A first complete catalog of highly expressed genes in eight chicken tissues reveals uncharacterized gene families specific for the chicken testis.

Based on next-generation sequencing, we established a repertoire of differentially overexpressed genes (DoEGs) in eight adult chicken tissues: the testis, brain, lung, liver, kidney, muscle, heart, and intestine. With 4,499 DoEGs, the testis had the highest number and proportion of DoEGs compared with the seven somatic tissues. The testis DoEG set included the highest proportion of long noncoding RNAs (lncRNAs; 1,851, representing 32% of the lncRNA genes in the whole genome) and the highest proportion of protein-coding genes (2,648, representing 14.7% of the protein-coding genes in the whole genome). The main significantly enriched Gene Ontology terms related to the protein-coding genes were "reproductive process," "tubulin binding," and "microtubule cytoskeleton." Using real-time quantitative reverse transcription-polymerase chain reaction, we confirmed the overexpression of genes that encode proteins already described in chicken sperm [such as calcium binding tyrosine phosphorylation regulated (CABYR), spermatogenesis associated 18 (SPATA18), and CDK5 regulatory subunit associated protein (CDK5RAP2)] but whose testis origin had not been previously confirmed. Moreover, we demonstrated the overexpression of vertebrate orthologs of testis genes not yet described in the adult chicken testis [such as NIMA related kinase 2 (NEK2), adenylate kinase 7 (AK7), and CCNE2]. Using clustering according to primary sequence homology, we found that 1,737 of the 2,648 (67%) testis protein-coding genes were unique genes. This proportion was significantly higher than the somatic tissues except muscle. We clustered the other 911 testis protein-coding genes into 495 families, from which 47 had all paralogs overexpressed in the testis. Among these 47 testis-specific families, eight contained uncharacterized duplicated paralogs without orthologs in other metazoans except birds: these families are thus specific for chickens/birds.NEW & NOTEWORTHY Comparative next-generation sequencing analysis of eight chicken tissues showed that the testis has highest proportion of long noncoding RNA and protein-coding genes of the whole genome. We identified new genes in the chicken testis, including orthologs of known mammalian testicular genes. We also identified 47 gene families in which all the members were overexpressed, if not exclusive, in the testis. Eight families, organized in duplication clusters, were unknown, without orthologs in metazoans except birds, and are thus specific for chickens/birds.

Expanding duplication of the testis PHD Finger Protein 7 (PHF7) gene in the chicken genome.

Gene duplications increase genetic and phenotypic diversity and occur in complex genomic regions that are still difficult to sequence and assemble. PHD Finger Protein 7 (PHF7) acts during spermiogenesis for histone-to-histone protamine exchange and is a determinant of male fertility in Drosophila and the mouse. We aimed to explore and characterise in the chicken genome the expanding family of the numerous orthologues of the unique mouse Phf7 gene (highly expressed in the testis), observing the fact that this information is unclear and/or variable according to the versions of databases. We validated nine primer pairs by in silico PCR for their use in screening the chicken bacterial artificial chromosome (BAC) library to produce BAC-derived probes to detect and localise PHF7-like loci by fluorescence in situ hybridisation (FISH). We selected nine BAC that highlighted nine chromosomal regions for a total of 10 distinct PHF7-like loci on five Gallus gallus chromosomes: Chr1 (three loci), Chr2 (two loci), Chr12 (one locus), Chr19 (one locus) and ChrZ (three loci). We sequenced the corresponding BAC by using high-performance PacBio technology. After assembly, we performed annotation with the FGENESH program: there were a total of 116 peptides, including 39 PHF7-like proteins identified by BLASTP. These proteins share a common exon-intron core structure of 8-11 exons. Phylogeny revealed that the duplications occurred first between chromosomal regions and then inside each region. There are other duplicated genes in the identified BAC sequences, suggesting that these genomic regions exhibit a high rate of tandem duplication. We showed that the PHF7 gene, which is highly expressed in the rooster testis, is a highly duplicated gene family in the chicken genome, and this phenomenon probably concerns other bird species.

Effect of cumulus cell removal and sperm pre-incubation with progesterone on in vitro fertilization of equine gametes in the presence of oviductal fluid or cells.

