Abstract Ensuring boar sperm quality before insemination is crucial for maximizing field fertility and efficient pig production. The computer-assisted sperm analysis (CASA) and fluorescence probes combined with flow cytometry (FC) are commonly used techniques for evaluating sperm kinematics and functions, closely related to animal fertility. However, their high cost and complex operations make it challenging to apply them in laboratories or pig breeding farms with limited resources. Here, our aim was to develop a new protocol using a resazurin redox dye to assess boar sperm quality for practical application. We first created simulated semen samples with different levels of sperm quality (0, 20, 40, 60, 80, 100%) at concentrations of 300 and 150 × 106 cells/mL. Subsequently, the simulated semen was used to establish an optimal standard protocol based on the results of the resazurin colorimetric assay. Finally, the condition that showed the strongest correlation between resazurin redox rate and sperm parameters was selected to perform a linear regression test. Two optimal working conditions were identified, involved incubating 10 µL of resazurin reagent with 100 µL of semen for either 20 or 40 min, depending on the sperm concentration of either 300 or 150 × 106 cells/mL. We subsequently conducted a linear regression analysis using data that included the resazurin reaction rate and measurements of sperm parameters. Finally, we obtained two sets of five equations, allowing directly convert the absorbance of the resazurin assay into values for sperm quality parameters. These parameters include total motility, progressive motility, viability, acrosome integrity, and mitochondrial activity. This new protocol is valuable for sperm evaluation because it is cost-effective, time-efficient, and labor-saving.

Abstract The aim of this study was to investigate the effects of modulating reactive oxygen species (ROS) in vitrified bovine in vitro produced (IVP) embryos. In experiment I we compared ROS production in fresh and vitrified-warmed blastocysts. In experiment II we evaluated the effects of antioxidant supplementation (100 μM of 2-mercaptoethanol; BME; 0 h to 2 h during warming) on ROS levels in vitrified-warmed blastocysts, and in experiment III we compared the development of fresh and vitrified-warmed blastocysts in the presence (BME) or absence (Control) of antioxidant (100 μM BME; 0 h to 48 h during warming). Higher ROS production (Fresh: 68.48 ± 7.92 vs Vitrified: 123.53 ± 13.15; P<0.05) and lower cell number was observed in vitrified compared to fresh embryos (Fresh: 123.01 ± 5.67 vs Vitrified: 103.04 ± 4.25; P<0.05). Antioxidant supplementation reduced ROS levels (Vitrified: 38.24 ± 1.27 vs. Vitrified/BME: 33.54 ± 1.08; P<0.05) and increased cell number in treated embryos (Vitrified: 100.65 ± 3.98 vs. Vitrified/BME: 112.95 ± 3.72; P<0.05). No differences were observed in the re-expansion rates of vitrified embryos cultured in the absence and presence of BME at 0, 2, and 4 h after warming (P>0.05). The embryo hatching rate did not differ (P>0.05) among embryos from the fresh, vitrified and vitrified/BME groups. However, the total cell numbers were higher (P<0.05) in vitrified embryos supplemented with BME (143.02 ± 6.97) than in vitrified embryos without BME (113.25 ± 5.09) but similar (P>0.05) to that observed in fresh embryos cultured with (150.54 ± 8.99) and without BME (142.71 ± 13.60). It was concluded that the vitrification and warming processes increased ROS levels in blastocysts and its attenuation with BME antioxidant improved embryonic quality.

