Acetoacetate is a trigger of NLRP3 inflammasome activation in bovine peripheral blood mononuclear cells.

Repeat breeding, which is non-pregnancy following three or more breeding attempts, is a serious reproductive disorder in cattle. In the present study, metabolomic profiling was used to identify metabolites in the blood plasma of repeat breeder cows (RBCs) and non-RBCs. Metabolomic analysis showed that acetoacetate (AcAc), a ketone body, was detected in RBCs, but not in non-RBCs. In contrast, ß-hydroxybutyrate (BHB) was at similar levels in both RBCs and non-RBCs. We hypothesized that an imbalance of AcAc and BHB induces abnormal inflammatory conditions, especially the NLRP3 inflammasome, which regulates sterile inflammation to control interleukin (IL)-1ß secretion, and may be associated with repeat breeding in cattle. To investigate this hypothesis, blood samples were collected from both non-RBCs and RBCs on day 7 of the estrous cycle. The mRNA expression of IL1B in peripheral blood mononuclear cells (PBMCs) was observed to be higher in RBCs than in non-RBCs. To test the effects of AcAc and BHB on inflammatory responses, blood samples were collected from healthy cows and PBMCs were isolated. PBMCs were treated with AcAc and BHB to investigate the activation of the NLRP3 inflammasome (complex of NLRP3, ASC, and caspase-1) and IL-1ß secretion. AcAc treatment resulted in higher protein and/or mRNA expression of NLRP3 and IL-1ß in PBMCs. Moreover, AcAc increased the co-localization of NLRP3 and ASC and stimulated caspase-1 activation, indicating the formation of the platform of the NLRP3 inflammasome. Addition of specific NLRP3 inhibitor, MCC950, suppressed AcAc stimulation-induced IL-1ß secretion. Contrary to the effects of AcAc, BHB treatment suppressed the activation of NLRP3 inflammasome and IL-1ß secretion in response to AcAc and typical NLRP3 inflammasome triggers. These findings demonstrate that AcAc can potentially trigger NLRP3 inflammasome activation, resulting in IL-1ß secretion, and that these inflammatory responses are suppressed by BHB in bovine PBMCs. In addition, the imbalance between AcAc and BHB with higher levels of IL-1ß may be associated with repeat breeding in cattle.

Production, dissemination activities, and supplying-system development for in vitro fertilized bovine embryo.

This review discusses the production and sale of fertile oocytes for in vitro fertilization technology, calf production through transplantation and delivery, and the current circulation of calves produced by in vitro production (IVP) embryos.

Metabolomic profiles of plasma and uterine luminal fluids from healthy and repeat breeder Holstein cows.

BACKGROUND: Repeat breeding is a critical reproductive disorder in cattle. The problem of repeat breeder cattle remains largely unmanageable due to a lack of informative biomarkers. Here, we utilized metabolomic profiling in an attempt to identify metabolites in the blood plasma and uterine luminal fluids. We collected blood and uterine fluid from repeat breeder and healthy cows on day 7 of the estrous cycle. RESULTS: Metabolomic analysis identified 17 plasma metabolites detected at concentrations that distinguished between the two groups, including decreased various bile acids among the repeat breeders. However, no metabolites that varied significantly were detected in the uterine luminal fluids between two groups. Among the plasma samples, kynurenine was identified as undergoing the most significant variation. Kynurenine is a metabolite produced from tryptophan via the actions of indoleamine 2,3-dioxygenase (IDO). As IDO is key for maternal immune tolerance and induced in response to interferon tau (IFNT, ruminant maternal recognition of pregnancy factor), we examined the responsiveness to IFNT on peripheral blood mononuclear cells (PBMC) isolated from healthy and repeat breeder cows. The mRNA expression of IFNT-response makers (ISG15 and MX2) were significantly increased by IFNT treatment in a dose-dependent manner in both groups. Although treatment with IFNT promoted the expression of IDO in PBMCs from both groups, it did so at a substantially reduced rate among the repeat breeder cows, suggesting that decreased levels of kynurenine may relate to the reduced IDO expression in repeat breeder cows. CONCLUSIONS: These findings provide valuable information towards the identification of critical biomarkers for repeat breeding syndrome in cattle.

Adverse reproductive effects of S100A9 on bovine sperm and early embryonic development in vitro.