In spite of many attempts to establish an in vitro fertilization (IVF) technique in the equine, no efficient conventional IVF technique is available. The presence of oviductal fluid or oviductal cells during IVF helps to improve embryo production in vitro but is not sufficient to reach high fertilization rates. Thus, our aim was to perform equine IVF either after sperm pre-incubation with oviductal fluid or in the presence of oviductal cells, and to evaluate the effect of cumulus removal from the oocyte or sperm pre-incubation with progesterone. In experiments 1 and 2, IVF was performed in the presence of porcine oviduct epithelial cells. The removal of cumulus cells from equine oocytes after in vitro maturation tended to increase the percentage of fertilization when fresh sperm was used (1/33 vs. 4/31, p > 0.05) but had no effect when frozen sperm was used (1/32 vs. 1/32). Equine sperm pre-incubation with progesterone did not significantly influence the fertilization rate when fresh or frozen sperm was used (2/14 vs. 2/18 for fresh, 1/29 vs. 1/25 for frozen). In experiments 3 and 4, IVF was performed after pre-incubation of sperm with porcine oviductal fluid. The removal of cumulus cells tended to increase the percentage of fertilization when fresh sperm was used (1/24 vs. 3/26, p > 0.05). Sperm pre-incubation with progesterone did not significantly influence the fertilization rate when fresh or frozen sperm was used (2/39 vs. 2/36 for fresh, 2/37 vs. 1/46 for frozen), but two 3-4 cell stage zygotes were obtained with fresh sperm pre-incubated with progesterone. This is an encouraging result for the setting up of an efficient IVF procedure in equine.

First attempts for vitrification of immature oocytes in donkey (Equus asinus): Comparison of two vitrification methods.

Most wild donkey breeds are severely threatened by poaching for meat, habitat loss, and competition with livestock for food resources. Moreover, due to the mechanization in agriculture and in transport, most domestic donkey breeds are at risk of extinction. Considering the importance of biodiversity and preservation of genetic resources, the creation of genetic banks for endangered donkey breeds is urgently needed. Cryopreservation of immature jennies oocytes would be an efficient tool to allow storage of female genetics. The aim of the present study was to establish conditions for immature donkey oocyte vitrification, using equine oocytes as a control. Asine and equine immature cumulus-oocyte complexes were collected by transvaginal ultrasound-guided follicular aspiration and flushed to obtain oocytes surrounded by only corona radiata. Oocytes were vitrified after exposure to increasing concentrations of dimethyl sulfoxide, ethylene glycol and sucrose as cryoprotectants in a solution of INRA-Freeze™ medium or TCM199-Hepes supplemented with bovine serum albumin. Oocytes were warmed in decreasing concentrations of sucrose and processed for in vitro maturation. The recovery rate was 48% for jennies oocytes (4.8 oocyte per female) and 42% for mares oocytes (3.5 oocyte per female). When oocytes were exposed to cryoprotectants in INRA-Freeze™ medium none of the jennies re-warmed oocytes matured, whereas 24% of the mares re-warmed oocytes reached metaphase II after in vitro maturation. When oocytes were exposed to cryoprotectants in TCM199-Hepes-BSA medium, 33% of the jennies re-warmed oocytes matured. In conclusion, we developed a method for the vitrification of immature oocytes from jennies that allows in vitro maturation of the vitrified-warmed asine oocytes. Their competence for fertilization and development has to be ascertain.

Exposure to follicular fluid during oocyte maturation and oviductal fluid during post-maturation does not improve in vitro embryo production in the horse.

Most wild equids and many domestic horse breeds are at risk of extinction, so there is an urgent need for genome resource banking. Embryos cryopreservation allows the preservation of genetics from male and female and is the fastest method to restore a breed. In the equine, embryo production in vitro would allow the production of several embryos per cycle. Intracytoplasmic sperm injection (ICSI) is used to generate horse embryos, but it requires expensive equipment and expertise in micromanipulation, and blastocyst development rates remain low. No conventional in vitro fertilization (IVF) technique for equine embryo production is available. The development of culture conditions able to mimic the maturation of the oocyte in preovulatory follicular fluid (pFF) and the post-maturation in oviductal fluid (OF) may improve embryo production in vitro. Our aim was to analyse the effect of in vitro maturation in pFF and incubation in OF on in vitro maturation of equine oocytes, fertilization using conventional IVF or ICSI, and embryo development after culture in synthetic oviductal fluid (SOF) or DMEM-F12. Oocytes collected from slaughtered mares or by ovum pick up were matured in vitro in pFF or semi-synthetic maturation medium (MM). The in vitro maturation, fertilization and development rates were not statistically different between pFF and MM. After in vitro maturation, oocytes were incubated with or without OF. Post-maturation in OF did not significantly improve the fertilization and development rates. Thus, in our study, exposure to physiological fluids for oocyte maturation and post-maturation does not improve in vitro embryo production in the horse.