Abstract More than 90% of spermatozoa of boars in pork producing countries is stored in liquid at 17 °C; however, the quality of these spermatozoa is affected by bacterial breeding and oxidative damage. This study analyzed sperm quality and sperm capacitation after storage to study the effects of the effects of ZnO nanoparticles (ZnO NPs) supplementation on seminal plasma (SP)-free sperm preservation. We investigated the effects of adding 20, 50, 100 and 200 μg/mL of ZnO NPs to a seminal free boar sperm diluent over a 7-day period at 17 °C to assess the changes in non-capacitated/capacitated sperm quality parameters, antioxidant capacity, ATP content and extent of protein tyrosine phosphorylation. The addition of different doses of ZnO NPs to stored sperm did not induce significant effects on the sperm motility and ATP content when compared to the sperm without ZnO NPs treatment. However, the addition of 50, 100, 200 μg/mL ZnO NPs to stored sperm improved total antioxidant capacity (T-AOC) and CuZn-superoxide dismutase (CuZn-SOD) (p < 0.05). ZnO NPs also reduced the malondialdehyde (MDA) content of the preserved sperm (p < 0.05). Moreover, our results indicate that the supplementation of 50 μg/mL ZnO NPs to preserved sperm improved the sperm membrane integrity (p < 0.05). ZnO NPs exerted protective effects on protein tyrosine phosphorylation, especially with regards to membrane proteins. Following incubation and capacitation, sperm exhibited good levels of protein tyrosine phosphorylation and ATP levels with high T-AOC and CuZn-SOD activity and low MDA content. ZnO NPs exerted protective capacity to a preservation extender used for SP-free boar sperm during storage at 17 °C. The optimal concentration of ZnO NPs for preservation extender was 50 μg/mL.

Abstract This study aimed to develop a non-surgical method to neutralize reproduction in female dogs. Female Beagle puppies, aged 6 days, were treated with pellets designed to release estradiol benzoate (EB; 1.0 mg) and progesterone (P4; 5.0 mg) over approximately 3 weeks. Their estrous cycles were monitored from 6 to 34 months of age by examining their vulvas daily and measuring their serum P4 levels once a month. Vulvar edema and discharge, followed by a serum P4 level above 5 ng/ml, indicated the potential estrus. Each time a dog showed these signs, breeding was attempted by housing with a proven male Beagle. All the treated dogs displayed cyclic progesterone surges with 5 to 6-month-long anestrous intervals. Surprisingly, none exhibited sexual behaviors, and no mating occurred (i.e., no intromission and copulatory tie), resulting in no pups being born. This phenomenon was further explored in laboratory animals. Neonatal female rats were treated with microspheres containing smaller doses of the same steroids (0.3 mg EB + 3.0 mg P4) at 1 or 2 days old. At 3 months old, the rats were ovariectomized, chemically stimulated to exhibit estrus behaviors using a standard protocol and tested for receptivity to proven male rats. Untreated control rats showed normal receptivity (i.e., lordosis) and allowed males to mate. However, rats treated with EB+P4 did not exhibit lordosis or allow mating. These results indicate that the combined use of estrogen and progesterone could be an effective non-surgical method for inhibiting mating behavior and, consequently, neutralizing female dog reproduction.

Abstract Somatic cell nuclear transfer (SCNT), or cloning, is used to reprogram cells and generate genetically identical embryos and animals. However, the cloning process is inefficient, limiting its application to producing valuable animals. In swine, cloning is mainly utilized to produce genetically modified animals. Indeed, recombinant DNA technologies have evolved considerably in recent years, with homologous recombination and gene editing technologies becoming more efficient and capable of recombining both alleles in a single cell. The selection of appropriate cells and their use as nuclear donors for SCNT is the most common method for generating edited and genetically modified animals for commercial and research purposes. This article reviews current applications of swine cloning and shares our personal experiences with the procedure in this species.

Abstract This study aimed to compare the effects of nandrolone decanoate on the morphology and physiology of ovarian tissues in two experimental models, Zebrafish and rats, after in vitro cultivation. A total of 136 animals were used (Wistar rats, n=36, and Zebrafish, n=100). In both experiments, the animals were divided into two groups (Control and Deca) and were exposed to nandrolone decanoate for seven weeks. At the end of the administrations, the animals were euthanized, and the tissues were collected for morphological and biochemical analyses. Data were expressed as mean ± SEM. Tukey and Shapiro-Wilk tests were used. ANOVA and chi-square tests were applied for group comparisons. Differences were considered significant when P<0.05. The results showed an increase in body weight in Wistar rats, while Zebrafish body weight was decreased. In both experiments, the number of atretic follicles increased throughout the in vitro culture, from day 0 to day 7, in the Control group (CTRLr and CTRLz), while in the DECA group (DECAr and DECAz), atretic follicles were reduced from D0 to D7. The antioxidant environment, represented by increased the thiol content, which was significantly higher on day zero in CTRLz compared to CTRLr. SOD activity increased in Zebrafish (group DECAz), while CAT activity decreased in both models (group DECAr and DECAz). In conclusion, the study demonstrated similarity in ovarian physiology between the models exposed or not exposed to nandrolone decanoate, suggesting that, when convenient, researchers could consider changing the experimental model.

