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 number of antral follicles is considered an important fertility trait because animals with a high follicle count (HFC) produce more oocytes and embryos per cycle. Identification of these animals by genetic markers such as single nucleotide polymorphisms (SNPs) can accelerate selection of future generations. The aim of this study was to perform a genome wide association study (GWAS) on Nelore and Angus heifers with HFC and low (LFC) antral follicle counts. The groups HFC and LFC for genotyping were formed based on the average of total follicles (≥ 3 mm) counted in each breed consistently ± standard deviation. A total of 72 Nelore heifers (32 HFC and 40 LFC) and 48 Angus heifers (21 HFC and 27 LFC) were selected and the DNA was extracted from blood and hair bulb. Genotyping was done using the Illumina Bovine HD 770K BeadChip. The GWAS analysis showed 181 and 201 SNPs with genotype/phenotype association (P ≤ 0.01) in Nelore and Angus heifers, respectively. Functional enrichment analysis was performed on candidate genes that were associated with SNPs. A total of 97 genes were associated to the 181 SNPs in the Nelore heifers and the functional analysis identified genes (ROBO1 and SLIT3) in the ROBO-SLIT pathway that can be involved in the control of germ cell migration in the ovary as it is involved in lutheal cell migration and fetal ovary development. In the Angus heifers, 57 genes were associated with the 201 SNPs, highlighting Fribilin 1 (FBN1) gene, involved in regulation of growth factors directly involved in follicle activation and development. In summary, GWAS for Nelore and Angus heifers showed SNPs associated with higher follicle count phenotype. Furthermore, these findings offer valuable insights for the further investigation of potential mechanism involved in follicle formation and development, important for breeding programs for both breeds. (HFC cycle (SNPs generations (GWAS (LFC counts ≥ ( mm deviation 7 32 (3 4 21 (2 2 bulb K BeadChip 18 20 genotypephenotype genotype phenotype P 0.01 001 0 01 respectively 9 ROBO1 ROBO (ROBO SLIT3 SLIT ROBOSLIT development 5 FBN1 FBN (FBN1 gene summary Furthermore breeds 0.0 00 (FBN 0.

Abstract Cryoprotectants are required to reduce damage caused to the cells due to low temperatures during the cryopreservation. Antifreeze proteins (AFP) have a well-known role in cell membrane protection, while resveratrol is a potent antioxidant. This study assessed the effect of the association of resveratrol concentrations and AFP I in a ram semen extender. Pooled semen of four rams was allocated into six treatments in a factorial arrangement: (CONT, only the semen extender); only AFP I (ANT: 0.1 µg/mL of AFP I), only resveratrol, one treatment with two levels (10 µM/mL or 50 µM/mL of resveratrol); and two treatments with the interactions, with one AFP I and one of the two levels of resveratrol (0.1 µg/mL of AFP I with 10 µM/mL resveratrol; 0.1 µg/mL of AFP I with 50 µM/mL resveratrol). No interaction between factors was observed on sperm kinetics, plasma membrane integrity, hypo-osmotic test, and mitochondrial activity parameters. There was a high probability (P = 0.06) of reducing sperm cells with functional membrane percentage in the hypo-osmotic test and increasing the percentage of sperm with high mitochondrial activity (P = 0.07) was observed in AFP presence. An interaction of AFP and resveratrol was observed in non-capacitated sperm (P = 0.009), acrosomal reaction (P = 0.034), and sperm binding (P = 0.04). In conclusion, the association of resveratrol and AFP did not improve the quality of frozen-thawed semen and even promoted deleterious effects compared to their single addition in the semen extender. The supplementation of 50 µM/mL of resveratrol improved the outcomes of frozen-thawed ram sperm, being a potential cryoprotectant. cryopreservation (AFP wellknown well known protection antioxidant extender arrangement CONT, CONT (CONT extender) ANT (ANT 01 0 1 0. µgmL µg mL I, , I) (1 µMmL µM 5 resveratrol) interactions (0. resveratrol. . kinetics integrity hypoosmotic hypo osmotic parameters P 0.06 006 06 0.07 007 07 presence noncapacitated non capacitated 0.009, 0009 0.009 009 0.009) 0.034, 0034 0.034 034 0.034) 0.04. 004 0.04 04 0.04) conclusion frozenthawed frozen thawed cryoprotectant ( (0 0.0 00 000 0.00 003 0.03 03