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes.

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previously developed in mice. Specifically, the chromosomes and the centromeres of metaphase II (MII) oocytes were fluorescently labeled and counted on sequential focal plans after confocal laser microscope scanning. This analysis revealed a higher incidence in the aneuploidy rate when immature oocytes were collected from the follicles and matured in vitro compared to in vivo. Immunostaining for tubulin and the acetylated form of histone four at specific lysine residues also revealed differences in the morphology of the meiotic spindle and in the global pattern of histone acetylation. Finally, the expression of mRNAs coding for histone deacetylases (HDACs) and acetyl-transferases (HATs) was investigated by reverse transcription and quantitative-PCR (q-PCR). No differences in the relative expression of transcripts were observed between in vitro and in vivo matured oocytes. In agreement with a general silencing of the transcriptional activity during oocyte maturation, the analysis of the total transcript amount can only reveal mRNA stability or degradation. Therefore, these findings indicate that other translational and post-translational regulations might be affected. Overall, the present study describes an experimental approach to morphologically and biochemically characterize the horse oocyte, a cell type that is extremely challenging to study due to low sample availability. However, it can expand our knowledge on the reproductive biology and infertility in monovulatory species.

In vitro maturation affects chromosome segregation, spindle morphology and acetylation of lysine 16 on histone H4 in horse oocytes.

Implantation failure and genetic developmental disabilities in mammals are caused by errors in chromosome segregation originating mainly in the oocyte during meiosis I. Some conditions, like maternal ageing or in vitro maturation (IVM), increase the incidence of oocyte aneuploidy. Here oocytes from adult mares were used to investigate oocyte maturation in a monovulatory species. Experiments were conducted to compare: (1) the incidence of aneuploidy, (2) the morphology of the spindle, (3) the acetylation of lysine 16 on histone H4 (H4K16) and (4) the relative amount of histone acetyltransferase 1 (HAT1), K(lysine) acetyltransferase 8 (KAT8, also known as MYST1), histone deacetylase 1 (HDAC1) and NAD-dependent protein deacetylase sirtuin 1 (SIRT1) mRNA in metaphase II stage oocytes that were in vitro matured or collected from peri-ovulatory follicles. The frequency of aneuploidy and anomalies in spindle morphology was increased following IVM, along with a decrease in H4K16 acetylation that was in agreement with our previous observations. However, differences in the amount of the transcripts investigated were not detected. These results suggest that the degradation of transcripts encoding for histone deacetylases and acetyltransferases is not involved in the changes of H4K16 acetylation observed following IVM, while translational or post-translational mechanisms might have a role. Our study also suggests that epigenetic instabilities introduced by IVM may affect the oocyte and embryo genetic stability.

Influence of transvaginal ultrasound-guided follicular punctures in the mare on heart rate, respiratory rate, facial expression changes, and salivary cortisol as pain scoring.

Transvaginal ultrasound-guided follicular punctures are widely used in the mare for diagnosis, research, and commercial applications. The objective of our study was to determine their influence on pain, stress, and well-being in the mare, by evaluating heart rate, breath rate, facial expression changes, and salivary cortisol before, during, and after puncture. For this experiment, 21 pony mares were used. Transvaginal ultrasound-guided aspirations were performed on 11 mares. After injections for sedation, analgesia, and antispasmodia, the follicles from both ovaries were aspirated with a needle introduced through the vagina wall into the ovary. In the control group, 10 mares underwent similar treatments and injections, but no follicular aspiration. Along the session, heart rate and breath rate were evaluated by a trained veterinarian, ears position, eyelid closure, and contraction of facial muscles were evaluated, and salivary samples were taken for evaluation of cortisol concentration. A significant relaxation was observed after sedative injection in the punctured and control mares, according to ear position, eyelid closure, and contraction of facial muscles, but no difference between punctured and control animals was recorded. No significant modification of salivary cortisol concentration during puncture and no difference between punctured and control mares at any time were observed. No significant modification of the breath rate was observed along the procedure for the punctured and the control mares. Heart rate increased significantly but transiently when the needle was introduced in the ovary and was significantly higher at that time for the punctured mares than that for control mares. None of the other investigated parameters were affected at that time, suggesting discomfort is minimal and transient. Improving analgesia, e.g., through a multimodal approach, during that possibly more sensitive step could be recommended. The evaluation of facial expression changes and heart rate is easy-to-use and accurate tools to evaluate pain and well-being of the mare.