Abstract Early puberty is associated with improved long-term reproductive performance. Predicting who will achieve early puberty is limited to intensive, invasive serial blood collections for measurement of reproductive hormones. The vaginal genome during pubertal development has potential as biomarkers of early estrus in the pre-pubertal period. Pre-pubertal gilts (n =13) were followed from d74 ± 3 of age until first estrus or d214 ± 1 of age. Blood, vaginal epithelia, and anogenital distance were collected at five timepoints during reproductive development (d74, d104, d130, d160 and first estrus or end of trial). Total RNA was isolated from vaginal epithelia and relative gene expression of two toll-like receptors (TLR-4 and TLR-5), tacykinin precursor-3 (TAC-3), insulin-like growth factor-1 (IGF-1), and estrogen receptor (ERα)-alpha was quantified by real time RT-PCR, relative to expression of RPLP0. Four gilts exhibited estrus early (< d184), 3 were average (d194 to 195), 3 were late (d203 to 213), and 3 were deemed anestrus. Comparison of expression of each gene relative to d70 was performed using the PCR package in RStudio (version 1.2.5025) and Fisher’s exact t-test for TLR-4, TLR-5 and TAC-3, and ANOVA for ER-alpha and IGF-1. Correlation analysis examined the relationship between anogenital distance and age at first estrus. A single blood draw for serum progesterone was obtained 8 days after recorded first estrus or end of trial; the presence of serum progesterone supports the visual identification of standing estrus. Expression of IGF-1 and TAC-3 were up-regulated 9- and 7-fold, respectively at d160 (P < 0.05). Expression of ERα tended to be upregulated 3-fold at d104 (P = 0.08) and expression of TLR-4 and TLR-5 was not detected until first estrus. Anogenital distance was positively correlated to the first estrus. These transcripts associated with reproduction warrant further investigation into use as biomarkers to detect early estrus. longterm long term performance intensive hormones prepubertal pre period Prepubertal Pre n =13 13 d d7 d21 Blood d74, (d74 d130 d16 trial. trial . trial) tolllike toll like TLR4 TLR 4 (TLR- TLR5, TLR5 5 , TLR-5) precursor3 precursor precursor- TAC3, TAC3 TAC (TAC-3) insulinlike insulin factor1 factor factor- IGF1, IGF1 IGF (IGF-1) ERαalpha alpha RTPCR, RTPCR RT PCR, RT-PCR RPLP0 RPLP ( d184, d184 d184) d194 (d19 195, 195 195) d203 (d20 213, 213 213) anestrus version 1.2.5025 125025 2 5025 Fishers Fisher s ttest t test TLR4, 4, TLR- 3, ERalpha ER IGF1. 1. IGF- TAC- up regulated 9 7fold, 7fold fold 7 fold, 7-fold P 0.05. 005 0.05 0 05 0.05) 3fold d10 0.08 008 08 =1 d2 (d7 d13 d1 (TLR (TAC-3 (IGF-1 d18 d19 (d1 19 d20 (d2 21 1.2.502 12502 502 00 0.0 (d (TAC- (IGF- 1.2.50 1250 50 0. (TAC (IGF 1.2.5 125 1.2. 12 1.2

