipid peroxidation (LPO), and mitochondrial membrane potential (ΔΨM) were evaluated by flow cytometry. Linear models were adjusted and the comparison of the means was carried out by the Duncan test. A sperm motility index (SMI) was calculated and regression analyses were performed. SILYD20 increased total motility (TM) at 72 h of cooling, and SILYD10, SILYD20 and SILYD40 improved progressive motility (PM) at 72 h of cooling, compared to the control (p < 0.05). Likewise, all treatments with SILYD increased the beat cross frequency (BCF) after 24 h (p < 0.05); and SILYD10 and SILYD40 increased rapid spermatozoa (RS) after 72 h of cooling (p < 0.05). SILYD10 reached the maximum SMI. In addition, the regression analysis showed that SILYD20 had the lowest rate of decline of SMI during the cooling period (p < 0.05). At 96 h of cooling, all SILYD treatments reduced ROS production. Likewise, SILYD10 and SILYD60 increased the TAC assessed by the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay at 0 h, and by the ferric reducing antioxidant power (FRAP) assay at 96 h of cooling, respectively (p < 0.05). At 0 h of cooling SILYD10 produced an increase in FMI (p < 0.05), and a decrease in sperm with non-intact membrane and non-peroxidized lipids (p < 0.05). It is concluded that silydianin improves the quality of cooled porcine semen, and reduces oxidative stress by lowering ROS production and enhancing antioxidant capacity.

The ZyMōt system is one of the commercially available sperm selection tools based on the microfluidic method. The aim of this study was to evaluate the potential of the ZyMōt system for the selection of canine sperm. Semen samples from six dogs were subjected to selection. Spermatozoa were assessed using basic and advanced methods of semen analysis, including computer-assisted sperm analysis and flow cytometry. Sperm viability, morphology, motility and morphostructural features, such as cell membrane integrity, acrosome integrity and mitochondrial membrane potential, were assessed. The results revealed that the ZyMōt membrane is able to retain dead, slow-moving, immotile and morphologically abnormal spermatozoa. However, no significant improvement was observed in any of the evaluated parameters in the selected sperm population. Moreover, the selection process resulted in substantial losses in the total number of sperm. For this reason, the ZyMōt system is not applicable for dog sperm selection under the currently developed protocol. Further research is necessary to adapt this method for use in this species.

Male infertility poses a significant challenge in animal breeding, impacting genetic progress and reproductive efficiency. This study investigated the effects of L-carnitine supplementation (300 mg/kg basal diet for 105 days) on semen quality and underlying molecular mechanisms in Malabari bucks using a transcriptomic approach. Eight mature bucks (2-3 years) were randomly divided into control and treatment groups (n = 4 each). Semen parameters like semen volume (mL), concentration (10[6]/mL), viability (%), abnormality (%), acrosome integrity (%), plasma membrane integrity (%) and oxidative stress via the Nitro Blue Tetrazolium (NBT) assay were evaluated. For transcriptomic analysis, sperm RNA was isolated, sequenced on the Illumina NovaSeq 6000 platform (150 bp paired-end), and differentially expressed genes (DEGs) were identified using DESeq2. Functional annotation was performed through GO enrichment and KEGG pathway analysis, whereas hub genes were identified via protein-protein interaction (PPI) analysis using STRING and Cytoscape. A total of 425 DEGs were identified, with 258 upregulated and 167 downregulated. Significant improvements were observed in acrosomal integrity, plasma membrane integrity, and oxidative stress markers following L-carnitine supplementation. GO enrichment analysis suggested upregulation of biological processes associated with meiotic cell division, sperm structural integrity, DNA repair, and spermatogenesis, alongside downregulation of carbohydrate metabolism pathways and a possible shift toward fatty acid oxidation. KEGG pathway analysis indicated upregulated Polycomb repressive complex pathway, and downregulation of insulin resistance and oestrogen signalling pathways, potentially creating a favourable endocrinological environment for male fertility. The hub genes identified, namely PMS1, ZFAND4, MYSM1, EEFSEC and CEP126, were found to be associated with key processes in male fertility. These findings provide preliminary mechanistic insights into L-carnitine's potential therapeutic role in small ruminant reproduction, and further functional validation of the identified hub genes is warranted.

Reproduction can be avoided using immunocontraception. This research aims to study the effect of the anti-GnRH vaccine Improvac on clinical and reproductive function in colony-housed adult queens (Felis catus). Short-hair mixed-breed queens (n = 12) were housed together under a 14-h photoperiod. The treated group (TRT, n = 9) received 0.5 mL of Improvac, and the control group (CON, n = 3) received 0.5 mL of saline solution. Queens received two doses 30 days (D) apart. Animals were divided into 4 groups (G). Control queens (G1: n = 3) were ovariohysterectomized at 90 D. Treated queens were ovariohysterectomized at 120 D (G2, n = 3), at 270 D (G3, n = 3) and at 330 D (G4, n = 3). Data were analysed with GLIMMIX. The estrus was determined by behavioural observation and vaginal cytology. All CON queens displayed signs of estrus every 12-20 days. In the TRT group, seven queens (n = 7/9) did not display signs of estrus until the day of ovariohysterectomy (120 D [n = 2], 270 D [n = 2], 330 D [n = 3]). However, two queens (120 D [n = 1], 270 D [n = 1]) displayed signs of estrus after receiving the vaccine, at G1 on D10 and G2 on D45. The clinical, haematological and biochemical parameters of treated queens remained within the reference range for the species, supporting short-term tolerability of the vaccine. The TRT queens developed painless, soft tissue masses in the subcutaneous tissue at the vaccination sites. The masses appeared after the first dose but were not detectable at the end of the study. No histological abnormalities were detected in the endometrium or ovaries of control queens at 90 D. However, TRT animals exhibited altered follicular development, characterised by ovarian follicular cysts, while the endometrium predominantly showed atrophic changes. This vaccine is a promising alternative for reproductive control in adult queens.

Sex-specific proteins are unique markers found on either X- or Y-chromosome bearing spermatozoa, acting like molecular signatures, and can be used as a key target to distinguish X- or Y-bearing cells. Towards this goal, by reviewing the literature, a total of 6 proteins were selected from the selected, of which 2, CLRN3 and SCAMP1, were finally chosen for the immunoprecipitation study. For the finally selected proteins, predictions of subcellular localisation and transmembrane topology were generated using online tools such as BUSCA, DeepLoc 2.0, DeepTMHMM, and TMHMM. Additionally, several online platforms like SEMMA 2.0 and IEDB were employed to identify potential immunogenic targets. Following this, commercially available antibodies against the 2 identified proteins were procured for subsequent use in multiple immunoprecipitation-based techniques, such as Western blotting, immunocytochemistry, and ELISA (Enzyme-linked immunosorbent assay). Targeting these proteins revealed their binding intensity on spermatozoa. In this study, the CLRN3 antibody targeting X-spermatozoa showed stronger interaction with X-sorted samples than the Y-specific SCAMP1 antibody in the cell-based ELISA. Results from the study suggest targeting sex-specific surface proteins could enable the development of an immune-based sperm-sorting technique in bovines. Overall, the results support the potential of CLRN3 as an X-specific surface marker that needs further validation for the development of a novel immune-based sperm sorting technique.