Establishment of conditions for ovum pick up and IVM of jennies oocytes toward the setting up of efficient IVF and in vitro embryos culture procedures in donkey (Equus asinus).

Most wild and domestic donkey breeds are currently endangered or threatened. Their preservation includes the creation of a Genome Resource Bank. Embryos cryopreservation allows the preservation of genetics from both male and female and is the fastest method to restore a breed. Because embryo production in vivo is limited in equids, our objective was to establish conditions for in vitro production of embryos in donkey using ovum pick up (OPU), IVM, IVF, and in vitro culture of zygotes. Donkey cumulus-oocyte complexes (COCs) were collected by transvaginal ultrasound-guided aspirations OPU in adult cyclic jennies and in vitro matured in tissue culture medium 199 supplemented with fetal calf serum and epidermal growth factor for 24, 30, 34, or 38 hours. They were preincubated with oviductal fluid for 30 minutes, coincubated with frozen-thawed donkey semen treated with procaine for 18 hours, and cultured for 30 hours in Dulbecco's Modified Eagle Medium-F12 supplemented with NaHCO3, fetal calf serum, and gentamycin. From the five OPU sessions, we collected 92 COCs in 193 follicles (48%) with an average of 4.2 COCs per jenny. All COCs were expanded after more than 24-hour IVM. At collection, jennies oocytes contained a germinal vesicle. Metaphase 1 oocytes were observed after 30-hour IVM and 44% were in metaphase 2 after 34-hour IVM. In our conditions, IVM of donkey oocytes was slower than IVM of equine oocytes and optimal duration for donkey oocytes IVM may be 34 hours. Only 15% of jennies oocytes contained two pronuclei after coincubation with donkey spermatozoa and none of them developed further after 48 hours post-IVF. Moreover, some parthenogenetic activation occurred. Thus, the treatment of donkey sperm with procaine may not be efficient for IVF. In conclusion, we established for the first time conditions for OPU in jennies with high recovery rates. We reported that IVM of jennies oocytes can produce 44% of metaphase 2 oocytes after 34 hours in culture and we described for the first time the chronology of IVM of donkey oocytes. Further studies are in progress to establish efficient conditions for IVF and development of donkey zygotes.

Differential expression and localization of glycosidic residues in in vitro- and in vivo-matured cumulus-oocyte complexes in equine and porcine species.

Glycoprotein oligosaccharides play major roles during reproduction, yet their function in gamete interactions is not fully elucidated. Identification and comparison of the glycan pattern in cumulus-oocyte complexes (COCs) from species with different efficiencies of in vitro spermatozoa penetration through the zona pellucida (ZP) could help clarify how oligosaccharides affect gamete interactions. We compared the expression and localization of 12 glycosidic residues in equine and porcine in vitro-matured (IVM) and preovulatory COCs by means of lectin histochemistry. The COCs glycan pattern differed between animals and COC source (IVM versus preovulatory). Among the 12 carbohydrate residues investigated, the IVM COCs from these two species shared: (a) sialo- and ßN-acetylgalactosamine (GalNAc)-terminating glycans in the ZP; (b) sialylated and fucosylated glycans in cumulus cells; and (c) GalNAc and N-acetylglucosamine (GlcNAc) glycans in the ooplasm. Differences in the preovulatory COCs of the two species included: (a) sialoglycans and GlcNAc terminating glycans in the equine ZP versus terminal GalNAc and internal GlcNAc in the porcine ZP; (b) terminal galactosides in equine cumulus cells versus terminal GlcNAc and fucose in porcine cohorts; and (c) fucose in the mare ooplasm versus lactosamine and internal GlcNAc in porcine oocyte cytoplasm. Furthermore, equine and porcine cumulus cells and oocytes contributed differently to the synthesis of ZP glycoproteins. These results could be attributed to the different in vitro fertilization efficiencies between these two divergent, large-animal models.