Abstract The relationships between members of the groups include behaviors related to affiliation, dispute for dominant positions, parental care, and facing disputes for food and territory. All these activities are under hormone modulation and those of a steroidal nature are heavily involved. Despite this, only few data are available on steroid hormones in free-ranging marmosets of the Callithrix genus, which limits the understanding of the physiological functioning and modulation of the socio-sexual behavior by steroid hormones of this taxon. In this study, we characterized fecal concentrations of progesterone, estrogens, and glucocorticoids of six breeding and non-breeding females from two groups of free-ranging Callithrix penicillata (É. Geoffroy, 1812). The concentration of progesterone was significantly higher in females which gave birth, compared to non-breeding females. The levels of fecal estrogens and glucocorticoids did not differ between breeding and non-breeding females. The data are in agreement with the few studies on steroid values of wild and captive marmosets. This study shows the concentrations of progesterone and glucocorticoids in free-ranging C. penicillata for the first time, and it is the only study reporting the concentration of fecal estrogens in wild marmosets. Overall, the high levels of progesterone associated with pregnancy in free-ranging C. penicillata as well as levels of estrogens and glucocorticoids close to those reported for other species, suggest a conserved pattern of hormonal secretion between Callithrix species that have been studied in captivity. affiliation positions care territory involved freeranging free ranging genus sociosexual socio sexual taxon nonbreeding non É. É (É Geoffroy 1812. 1812 . 1812) birth C time Overall captivity 181 18 1

Abstract Anticancer therapy often leads to premature ovarian insufficiency (POI) and infertility due to the extreme sensitivity of the ovarian follicle reserve to the effects of chemotherapy. Withanolides are known for their cytotoxic effect on cancer cells and low cytotoxicity on non-malignant or healthy cells. Therefore, this study aimed to investigate the in vivo effects of three withanolides derivatives: 27-dehydroxy-24,25-epoxywithaferin A (WT1), 27-dehydroxywithaferin A (WT2), and withaferin A (WTA) on fertility, and the ovarian preantral follicles of young female mice. To achieve this, mice received 7 intraperitoneal doses of WT1, WT2, or WTA at a concentration of 2 mg/kg (Experiment I) and 5 or 10 mg/kg (Experiment II) over 15 alternate days. In experiment I, two days after administration of the last dose, half of the mice were mated to evaluate the effects of withanolides on fertility. The other half of the mice, as well as all mice from experiment II, were sacrificed for histological, inflammation, senescence, and immunohistochemical analyses of the follicles present in the ovary. Regardless of the administered withanolide, the concentration of 2 mg/kg did not show toxicity on the follicular morphology, ovarian function, or fertility of the mice. However, at concentrations of 5 and 10 mg/kg, the three derivatives (WT1, WT2, and WTA) increased follicular activation, cell proliferation, and ovarian senescence without affecting inflammatory cells. Furthermore, at a concentration of 10 mg/kg, the three withanolides showed intensified toxic effects, leading to DNA damage as evidenced by the labeling of γH2AX, activated Caspase 3, and TUNEL. We conclude that the cytotoxic effect of the tested withanolide derivatives (WT1, WT2, and WTA) in the concentration of 2 mg/kg did not show toxicity on the ovary. However, in higher concentrations, such as 10 mg/kg, toxic effects are potentiated, causing DNA damage. POI (POI chemotherapy nonmalignant non malignant Therefore 27dehydroxy24,25epoxywithaferin 27dehydroxy2425epoxywithaferin dehydroxyepoxywithaferin 27 dehydroxy 24,25 epoxywithaferin 24 25 WT1 WT , (WT1) 27dehydroxywithaferin dehydroxywithaferin WT2 (WT2) (WTA mgkg mg kg Experiment I 1 II dose histological inflammation ovary morphology function However (WT1 activation proliferation Furthermore γH2AX γHAX γH AX 3 TUNEL potentiated 27dehydroxy24 25epoxywithaferin 2425 24,2 (WT2 (WT 27dehydroxy2 242 24, 27dehydroxy