The phenomenon of aging arises from multiple, complex interactions causing dysfunction in cells and organs. In particular, fertility drastically decreases with age. Previously, we have demonstrated that the functional characteristics of the bovine oviduct and uterus change with the age-dependent upregulation of inflammation and noted that S100A9 triggers inflammatory responses in oviduct epithelial cells. In the present study, we investigated the hypothesis that S100A9 affects reproductive events to aspect such as sperm function, fertilization, and the development of the embryo in cows. To investigate the effect of S100A9 on bovine sperm, we incubated sperms in vitro with S100A9 for 5 h and observed significantly decreased sperm motility and viability. During in vitro fertilization, S100A9 treatment for 5 h did not affect the rate of fertilization, time of first division of embryos, or embryo development to blastocyst stage. Treatment of 2-cell stage embryos with S100A9 for 5 h significantly reduced the proportion of cells undergoing normal division (4-8 cell embryos) and embryo development to the blastocyst stage. In experiment involving 24 h treatment of 2-cell embryos, the development of all embryos stopped at the 2-cell stage in the S100A9-treated group. In blastocyst-stage embryos, S100A9 treatment significantly stimulated the expression of endoplasmic reticulum (ER) and the mRNA expression of ER stress markers, and activated caspase-3 with subsequent nuclear fragmentation. Pre-treatment with an ER stress inhibitor significantly suppressed caspase-3 activation by the S100A9 treatment, suggesting that S100A9 induces blastocyst dysfunction by apoptosis (via caspase-3 activation) depending on ER stress. These results indicate that direct exposure to S100A9 exerted adverse effects on sperm function and embryo development. These findings suggest that excessive dose of S100A9 may have an adverse effect to the reproductive machinery by inducing inflammation and tissue dysfunction.

Expression of genes encoding mineralocorticoid biosynthetic enzymes and the mineralocorticoid receptor, and levels of mineralocorticoids in the bovine follicle and corpus luteum.

Unlike sex steroids, mineralocorticoids have attracted limited attention in ovarian physiology. Recent studies on primates have indicated possible local synthesis and action of mineralocorticoids in the ovary. Here, we examined developmental changes in the levels of mineralocorticoids and expression of genes encoding their biosynthetic enzymes and receptor in the bovine ovary. The follicles and corpora lutea (CL) were collected from F1 heifers. Expression levels of 21α-hydroxylase (CYP21A2), 11ß-hydroxylase-1 (CYP11B1), and the mineralocorticoid receptor (NR3C2) in granulosa cells (GC), thecal layers (TL), and CL tissues were quantified by real-time PCR, whereas mineralocorticoids in the follicular fluid were measured by enzyme immunoassay (EIA). TL and GC expressed CYP21A2 and NR3C2, whereas CYP11B1 was expressed at very low or undetectable levels. The expression levels of these genes were not significantly different among small/large and healthy/atretic follicles but were higher in TL than in GC. CYP21A2 and NR3C2 were expressed in all CL stages with higher expression observed in the mid-stage. CYP11B1 expression was only apparent in the mid-stage CL. Aldosterone was detected in all follicles, and its concentration was not significantly different among the follicular groups. In paired large-healthy/atretic follicles, the concentration of deoxycorticosterone, a precursor of aldosterone, was approximately ten-fold higher than that of aldosterone and not significantly different between healthy and atretic follicles. In conclusion, the presence of mineralocorticoids and expression of NR3C2 in the bovine follicle together with the developmental change in the expression of CYP21A2, CYP11B1, and NR3C2 in the CL suggest possible endocrine/paracrine/autocrine roles of mineralocorticoids in the bovine ovary.

The transfer of parthenogenetic embryos following artificial insemination in cows can enhance pregnancy recognition via the secretion of interferon tau.

Repeat breeding is a reproductive disorder in cattle. Embryo transfer following artificial insemination (AI) improves pregnancy rate by replenishing interferon tau (IFNT), but it results in a notably higher rate of twin occurrence. This study hypothesized that parthenogenetic (PA) embryo transfer following AI (AI + PA) could improve the conception rate because that PA embryo become as a supplemental source of IFNT without twins. PA embryos showed higher IFNT mRNA expression than in vitro fertilization (IVF) embryos. An examination of the effect of the cultured conditioned media (CM) of PA or IVF embryos on Madin-Darby bovine kidney cells with stably introduced promoter-reporter constructs of interferon-stimulated gene 15 (ISG15, marker of IFN response) showed higher stimulation levels of ISG15 promoter activity with PA than with IVF embryo. We investigated in vivo the effect of AI + PA on healthy Japanese Black cattle. Cattle transferred with PA embryo alone were non-fertile, but those that underwent AI + PA showed a pregnancy rate of 53.3%, the similar as that with AI alone (60%). In pregnant cattle in AI + PA group, adding the PA embryo upregulated the expression of ISGs and plasma progesterone concentration. No twin were generated in AI only and AI + PA groups. Using repeat breeding Holstein cows that did not become pregnant with 4-9 times of AI, transfer of PA embryo following AI resulted in a higher pregnancy rate than that of control (AI only). We suggest that AI + PA may be beneficial for improving maternal pregnancy recognition in repeat breeder cattle while avoiding twin generation.