Abstract Germ cell transplantation in fish is a promising technique for surrogate broodstock parents with broader application in aquaculture and conserving endangered and valuable genetic resources. Herein, we describe the establishment of an intrapapillary xenogeneic transplant of germ cells from sexually mature goldfish (C. auratus) males into common carp (C. carpio) males cytoablated with a thermochemical treatment (two doses of busulfan at 40 mg/kg at 35°C). To analyze the presence and development of donor germ cells in recipient testes, donor germ cells were labeled with PKH26, a fluorescent cell membrane dye, before transplantation. Our results demonstrated that thermochemical treatment caused effective spermatogenesis suppression and pronounced germ cell loss. Moreover, transplanted spermatogonial cells were able to colonize the recipients’ testes, resume spermatogenesis, and generate spermatozoa within eight weeks after germ cell transplantation. These findings suggested that recipient testes provided suitable conditions for the survival, colonization, proliferation, and differentiation of donor spermatogonia from a related species. This study indicated that recipients’ testes exhibited a high degree of plasticity to accept and support xenogeneic donor germ cells, which were able to form sperm in a short time frame. This approach has significant implications for assisted animal reproduction, biotechnology, conservation, and the production of valuable genetic resources and endangered fish species. Herein C. C (C auratus carpio two 4 mgkg mg kg 35°C. 35C 35°C . 35 35°C) PKH26 PKH dye loss Moreover recipients survival colonization proliferation species frame reproduction biotechnology conservation 3 PKH2

Abstract Reproductive control is one of the biggest challenges in tilapia production and triploidy was developed as an alternative to sterilization. In general, polyploids present chromosomal instability but for triploid Nile tilapia it has yet to be reported. This study evaluated the chromosomal instability from juveniles to adulthood, growth performance and gonadal status of tilapia hatched from eggs submitted or not to heat shock for triploid induction. Nile tilapia oocytes were fertilized (1,476 oocytes), half of the eggs were subjected to a four-minute shock in 41 °C water four minutes after fertilization and the other half were not (Control group). The eggs were incubated (at 27°C) and 160 larvae from the treated group hatched and survived after yolk sac absorption. The determination of ploidy was performed by flow cytometry at 85th (juveniles) and 301st (adults) days of age post yolk sac absorption. At the time of the first cytometry analysis there were 73 surviving juveniles from the treated group, and only 14 were confirmed triploid. However, at the analysis of adult ploidy, one out of 8 surviving adult tilapias from the 14 confirmed triploid juveniles remained triploid. Gonadal histology showed that the non-remaining triploids continued to produce gametes. The growth performance of triploid tilapia was initially superior to that of diploid tilapia during the juvenile phase, but similar in adults. Once the chromosome sets are lost and the tilapias become diploid again, at least in tissues with a high proliferation rate, such as the hematopoietic tissue that was analyzed (and possibly in gonads), all possible advantages of triploids are probably lost. Thus, our results suggest that, due to genomic instabilities, the triploid generation of tilapia has low efficiency. sterilization general reported adulthood induction 1,476 1476 1 476 (1,47 oocytes, , oocytes) fourminute minute 4 C Control group. . group) 27°C 27C 27 16 absorption th (juveniles st adults (adults 7 However nonremaining non remaining gametes phase again rate gonads, gonads gonads) Thus instabilities efficiency 1,47 147 47 (1,4 2 1,4 (1, 1, (1 (