Abstract This study was conducted to evaluate manila duck’s (Cairina moschata) frozen semen quality after cryopreservation in lactated ringer's egg yolk-astaxanthin (LREY-A) with 5 different concentrations of dimethyl sulfoxide (DMSO). Methodology: Semen was collected from 3 manila ducks (Cairina moschata) using the cloaca massage technique twice a week. Fresh semen was evaluated macro and microscopically then polled and divided into 5 tubes of treatments. Each tube was diluted in DMSO4, DMSO6, DMSO8, DMSO10, and DMSO12. The semen of each treatment was loaded into a 0.25 mL straw and equilibrated at 5 °C for 2 h. Freeze above nitrogen vapor and stored a container of liquid nitrogen at -196 °C, then semen thawed in a water bath at 37 °C for 30 sec. Data were analyzed using One-Way ANOVA Analysis. Results of this showed that post-equilibration sperm motility and sperm viability have differed significantly (P<0.05) for each treatment, with the highest % sperm motility DMSO8 and DMSO6, this is also shown in post-thawing sperm motility and viability which have differed significantly (P<0.05) and the highest % sperm viability were DMSO8 and DMSO6. In conclusion, Frozen semen extender formulation of DMSO8 and DMSO6 which are used in manila duck semen cryopreservation was the best to other treatments to maintain % sperm motility and % sperm viability in post-equilibration and post-thawing. The highest sperm motility recovery rate was in DMSO8. The lowest sperm live and dead abnormality was in DMSO8. It is concluded that the combination of DMSO8 was the best in maintaining the quality of manila duck frozen semen. s Cairina moschata ringers ringer yolkastaxanthin yolk astaxanthin LREYA LREY A (LREY-A DMSO. DMSO . (DMSO) Methodology week DMSO4 DMSO10 DMSO12 025 0 25 0.2 C h 196 -19 sec OneWay One Way Analysis postequilibration post equilibration P<0.05 P005 P 05 (P<0.05 postthawing thawing conclusion postthawing. thawing. (DMSO DMSO1 02 0. 19 -1 P<0.0 P00 (P<0.0 1 - P<0. P0 (P<0. P<0 (P<0 P< (P< (P

Abstract Deslorelin is a GnRH agonist used in veterinary medicine to temporarily inhibit reproduction in domestic animals and is sometimes tested in captive species in zoo to control population or tame aggressive behaviours in males. However, some studies have revealed the inefficacy of deslorelin specifically in males, contrary to females that follow a classic long-term inhibition of the reproductive hypothalamic-pituitary axis through sexual steroid negative feedback. We implanted 5 males and 6 females grey mouse lemurs (Microcebus murinus), long-day breeders that display a complete inhibition of the reproductive system during winter, at the end of the short-day period, a few weeks before the breeding season. Contrary to females, which exhibited a classic inhibitory response to deslorelin, males’ testosterone levels increased as well as their testis size, which suggests a sex-specific sensitivity to the negative feedback of sexual steroids before the mating period. We propose that this sex-imbalance is related to the different life-history of males as opposed to females concerning reproductive tasks and behaviour. However longterm long term hypothalamicpituitary hypothalamic pituitary Microcebus murinus, murinus , murinus) longday day winter shortday short period season size sexspecific sex specific seximbalance imbalance lifehistory life history behaviour

Abstract Embryo transfer in cattle is an increasingly important technique for cattle production. Full attainment of the benefits of the technology will depend on overcoming hurdles to optimal performance using embryos produced in vitro. Given its importance, embryo technology research should become a global research priority for animal reproduction science. Among the goals of that research should be developing methods to increase the proportion of oocytes becoming embryos through optimization of in vitro oocyte maturation and in vitro fertilization, producing an embryo competent to establish and maintain pregnancy after transfer, and increasing recipient fertility through selection, management and pharmacological manipulation. The embryo produced in vitro is susceptible to epigenetic reprogramming and methods should be found to minimize deleterious epigenetic change while altering the developmental program of the resultant calf to increase its health and productivity. There are widening opportunities to rethink the technological basis for much of the current practices for production and transfer of embryos because of explosive advances in fields of bioengineering such as microfluidics, three-dimensional printing of cell culture materials, organoid culture, live-cell imaging, and cryopreservation. importance science fertilization selection manipulation productivity microfluidics threedimensional three dimensional materials livecell live imaging cryopreservation