Improvement of fertility in repeat breeder dairy cattle by embryo transfer following artificial insemination: possibility of interferon tau replenishment effect.

Repeat breeder cattle do not become pregnant until after three or more breeding attempts; this represents a critical reproductive disorder. Embryo transfer (ET) following artificial insemination (AI) in repeat breeder cattle reportedly improves pregnancy rate, leading to speculation that interferon tau (IFNT) is associated with this phenomenon. However, the reason why the conception rate improves remains unknown. We investigated the effect of ET following AI on repeat breeder cattle in field tests, and determined whether adding an embryo affects the maternal immune cells detected by interferon-stimulated genes (ISGs), marker genes of IFN response. In total, 1122 repeat breeder cattle were implanted with in vitro fertilization (IVF) embryos after previous AI. ET following AI resulted in pregnancy rates of 46.9% in repeat breeder dairy cattle. In basic in vivo tests, to investigate the effect of adding embryos, ISGs mRNA expression levels were significantly higher in the AI + ET group than in the AI + sham group (transfer of only embryonic cryopreservation solution). Then, we examined the effect of cultured conditioned media (CM) of IVF embryos on splenic immune cells and Madin-Darby bovine kidney (MDBK) cells with stably introduced ISG15 promoter-reporter constructs. These cells exhibited a specific increase in ISG15 mRNA expression and promoter activity when treated with the CM of IVF embryos, suggesting that IVF embryos have the potential to produce and release IFNT. In conclusion, ET following AI is beneficial for improving conception in repeat breeder cattle. Added embryos may produce and secrete IFNT, resulting in the increased expression of ISGs.

Clinical study report on milk production in the offspring of a somatic cell cloned Holstein cow.

This study examined two female offspring of a somatic cell cloned Holstein cow that had reproduction problems and milk production performance issues. The two offspring heifers, which showed healthy appearances and normal reproductive characteristics, calved on two separate occasions. The mean milk yields of the heifers in the first lactation period were 9,037 kg and 7,228 kg. The relative mean milk yields of these cows were 111.2% and 88.9%, respectively, when compared with that of the control group. No particular clinical abnormalities were revealed in milk yields and milk composition rate [e.g., fat, protein and solids-not-fat (SNF)], and reproductive characteristics of the offspring of the somatic cell cloned Holstein cow suggested that the cloned offspring had normal milk production.

Effect of bovine cumulus-oocyte complexes-conditioned medium on in-vitro maturation of canine oocytes.

PURPOSE: To investigate the ability of medium conditioned with bovine cumulus-oocyte complexes (COCs) to support nuclear maturation of canine oocytes recovered from domestic dog ovaries. METHODS: Cumulus-oocyte complexes were obtained from ovaries of domestic bitches (8 months old to 7 years old), and in-vitro maturation was evaluated in TCM-199 supplemented with different concentrations (0, 20, 30 or 50%) of bovine COCs-conditioned medium (BCM). The canine COCs were cultured for 72 or 96 h at 38.5°C in 5% CO2, 5% O2 and 90% N2. The bovine COCs-conditioned medium was obtained from culture of bovine COCs with TCM-199 supplemented with 5% FCS for 22 h at 38.5°C in 2% CO2, 98% air. RESULTS: The proportion of germinal vesicle breakdown (GVBD) after 72 h was significantly higher (P < 0.05) in medium supplemented with 30% BCM (20.7%) compared with the control group (13.4%). The rates of GVBD-MII stage were significantly higher (P < 0.05) when oocytes were matured with BCM at concentration of 30% (41.5%) compared with control (26.6%) after 72 h in-vitro culture. After 96 h in-vitro culture, the oocytes matured in medium supplemented with 30% BCM (5.5%) showed a significant increase (P < 0.05) in the proportion of MII compared with control (0.7%). However, increasing the cultivation time from 72 to 96 h resulted in an increase in oocyte degeneration rate. CONCLUSIONS: The results suggested that bovine COCs-conditioned medium supplementation significantly increased nuclear maturation of canine oocytes.