Abstract We hypothesized that the hCG modulates the expression of IFNT-pathway and ISGs in bovine endometrium during early pregnancy. The aim of the current study is to evaluate the effect of hCG on IFNT-pathway signals and ISGs expression in endometrial cells. For this, 29 non-lactating cross-bread cows were used in the study and submitted to a 9-day fixed-time artificial insemination (FTAI) protocol. The day of the AI was considered Day 0 (D0), and five days (D5) after the FTAI, the cows were allocated into two groups: Control and hCG group, when a hCG group received a single dose of 2.500UI of hCG. On day 18 after FTAI (D18) cows were slaughtered and endometrial tissue samples were collected. There was no difference between the embryo recovery rate of the cows in C compared to the hCG. The hCG group increased the accessory corpus luteum formation rate. The hCG resulted in greater serum progesterone concentration in the hCG group compared to the C on Day 14. Only the expression of IFNAR2 and STAT1 were upregulated on pregnant cows of the hCG group compared to the C group. The pathway genes (JAK1, STAT2, and IRF9) were not regulated. The mRNA abundance of ISG15, MX1, MX2, and OAS1 was upregulated in pregnant cows for hCG group, compared to C group. The results show that the administration of hCG, 5 days after AI, in addition to increasing the serum progesterone, modulates the expression of IFNT-pathway and ISGs on bovine endometrium on Day 18 of pregnancy. IFNTpathway IFNT pregnancy cells this 2 nonlactating non lactating crossbread cross bread 9day 9 fixedtime fixed time (FTAI protocol D0, D0 D , (D0) D5 (D5 groups 2500UI UI 500UI 1 D18 (D18 collected 14 IFNAR STAT JAK1, JAK1 JAK (JAK1 STAT2 IRF9 IRF regulated ISG15 ISG MX1 MX MX2 OAS (D0 (D D1 (D1 (JAK ISG1

Abstract Since the 1970s, maternal corticosteroid therapy has been used successfully to induce labor. This allows for better monitoring of parturients and provision of first aid to neonates, improving neonatal viability, as this treatment induces maturation in a variety of fetal tissues, thereby reducing morbidity and mortality. Although the effects of corticosteroids are well known, few studies have investigated the influence of this therapy in Santa Inês sheep. This study aimed to evaluate the efficacy of dexamethasone at two doses (8 and 16 mg) to induce lambing in Santa Inês ewes at 145 days of gestation and assess its effects on neonatal vitality. For this study, 58 ewes raised in an extensive system were investigated. Pregnancy was confirmed after artificial insemination at a set time or after controlled mounting. Ewes were separated into three groups: an untreated control group (G1: 0 mg) and groups treated with two doses of dexamethasone (G2: 8 mg and G3: 16 mg). In total, 79 lambs were born. Their vitality was assessed based on their Apgar score, weight, temperature, and postnatal behavior. SAS v9.1.3 (SAS Institute, Cary, NC) was used to analyze data, considering a 5% significance level for all analyses. The births in the induced groups occurred 48.4 ± 22.1 h after induction, while the ewes that underwent non-induced labor gave birth 131.96 ± 41.9 h after placebo application (p < 0.05), confirming the efficacy of dexamethasone to induce and synchronize labor. The induced and non-induced neonates had similar Apgar scores, temperatures, weights, and postnatal behavioral parameters (p > 0.05). This study showed that inducing labor in Santa Inês ewes at 145 days of gestation with a full (16 mg) or half dose (8 mg) of dexamethasone is an effective technique and does not compromise neonate vitality. 1970s s viability tissues mortality known sheep ( 1 14 5 mounting G1 G (G1 G2 (G2 G3 mg. . total 7 born score weight temperature behavior v913 v v9 3 v9.1. Institute Cary NC data analyses 484 48 4 48. 221 22 22. induction noninduced non 13196 131 96 131.9 419 41 9 41. p 0.05, 005 0.05 , 05 0.05) scores temperatures weights 0.05. (1 (G v91 v9.1 2 1319 13 131. 00 0.0 v9. 0.

Abstract Reviewing the current state of knowledge on reproductive performance and productive traits in rams has many advantages. First, the compilation of this information will serve as a literature resource for scientists conducting research around the world and will contribute to the understanding of the data collected and interpreted by researchers on the different hormonal strategies used to improve reproductive performance in rams. Second, it will allow scientists to identify current knowledge gaps and set future research priorities in ram reproduction. Rams play an important role in the global flock economy, but their reproductive analysis has been limited in the use of hormonal technologies to increase the productivity of sheep flocks. In this review, we cite the most important works on six hormones that, in one way or another, modify the hypothalamus-pituitary-gonadal axis, at different doses, in and out of the reproductive season, breeds, application methods, among other factors. The overall aim is to increase the reproductive efficiency of rams in different scenarios and, in some cases, of other species due to the lack of limited information on rams. advantages First Second reproduction economy flocks review that another hypothalamuspituitarygonadal hypothalamus pituitary gonadal axis doses season breeds methods factors cases