Abstract Over the past 40 years, assisted reproductive technologies (ARTs) have grown significantly in scale and innovation, from the bovine embryo industry’s shift from in vivo derived to in vitro produced embryos and the development of somatic cell-based approaches for embryo production. Domestic animal models have been instrumental in the development of ARTs for wildlife species in support of the One Plan Approach to species conservation that integrates in situ and ex situ population management strategies. While ARTs are not the sole solution to the biodiversity crisis, they can offer opportunities to maintain, and even improve, the genetic composition of the captive and wild gene pools over time. This review focuses on the application of sperm and embryo technologies (artificial insemination and multiple ovulation/in vitro produced embryo transfer, respectively) in wildlife species, highlighting impactful cases in which significant progress or innovation has transpired. One of the key messages following decades of efforts in this field is the importance of collaboration between researchers and practitioners from zoological, academic, governmental, and private sectors. 4 years (ARTs industrys industry s cellbased cell based production strategies crisis maintain improve time artificial ovulationin ovulation transfer respectively transpired zoological academic governmental sectors

Abstract This conference celebrates the 40th anniversary of AETE. Over the past 40 years, AETE has served as a forum for scientists, practitioners, and students working in assisted animal reproduction in livestock species. AETE conferences have reflected developments in the field, from basic to applied science, as well as regulatory changes in assisted animal reproduction practices. Europe has led the way in these developments for many years, progressing from artificial insemination, embryo transfer, and cryopreservation to semen sexing, in vitro production of embryos, cloning by nuclear transfer, genomic selection, and the rescue of highly endangered species. These significant contributions were made possible by the support of funding agencies, both at the national and European levels, promoting cooperation between scientists and practitioners. Assisted reproduction, and animal breeding more generally, face opposition from various groups, including animal rights activists, vegetarians, proponents of organic farming, environmentalists, certain political parties, and increasing regulatory burdens. These challenges seriously affect funding for scientific research, the work of practitioners, and the breeding industry as a whole. It is crucial to invest time and resources in communication to remind the public, politicians, and regulators of the achievements in this field and the contributions made to the food supply chain and the care of the rural and natural environment. th 4 years practitioners species science practices insemination transfer sexing embryos selection agencies levels generally groups activists vegetarians farming environmentalists parties burdens research whole public politicians environment

Abstract This study explored the migration of follicular fluid (FF)-derived extracellular vesicles (EVs) of the uterine environment to the bloodstream and their interaction with neutrophils in vivo and in vitro. For the in vivo experiment, six Nellore heifers (Bos indicus) received an intrauterine infusion seven days after ovulation with 1X PBS only (sham group; n=1), 1X PBS stained with lipophilic dye PKH26 (control group; n=2), or FF-derived EVs stained with PKH26 (treated group; n=3). Plasma was collected at 0, 10, 30, 60-, 180-, 360-, 720-, and 1440-min post-infusion to obtained EVs for analysis by nano flow cytometry. Labeled EVs were present in the bloodstream at 30- and 60-min post-infusion in the treatment group. Additionally, plasma derived-EVs from all groups were positive for Calcein-AM, Alix, Syntenin, and Calnexin, which confirm the presence of EVs. The second experiment utilized the plasma-derived EVs from the heifers from 30 and 60 min timepoints to evaluate if neutrophils can uptake EVs in vitro. As results, it was possible to observe the presence of labeled EVs in neutrophils treated with plasma derived-EVs from the treatment group. In summary, our results suggest that labeled EVs can migrate from the uterine environment rapidly and interact with circulating immune cells in bovine. FFderived FF derived (EVs vitro Bos indicus X sham group n=1, n1 n n=1 , 1 n=1) PKH PKH2 control n=2, n2 n=2 2 n=2) n=3. n3 n=3 . 3 n=3) 0 10 60, 60- 180, 180 180- 360, 360 360- 720, 720 720- 1440min 1440 postinfusion post cytometry 60min Additionally derivedEVs CalceinAM, CalceinAM Calcein AM, AM Calcein-AM Alix Syntenin Calnexin plasmaderived 6 summary bovine n= 18 36 72 144 7 14