Gene expression of 11ß-HSD and glucocorticoid receptor in the bovine (Bos taurus) follicle during follicular maturation and atresia: the role of follicular stimulating hormone.

Glucocorticoids modulate ovarian function in cattle. However, their regulatory mechanisms have not been fully elucidated. In the present study, we examined gene expression of two glucocorticoid-metabolizing enzymes, a bidirectional 11ß-HSD type 1 (11HSD1) and a dehydrogenase 11ß-HSD type 2 (11HSD2), and glucocorticoid receptor (GR) in bovine follicles during follicular maturation and atresia. Granulosa cells (GCs) and theca interna layers (TIs) were harvested from follicles classified as small growing, dominant, preovulatory, early atretic and late atretic follicles. The expression levels of 11HSD1, 11HSD2 and GR mRNA were quantified by real-time PCR. In the healthy follicles, expression of 11HSD1 mRNA increased as follicles matured, both in GCs and TIs. A significant negative correlation was found between the concentration of cortisol in follicular fluid and the level of 11HSD1 mRNA in GCs. The expression of 11HSD2 and GR was either very low or largely unchanged during follicular maturation. In the atretic follicles, a drastic increase in the expression of 11HSD2 was observed both in GCs and TIs. To assess the effect of FSH on the expression of 11HSDs and GR, GCs were cultured with FSH (0-100 ng/ml) for up to 6 days. FSH increased 11HSD1 mRNA in a dose-dependent manner, but not 11HSD2, nor GR. Taken together, these results suggest that developmentally-regulated 11HSD1 plays a pivotal role in modulating the local glucocorticoid environment in maturing bovine follicles.

Classification of bovine follicles based on the concentrations of steroids, glucose and lactate in follicular fluid and the status of accompanying follicles.

A simple and clear means to identify the physiological status of follicles is essential for study of follicular biology. In the present study, we verified a novel classification procedure based on analysis of the follicular population and glucose concentration in follicular fluid (FF) as an alternative method to classify bovine follicles. Paired ovaries were collected from heifers, and the number of follicles and stage of the CL were recorded. Follicles were initially divided into the following 3 groups according to diameter and the ratio of E2 and P4 (E/P): E2 active (E-A: E/P>or=1), E2 inactive (E-I: E/P<1, >or=8.5 mm) and small follicles (E/P<1, <8.5 mm). E-A follicles were easily identified as E2-rich dominant follicles and were further classified according to diameter and stage of the CL as early dominant (EDF: <8.5 mm), dominant (DF: >or=8.5 mm, stages I-III) or preovulatory follicles (POF: >or=8.5 mm, stage IV). E-I follicles were classified as follows based on the status of the accompanying follicles: early atretic (EAF: without an E-A follicle), mid-atretic (MAF: with an EDF or DF) and late atretic follicles (LAF: with an EAF or POF). The follicular P4 concentrations of the MAF and LAF were significantly higher compared with that of the EAF, while follicular glucose concentration of the LAF was lower compared with those of EAF and MAF, indicating that this classification can be used to distinguish early atretic follicles from more advanced atretic follicles. Small follicles were classified as growing (GF: without E-A follicles) and suppressed small follicles (SSF: with E-A follicles). The SSF was easily identifiable by this procedure, but some GF populations likely contained SSF. To identify true GF, the ratio of E2 in the GF and accompanying EAF may be used. In conclusion, analysis of the follicular population in conjunction with biochemical indices such as steroid and glucose concentrations in FF provides a simple and accurate means of classifying bovine follicles.

Bovine oocytes and early embryos express mRNA encoding glycerol kinase but addition of glycerol to the culture media interferes with oocyte maturation.