Abstract In the current study, we aimed to assess the expression levels of two circulating microRNAs (miRNA) (oar-miR-485-5p and oar-miR-493-5p) in the ovine plasma during the peri-implantation. After mating, we collected the plasma samples from a total of 8 ewes on day 22 of pregnancy (P22; n = 4) and day 22 of the estrous cycle (C22; n=4). We used mature miRNA sequences for oar-miR-485-5p and oar-miR-493-5p out of one hundred fifty, which were retrieved from our microarray results of previous study. We showed that the miRNA expression of oar-miR-485-5p and oar-miR-493-5p were upregulated in P22 (P<0.05) when compared to C22. Those two miRNAs targeted 311 target genes in the peri-implantation period of pregnancy. Furthermore, we revealed 151 GO/pathway terms in biological process (BP) and 25 GO/pathway terms in molecular function (MF), while we demonstrated 13 GO/pathway terms in cellular component (CC). We revealed three hub genes as interleukin 2 (IL2), interleukin 18 (IL18), and C-X-C Motif Chemokine Ligand 10 (CXCL10). In conclusion, both miR-485-5p and oar-miR-493-5p have the potential to be a biomarker to understand peri-implantation of the ovine pregnancy in the aspect of pregnancy-reflected changes in maternal plasma. study (miRNA oarmiR4855p oarmiRp oar miR 485 5p p oarmiR4935p 493 periimplantation. periimplantation peri implantation. implantation mating P (P22 4 C22 C (C22 n=4. n4 n=4 . n=4) fifty P2 P<0.05 P005 0 05 (P<0.05 31 Furthermore 15 GOpathway GO pathway BP (BP MF, MF , (MF) 1 CC. CC (CC) IL2, IL2 IL (IL2) IL18, IL18 (IL18) CXC X CXCL10. CXCL10 CXCL (CXCL10) conclusion miR4855p miRp pregnancyreflected reflected oarmiR 48 49 (P2 C2 (C2 n= P<0.0 P00 (P<0.0 3 (MF (CC (IL2 IL1 (IL18 CXCL1 (CXCL10 (P (C P<0. P0 (P<0. (IL (IL1 (CXCL1 P<0 (P<0 (CXCL P< (P<

Abstract The objective of this study was to evaluate the effect of three doses of equine chorionic gonadotropin (eCG) for ovarian superstimulation on ovarian response, follicular development and cumulus-oocyte complexes (COCs) collection in llamas. For this purpose, eighteen multiparous non-lactating adult (4–7 yo) female llamas with an average body condition of 2.8 (BCS 1–5) were submitted to a follicular ablation (FA) to induce a new follicular wave emergence. Two days after FA (Day 0), synchronized llamas were randomly allocated to three treatment groups (n = 6/group) and given 500, 750 and 1000 IU of eCG (Novormon®, Syntex, Buenos Aires, Argentina) per animal respectively to induce ovarian superstimulation. Transrectal ultrasonography were performed on Days 2, 4, and 6; and ovum pick up (OPU) was performed on Day 6. Data was evaluated by one-way analysis of variance (ANOVA), repeated measures ANOVA, and 2-tailed Chi-square. The average size (mm) of follicles was greater (p≤ 0.05) in the 1000 IU group compared to the other groups. There was a greater (p≤ 0.05) number of follicles ≥ 7 mm in the 1000 IU group compared to the 500 IU group. Number of COCs collected on Day 6 and the COC recovery rate were not different among groups. In conclusion, a single dose of 1000 IU of eCG induced the best ovarian response resulting in larger and greater number of follicles at the time of OPU. (eCG cumulusoocyte cumulus oocyte (COCs purpose nonlactating non lactating 4–7 47 4 (4– yo 28 2 8 2. BCS 1–5 15 1 5 (FA emergence 0, 0 , 0) n 6/group 6group 75 100 Novormon®, Novormon (Novormon® Syntex Aires Argentina OPU (OPU oneway one way ANOVA (ANOVA) 2tailed tailed Chisquare. Chisquare Chi square. square Chi-square (mm p≤ p (p 0.05 005 05 50 conclusion 4– (4 1– 10 Novormon® (Novormon (ANOVA 0.0 00 ( 0.