Deleted in malignant brain tumor 1 is secreted in the oviduct and involved in the mechanism of fertilization in equine and porcine species.

Oviductal environment affects preparation of gametes for fertilization, fertilization itself, and subsequent embryonic development. The aim of this study was to evaluate the effect of oviductal fluid and the possible involvement of deleted in malignant brain tumor 1 (DMBT1) on IVF in porcine and equine species that represent divergent IVF models. We first performed IVF after pre-incubation of oocytes with or without oviductal fluid supplemented or not with antibodies directed against DMBT1. We showed that oviductal fluid induces an increase in the monospermic fertilization rate and that this effect is canceled by the addition of antibodies, in both porcine and equine species. Moreover, pre-incubation of oocytes with recombinant DMBT1 induces an increase in the monospermic fertilization rate in the pig, confirming an involvement of DMBT1 in the fertilization process. The presence of DMBT1 in the oviduct at different stages of the estrus cycle was shown by western blot and confirmed by immunohistochemical analysis of ampulla and isthmus regions. The presence of DMBT1 in cumulus-oocyte complexes was shown by western blot analysis, and the localization of DMBT1 in the zona pellucida and cytoplasm of equine and porcine oocytes was observed using immunofluorescence analysis and confocal microscopy. Moreover, we showed an interaction between DMBT1 and porcine spermatozoa using surface plasmon resonance studies. Finally, a bioinformatic and phylogenetic analysis allowed us to identify the DMBT1 protein as well as a DMBT1-like protein in several mammals. Our results strongly suggest an important role of DMBT1 in the process of fertilization.

Changes in histone H4 acetylation during in vivo versus in vitro maturation of equine oocytes.

Epigenetic modifications are established during gametogenesis and preimplantation embryonic development. Any disturbance of the normal natural environment during these critical phases could cause alterations of the epigenetic signature. Histone acetylation is an important epigenetic modification involved in the regulation of chromatin organization and gene expression. The present study was aimed to determine whether the proper establishment of post-translational histone H4 acetylation at lysine 8 (AcH4K8), 12 (AcH4K12) and 16 (AcH4K16) of equine oocytes is adversely affected during in vitro maturation (IVM) when compared with in vivo matured oocytes collected from naturally cycling mares not undergoing ovarian hyperstimulation. The acetylation patterns were investigated by means of indirect immunofluorescence staining with specific antibodies directed against the acetylated lysine residues. Our results indicate that the acetylation state of H4 is dependent on the chromatin configuration in immature germinal vesicle (GV) stage oocytes and it changes in a residue-specific manner along with the increase of chromatin condensation. In particular, the levels of AcH4K8 and AcH4K12 increased significantly, while AcH4K16 decreased significantly from the fibrillar to the condensed state of chromatin configuration within the GV. Moreover, during meiosis, K8 and K12 were substantially deacetylated without any differences between in vivo and in vitro conditions, while K16 displayed a strong acetylation in oocytes matured in vivo, and in contrast, it was markedly deacetylated following IVM. Although the functional meaning of residue-specific acetylation during oocyte differentiation and meiotic resumption needs further investigation, our results support the hypothesis that IVM conditions can adversely affect oocyte ability to regulate the epigenetic reprogramming, critical for successful meiosis and subsequent embryonic development.

The secretions of oviduct epithelial cells increase the equine in vitro fertilization rate: are osteopontin, atrial natriuretic peptide A and oviductin involved?