Glycerol plays multi-functional roles in cellular physiology. Other than forming the backbone molecule for glycerophospholipid and triglyceride (TG), glycerol acts as an energy substrate for glycolysis. Spermatozoa are known to utilize glycerol for energy production, but there are no reports of this in oocytes. In this study, the value of glycerol as an energy substrate for bovine oocyte maturation (Exp. 1) and the gene expression of glycerol kinase (GK), an enzyme crucial for cellular glycerol utilization, in bovine oocytes and early embryos (Exp. 2) were examined. In Exp. 1, in vitro maturation (IVM) was conducted using synthetic oviduct fluid supplemented with/without glucose (1.5 mM) and/or glycerol (1.0 mM), and maturation rate, degree of cumulus expansion, glucose consumption and lactate production by cumulus-oocyte complexes (COC) were examined. In Exp. 2, to examine the developmental expression of GK mRNA, cumulus cells, oocytes and embryos at the 2-, 8- and 16-cell, morula, expanded blastocyst and hatched blastocyst stages were obtained in separate experiments, and the expression of GK mRNA was quantified using a real-time PCR. Glycerol did not support oocyte maturation or cumulus expansion. Addition of glycerol to glucose-supplemented media significantly decreased the maturation rate. Expression of GK mRNA was very low in cumulus cells, whereas an appreciable level of the transcript was observed in the oocytes. GK mRNA was detected in embryos at all the stages examined, and its expression significantly increased at the morula stage. These results indicate that glycerol, at least at the present concentration, is not beneficial as a constituent of the medium for bovine oocyte maturation. However, the appreciable levels of GK mRNA found in the oocyte and embryo imply a physiological role for glycerol in bovine oocyte maturation and embryo development.

Fourier transform infrared spectroscopic analysis of the intact zona pellucida of the mammalian egg: changes in the secondary structure of bovine zona pellucida proteins during fertilization.

The zona pellucida is the acellular transparent envelope surrounding the mammalian oocyte. An analysis of the changes in the structures of zona pellucida proteins is essential for understanding the molecular mechanisms underlying the important physiological roles of the zona during fertilization and preimplantation. The hardening of the zona caused by the structural changes during fertilization is generally accepted to be responsible for blocking polyspermy. In this study, we analyzed changes in the secondary structure of the zona during fertilization by Fourier transform infrared (FTIR) spectroscopy and transmission electron microscopy. The predominance of beta-sheet structure in porcine ovarian egg zona proteins in water was ascertained using FTIR spectra. Alpha-helix structure was also present. The attenuated total reflection (ATR)-FTIR spectrum of intact, unsolubilized porcine zonae pellucidae from ovarian eggs indicated that the zona proteins in the native zona pellucida also have beta-structure as the main constituent. Attenuated total reflection-FTIR spectroscopy of intact bovine zona pellucida obtained from ovarian and fertilized eggs at the blastocyst stage revealed that the beta-structure content increased during fertilization. Furthermore, a reduction of the thickness of the zona during fertilization was observed using transmission electron microscopy. Therefore, the change in the zona architecture that causes hardening of the zona during fertilization is accompanied by changes in the secondary structure of the zona proteins.

Relationship between ovarian weight and follicular population in heifers.

The size of the ovary varies substantially among cattle. This variation may influence the potential of the ovary to produce follicles. In the present study, we examined whether a relationship exists between the weight of the ovary and the number of antral follicles >or=1 mm. Paired ovaries were obtained from Holstein x Japanese Black F1 heifers. Follicles were classified into three size categories (small: 1.0-<5.0 mm, medium: 5.0-<8.5 mm and large: >or=8.5 mm), and the number of follicles in each category was recorded. Large variations in the weight of ovaries and the number of follicles were observed among animals. Significant positive correlations (r>or=0.4, P<0.001) were found between the weight of intact ovaries and the number of follicles in all three categories for the ovary contralateral to CL (OCC) and in the small follicles for the ovary ipsilateral to CL (OIC). Significant positive correlations (r>0.4, P<0.0001) were also observed between the weight of ovaries devoid of CL and follicles and the number of small and medium follicles in both OIC and OCC, indicating that the correlation is not due to the increase in ovarian weight associated with the increase in follicular number. Paired ovaries contained a similar number of small and medium follicles, and significant positive correlations were observed between them (r>0.6, P<0.0001). There were significant positive correlations between the weight of OCC and the number of small and medium follicles in paired ovaries (r>0.4, P<0.0001). These results suggest that 1) the weight of an ovary reflects the potential of the ovary to produce antral follicles, and 2) a rough estimation of follicular population might be possible by using the weight of the ovary contralateral to CL in heifers.

In vitro attachment of bovine hatched blastocysts on fibronectin is mediated by integrin in a RGD dependent manner.