Abstract In vitro cell culture is a well-established technique present in numerous laboratories in diverse areas. In reproduction, gametes, embryos, and reproductive tissues, such as the ovary and endometrium, can be cultured. These cultures are essential for embryo development studies, understanding signaling pathways, developing drugs for reproductive diseases, and in vitro embryo production (IVP). Although many culture systems are successful, they still have limitations to overcome. Three-dimensional (3D) culture systems can be close to physiological conditions, allowing greater interaction between cells and cells with the surrounding environment, maintenance of the cells' natural morphology, and expression of genes and proteins such as in vivo. Additionally, three-dimensional culture systems can stimulated extracellular matrix generating responses due to the mechanical force produced. Different techniques can be used to perform 3D culture systems, such as hydrogel matrix, hanging drop, low attachment surface, scaffold, levitation, liquid marble, and 3D printing. These systems demonstrate satisfactory results in follicle culture, allowing the culture from the pre-antral to antral phase, maintaining the follicular morphology, and increasing the development rates of embryos. Here, we review some of the different techniques of 3D culture systems and their applications to the culture of follicles and embryos, bringing new possibilities to the future of assisted reproduction. wellestablished well established areas reproduction gametes embryos tissues endometrium cultured studies pathways diseases IVP. IVP . (IVP) successful overcome Threedimensional Three dimensional D (3D conditions environment morphology vivo Additionally threedimensional three produced drop surface scaffold levitation marble printing preantral pre phase Here (IVP

Abstract Steroids and gonadotrophins are essential for the regulation of late stages of preantral development and antral follicular development. Although the luteinizing hormone receptor (LHCGR) has been detected in the preantral follicles of rats, rabbits, and pigs, its expression, in bovine fetal ovary, has not been demonstrated. Based on this, we aimed to investigate the expression of the LHCGR and LHCGR mRNA binding protein (LRBP), as well as, to quantify bta-miR-222 (a regulatory microRNA of the LHCGR gene) during the development of bovine fetal ovary. In summary, LHCGR expression was observed in the preantral follicle in bovine fetal ovary, from oogonias to primordial, primary and secondary stages, and the mRNA abundance was lower on day 150 than day 60. However, the mRNA abundance of LRBP followed the opposite pattern. Similar to LRBP, the abundance of bta-miR-222 was higher on day 150 than day 60 or 90 of gestation. The LHCGR protein was detected in oogonia, primordial, primary, and secondary follicles. Moreover, both oocytes and granulosa cells showed positive immunostaining for LHCGR. In conclusion, we suggest the involvement of LHCGR/LRBP/bta-mir222 with mechanisms related to the development of preantral follicles in cattle. (LHCGR rats rabbits pigs ovary demonstrated this , (LRBP) btamiR222 btamiR bta miR 222 bta-miR-22 a gene summary primordial 15 However pattern 6 9 gestation oogonia Moreover conclusion LHCGR/LRBP/btamir222 LHCGRLRBPbtamir222 LHCGRLRBPbtamir LHCGR/LRBP/bta mir222 mir LHCGR/LRBP/bta-mir22 cattle (LRBP btamiR22 22 bta-miR-2 1 btamir222 btamir LHCGR/LRBP/btamir22 LHCGRLRBPbtamir22 LHCGRLRBPbta mir22 LHCGR/LRBP/bta-mir2 btamiR2 2 bta-miR- btamir22 LHCGR/LRBP/btamir2 LHCGRLRBPbtamir2 mir2 LHCGR/LRBP/bta-mir bta-miR btamir2 LHCGR/LRBP/btamir

Abstract The CRISPR/Cas9 system is a simpler and more versatile method compared to other engineered nucleases such as Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs), and since its discovery, the efficiency of CRISPR-based genome editing has increased to the point that multiple and different types of edits can be made simultaneously. These advances in gene editing have revolutionized biotechnology by enabling precise genome editing with greater simplicity and efficacy than ever before. This tool has been successfully applied to a wide range of animal species, including cattle, pigs, dogs, and other small animals. Engineered nucleases cut the genome at specific target positions, triggering the cell's mechanisms to repair the damage and introduce a mutation to a specific genomic site. This review discusses novel genome-based CRISPR/Cas9 editing tools, methods developed to improve efficiency and specificity, the use of gene-editing on animal models and translational medicine, and the main challenges and limitations of CRISPR-based gene-editing approaches. CRISPRCas9 CRISPRCas CRISPR Cas9 Cas CRISPR/Cas ZFNs (ZFNs ActivatorLike Activator Like TALENs, TALENs , (TALENs) discovery CRISPRbased based simultaneously before species cattle pigs dogs animals positions cells cell s site genomebased tools specificity geneediting medicine approaches (TALENs