BACKGROUND: Oviduct epithelial cells (OEC) co-culture promotes in vitro fertilization (IVF) in human, bovine and porcine species, but no data are available from equine species. Yet, despite numerous attempts, equine IVF rates remain low. Our first aim was to verify a beneficial effect of the OEC on equine IVF. In mammals, oviductal proteins have been shown to interact with gametes and play a role in fertilization. Thus, our second aim was to identify the proteins involved in fertilization in the horse. METHODS & RESULTS: In the first experiment, we co-incubated fresh equine spermatozoa treated with calcium ionophore and in vitro matured equine oocytes with or without porcine OEC. We showed that the presence of OEC increases the IVF rates. In the subsequent experiments, we co-incubated equine gametes with OEC and we showed that the IVF rates were not significantly different between 1) gametes co-incubated with equine vs porcine OEC, 2) intact cumulus-oocyte complexes vs denuded oocytes, 3) OEC previously stimulated with human Chorionic Gonadotropin, Luteinizing Hormone and/or oestradiol vs non stimulated OEC, 4) in vivo vs in vitro matured oocytes. In order to identify the proteins responsible for the positive effect of OEC, we first searched for the presence of the genes encoding oviductin, osteopontin and atrial natriuretic peptide A (ANP A) in the equine genome. We showed that the genes coding for osteopontin and ANP A are present. But the one for oviductin either has become a pseudogene during evolution of horse genome or has been not well annotated in horse genome sequence. We then showed that osteopontin and ANP A proteins are present in the equine oviduct using a surface plasmon resonance biosensor, and we analyzed their expression during oestrus cycle by Western blot. Finally, we co-incubated equine gametes with or without purified osteopontin or synthesized ANP A. No significant effect of osteopontin or ANP A was observed, though osteopontin slightly increased the IVF rates. CONCLUSION: Our study shows a beneficial effect of homologous and heterologous oviduct cells on equine IVF rates, though the rates remain low. Furthers studies are necessary to identify the proteins involved. We showed that the surface plasmon resonance technique is efficient and powerful to analyze molecular interactions during fertilization.

New insights into the mechanisms of fertilization: comparison of the fertilization steps, composition, and structure of the zona pellucida between horses and pigs.

The mechanism of fertilization remains largely enigmatic in mammals. Most studies exploring the molecular mechanism underlying fertilization have been restricted to a single species, generally the mouse, without a comparative approach. However, the identification of divergences between species could allow us to highlight key components in the mechanism of fertilization. In the pig, in vitro fertilization (IVF) and polyspermy rates are high, and spermatozoa penetrate easily through the zona pellucida (ZP). In contrast, IVF rates are low in the horse, and polyspermy is scarce. Our objective was to develop a comparative strategy between these two divergent models. First, we compared the role of equine and porcine gametes in the following five functions using intraspecific and interspecific IVF: ZP binding, acrosome reaction, penetration through the ZP, gamete fusion, and pronucleus formation. Under in vitro conditions, we showed that the ZP is a determining element in sperm-ZP attachment and penetration, whereas the capacity of the spermatozoa is of less importance. In contrast, the capacity of the spermatozoa is a key component of the acrosome reaction step. Second, we compared the composition and structure of the equine and porcine ZP. We observed differences in the number and localization of the ZP glycoproteins and in the mesh-like structure of the ZP between equine and porcine species. These differences might correlate with the differences in spermatozoal attachment and penetration rates. In conclusion, our comparative approach allows us to identify determining elements in the mechanism of fertilization.

The involvement of beta-1,4-Galactosyltransferase and N-Acetylglucosamine residues in fertilization has been lost in the horse.

BACKGROUND: In human and rodents, sperm-zona pellucida binding is mediated by a sperm surface Galactosyltransferase that recognizes N-Acetylglucosamine residues on a glycoprotein ZPC. In large domestic mammals, the role of these molecules remains unclear: in bovine, they are involved in sperm-zona pellucida binding, whereas in porcine, they are not necessary. Our aim was to clarify the role of Galactosyltransferase and N-Acetylglucosamine residues in sperm-zona pellucida binding in ungulates. For this purpose, we analyzed the mechanism of sperm-zona pellucida interaction in a third ungulate: the horse, since the Galactosyltransferase and N-Acetylglucosamine residues have been localized on equine gametes. METHODS: We masked the Galactosyltransferase and N-Acetylglucosamine residues before the co-incubation of gametes. Galactosyltransferase was masked either with an anti-Galactosyltransferase antibody or with the enzyme substrate, UDP Galactose. N-Acetylglucosamine residues were masked either with a purified Galactosyltransferase or with an anti-ZPC antibody. RESULTS AND DISCUSSION: The number of spermatozoa bound to the zona pellucida did not decrease after the masking of Galactosyltransferase or N-Acetylglucosamine. So, these two molecules may not be necessary in the mechanism of in vitro sperm-zona pellucida interaction in the horse. CONCLUSION: The involvement of Galactosyltransferase and N-Acetylglucosamine residues in sperm-zona pellucida binding may have been lost during evolution in some ungulates, such as porcine and equine species.