We investigated the effect of extracellular matrix protein on in vitro attachment and outgrowth of bovine hatched blastocysts. In vitro produced bovine hatched blastocysts were cultured on a fibronectin- or laminin-coated Petri dishes. Hatched blastocysts adhered and outgrew on the fibronectin-coated dish whereas no attachment was observed on the laminin-coated dish. The attachment and outgrowth on fibronectin were significantly inhibited in the presence of synthetic peptides containing the Arg-Gly-Asp (RGD) sequence, which interacts with the fibronectin receptor (integrin alpha5beta1), but were not inhibited by the control peptides containing the Arg-Gly-Glu (RGE) sequence. Addition of anti-fibronectin receptor (integrin alpha5beta1) antibody to the culture medium also inhibited the attachment and outgrowth on fibronectin-coated Petri dishes. Subsequently we examined mRNA expression and protein expression of alpha5 and beta1 integrin subunit in the hatched blastocyst by reverse transcription-polymerase chain reaction (RT-PCR) and immunostaining, respectively. Expression of both mRNA and protein were detected in blastocysts. These results indicate that trophectoderm cells of bovine hatched blastocysts have already acquired the ability to adhere and outgrow on fibronectin in vitro by an integrin- mediated manner.

Activity of exoglycosidases in ejaculated spermatozoa of boar and bull.

The activity of exoglycosidases in extracts from freshly ejaculated boar and bull spermatozoa with 0.2% Brij-35/2% acetic acid was measured. The results show that beta-N-acetylhexosaminidase, beta-galactosidase and alpha-mannosidase are the major glycosidases; much higher levels of activity were found in boar spermatozoa than in bull spermatozoa. When compared on a per spermatozoon basis, the ratios of the activities of beta-N-acetylhexosaminidase, beta-galactosidase and alpha-mannosidase in boar spermatozoon relative to those in bull spermatozoon were approximately 13000:1, 1700:1 and 400:1, respectively. Liberation of these glycosidases from bull spermatozoa by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC) was low, in contrast to liberation of alpha-mannosidase from boar spermatozoa previously found by the same means. The possibility that the exoglycosidases present in large amounts in boar spermatozoa play a role in the process of binding to the zona pellucida glycoprotein of the egg is discussed.

Possible role of interferon-tau on in vitro development of bovine embryos.

The effect of interferon-tau on in vitro development of bovine embryos was investigated. After in vitro fertilization, embryos developed to the morula stage were cultured for 3 days in TCM-199 or CR1 medium containing BSA or FCS supplemented with or without recombinant IFN-tau produced by a baculovirus expression system. Addition of baculovirus-expressed IFN-tau (100 ng/ml) significantly promoted development to the blastocyst stage in both culture media. Addition of E. coli expressed IFN-tau (2 microg/ml) also significantly promoted the embryonic development. Supplementation of BSA or FCS did not affect the growth-promoting effect of IFN-tau. To determine whether the growth-promoting effect of IFN-tau is related to the interferon type I receptors that bind to type I interferon such as IFN-alpha, embryos were cultured with IFN-alpha. Although IFN-alpha significantly promoted the development, a much higher concentration (25 microg/ml) was required than IFN-tau. A reverse transcription polymerase chain reaction analysis revealed the expression of mRNA encoded type-I IFN receptor subunit from morula to blastocyst stage embryos. The overall results suggest a novel function for IFNs in promoting embryonic development and the effect may be related to type-I IFN receptor expressed in the early stages of preimplantation embryos.

Localization of N-linked carbohydrate chains in glycoprotein ZPA of the bovine egg zona pellucida.

The zona pellucida, a transparent envelope surrounding the mammalian oocyte, consists of three glycoproteins, ZPA, ZPB and ZPC, and plays a role in sperm-egg interactions. In bovines, these glycoproteins cannot be separated unless the acidic N-acetyllactosamine regions of the carbohydrate chains are removed by endo-beta-Galactosidase digestion. Endo-beta-Galactosidase-digested ZPB retains stronger sperm-binding activity than ZPC. It is still unclear whether ZPA possesses significant activity. Recently, we reported that bovine sperm binds to Man5GlcNAc2, the neutral N-linked chain in the cow zona proteins. In this study, we investigated the localization of the sperm-ligand active high-mannose-type chain and the acidic complex-type chains in bovine ZPA. Three N-glycopeptides of ZPA, containing an N-glycosylation site at Asn83, Asn191 and Asn527, respectively, were obtained from endo-beta-Galactosidase-digested ZPA. Of these glycosylation sites, only Asn527 is present in the ZP domain common to all the zona proteins. The carbohydrate structures of the N-linked chains obtained from each N-glycopeptide were characterized by two-dimensional sugar mapping analysis, while considering the structures of the N-linked chains of the zona protein mixture reported previously. Acidic complex-type chains were found at all three N-glycosylation sites, while Man5GlcNAc2 was found at Asn83 and Asn191, but there was very little of this sperm-ligand active chain at Asn527 in the ZP domain of ZPA.