Abstract This study aims to investigate the gene expression of sperm-borne phospholipase C zeta (PLCζ), WW domain-binding protein 2N-Terminal Like (WBP2NL), and Tumor necrosis factor (TNF-α), as a negative control, in spermatozoa and their relationship with fertility and seminal quality in stallions. Ejaculates from 40 Criollo stallions were used, whose fertility was assessed on the basis of their pregnancy rate per cycle in at least two breeding seasons. Pregnancy rates ranged from 20% to 90% and were used to divide the stallions into two groups: High rates (≥ 50%) (n = 25), and Low rates (< 50%) (n = 15). A computer-assisted sperm analysis system - (CASA) analyzed semen after collection. Also were evaluated the physical and functional integrity of the plasmatic membrane and sperm morphology alterations. All stallions expressed PLCζ, WBP2NL, and TNF-α. PLCζ positively correlates with conception rate, total motility (TM), progressive motility (PM), plasmatic membrane functionality, and integrity. A simple linear regression was detected between pregnancy rate and PLCζ expression (P = 0.003), TM (P < 0.001) and PM (P < 0.001). PLCζ gene expression was higher (P = 0,012) in the High rates group than in the Low group. WBP2NL and TNF-α did not correlate with seminal quality and stallion’s fertility. It was concluded that PLCζ gene expression in the spermatozoa might be used as a biomarker of fertility and seminal quality in stallions. Parameters of sperm kinetics also showed, positive correlation between TM, PM and pregnancy rate. spermborne borne , (PLCζ) domainbinding domain binding 2NTerminal NTerminal 2N Terminal N WBPNL WBP NL (WBP2NL) TNFα, TNFα TNF α (TNF-α) control 4 seasons 20 90 groups ≥ ( 50% 50 n 25, 25 25) 15. 15 . 15) computerassisted computer assisted CASA (CASA collection alterations TNFα. α. (TM) PM, (PM) functionality P 0.003, 0003 0.003 0 003 0.003) 0.001 0001 001 0.001. 0,012 0012 012 stallion s showed (PLCζ (WBP2NL (TNF-α 2 9 5 1 (TM (PM 000 0.00 00 0,01 01 0.0 0,0 0. 0,

Abstract The adnexa fetal tissues are sources of mesenchymal stromal cells (MSCs) due to their noninvasive harvest, with all biological material discarded most of the time. MSCs are a promise regarding to their plasticity, self-renewal, differentiation potentials, immunomodulatory and anti-inflammatory properties, which have made clinical stem cell therapy a reality. The present study aimed to characterize and evaluate the immunomodulation ability of bovine mesenchymal cells collected from bovine amniotic fluid (bAFMSCs) isolated and subjected to sixth consecutive culture passages in vitro. The multilineage properties of the bAFMSCs collections confirmed the ability to undergo adipogenic, chondrogenic and osteogenic differentiation. The mesenchymal gene transcription CD106, CD73, CD29, CD90 and CD166 were detected in bAFMSCs, whereas CD34 and CD45 were not detected. Regarding cytokine mRNA expression, IL2, IL6, INFα, INFβ, INFγ, TNFα and TNFβ were downregulated, while IL10 was highly regulated in all studied passages. The present study demonstrated the immunological properties and multipotency of in vitro bAFMSCs collections, and thus, they can be tested in cattle pathological treatments or multiplication by nuclear transfer cloning. (MSCs harvest time plasticity selfrenewal, selfrenewal self renewal, renewal self-renewal potentials antiinflammatory anti inflammatory reality (bAFMSCs adipogenic CD106 CD CD73 CD29 CD9 CD16 CD3 CD4 expression IL2 IL IL6 INFα INFβ INFγ downregulated IL1 thus cloning CD10 CD7 CD2 CD1