Fertility-associated Antigen Research Articles

Utilization of bull fertility-associated antigen to improve the quality of frozen bull semen

Background and Aim:The implementation of artificial insemination (AI) is one of the strategies to use superior male semen optimally to improve the genetic quality of livestock. One of the factors that influence AI is a fertility-associated antigen (FAA). This research aimed to examine the effects of FAA extracted from the accessory sex glands of a bull from a slaughterhouse that was added in bull semen freezing medium to increase cattle (bull) fertilization.Materials and Methods:This research used a randomized complete block design. It consisted of two research phases, namely, explorative and experimental phases. The first phase involved determining the FAA molecular weight using the SDS-PAGE method, and the second phase consisted of laboratory and field testing, including testing the quality of frozen semen supplemented with FAA extracted from the accessory glands of a bull’s genital organ from a slaughterhouse with various doses (0, 5, 10, and 15 μg in every 200 million progressively motile spermatozoa).Results:The results showed that the percentages of bull sperm motility between the groups without and with the additional administration of FAA with a dose of 5 μg did not significantly differ. However, there was a difference between the groups without and with the additional administration of FAA with doses of 10 and 15 μg. After further testing, the highest percentage of sperm progressive motility occurred at a dose of 15 μg/200 million progressively motile spermatozoa (P3), which was equal to 2.59±46.88b (%).Conclusion:This research found that not all of the accessory glands (seminal vesicles) of bulls taken from the slaughterhouse contain the FAA. An FAA level between the accessory glands (seminal vesicles) of one cattle to another is different. The addition of the FAA protein from the accessory sex glands of a bull’s organ in cattle semen can improve fertility by increasing the percentage of viability, motility, intact plasma membrane of spermatozoa, and pregnancy rate of bulls and decreasing the sperm capacitation post-thawing.

Characterization of seminal plasma constituents of unimproved indigenous and Tankwa goats

The aim of the study was to characterize and compare seminal plasma constituents of unimproved indigenous and Tankwa goats. In this study, unimproved indigenous (11) and Tankwa (11) goats were used. Their average age was 3 ± 0.8 years and average bodyweights were 35.5 ± 2.1 kg and 59.8 ± 1.9 kg, respectively. Semen samples were collected with an electro ejaculator, and evaluated for macroscopic and microscopic semen characteristics and seminal plasma constituents. Both unimproved indigenous and Tankwa goats had normal semen characteristics for goats, with the exception of sperm cell concentration. However, Tankwa goats had higher straight-line velocity and average path velocity compared with unimproved indigenous goats. Unimproved indigenous goats had higher intact acrosome than Tankwa goats. Lipocalin-type prostaglandin-D synthase was one of the constituents that were detected, but it was below the detection limit of 0.5 mmol/L. Moreover, the semen of unimproved indigenous and Tankwa goats had the same levels of fertility-associated antigen, lactate dehydrogenase, sodium, potassium, magnesium, calcium, glucose, cholesterol, triglyceride, lipids and urea. In conclusion, unimproved indigenous and Tankwa goats had the same seminal plasma constituents. These constituents seem to be optimal for the sperm cells, as they resulted in acceptable semen characteristics, except for sperm cell concentration, which was low. Moreover, acceptable standards need to be set for all seminal plasma constituents, as they are not currently known. Keywords: Fertility-associated minerals, genetic variation, semen characteristics

Greater potentiality of sperm membrane bound fertility associated antigen to withstand oxidative stress ensuing improved sperm function of cryopreserved bull spermatozoa

Fertility associated antigen (FAA) are more abundant in semen of bulls with high fertility. The objective was to investigate relationship of FAA, either membrane bound and/or in seminal plasma, with that of markers of oxidative stress, mitochondrial activity and acrosome integrity of live cells, in cryopreserved spermatozoa. Grouping of bulls was done by electrophoretic profiling of proteins from seminal plasma (SP), and sperm membrane (SM), based on presence/absence of the FAA in SP and/or SM or both. Group I had detectable FAA in both SP and SM; Group II, FAA detected only in SM; Group III, detectable FAA only in SP and Group IV, undetectable FAA in both SP and SM. At post-thaw stage, the lipid peroxide levels were significantly lower (p Lass Than 0.05) and the activity of primary free radical scavengers (superoxide dismutase and catalase) was significantly higher (p Lass Than 0.05) in Groups I and II. Correspondingly, Group I and II showed significantly higher (p Lass Than 0.05) per cent of acrosome intact live spermatozoa and sperm cells with functionally active mitochondria. Collectively, this study showed that the presence of FAA on the sperm membrane is related to greater protection against oxidative stress ensuing improved acrosome intact live-sperm cells with functional mitochondria.

Presence of Fertility-associated antigen on sperm membrane corresponds to greater freezability potential of Frieswal bull semen

Several studies have shown relationship of semen freezability with Fertility-associated antigen (FAA) in purebreds. Objectives of present investigation were to determine presence of FAA in Frieswal bull semen and its relationship with freezability. Study involved separation of seminal plasma (SP, FAA – or +) and sperm membranes (SM, FAA – or +), electrophoresis, protein content and quality assessment (n=36).Five groups were: Group-I (19.4%, SP+ve/SM+ve); Group-II (22.2%, SP-ve/SM+ve); Group-III (25%, SP+ve/SM-ve); Group-IV (16.7%, SP-ve/SM-ve), and Group-V (16.7%, unprocessed, initial progressive motility ≤30%, negative control).At postthaw stage, Group-I and -II showed significantly higher (p< 0.001) freezabilitythan FAA-SM-ve groups. Moreover, Group-III with FAA in seminal plasma but not on membrane showed better post-thaw motility than Group-IV, which totally lacked FAA. Our results have shown that 28–30 kDa protein is present in SP and SM of majority of Frieswal bulls and presence of FAA in SM confers appreciable improvement in freezability.

Prediction of buffalo bull fertility on the basis of sperm motion traits, viability, membrane integrity, heat shock protein (HSP70) expression and fertility associated antigen (FAA)

The present study was conducted on 20 buffalo bulls to predict the fertility on the basis of: (a) Computer assisted semen analysis (CASA) based sperm motion traits, (b) viability, (c) membrane integrity, (d) expression for HSP70, and (e) assessment of fertility associated antigen (FAA). Six frozen semen straws from each buffalo bulls were analyzed for sperm motion traits, viz. individual motility, progressive motility, average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), amplitude of lateral head deviation (ALH), beat cross frequency (BCF), straightness (STR), linearity (LIN) and sperm size. Viability and hypoosmotic swelling tests (HOST) were also conducted. Heat shock protein 70 (HSP70) expression and quantification was conducted using a real time PCR. Fertility associated antigen (FAA) was assessed in fresh semen from buffalo bulls using test. Fertility trial was conducted on 250 normal cycling buffaloes following estrus synchronization with GnRH-PGF- GnRH protocol. It was concluded that buffalo bull fertility could be best predicted on the basis of sperm motion traits (VCL, VAP, VSL, ALH, LIN), motility, viability, HOST and HSP70 expression. However, FAA assessment in fresh semen did not indicate fertility.

Gamete Therapeutics: Recombinant Protein Adsorption by Sperm for Increasing Fertility via Artificial Insemination

A decrease in fertility can have a negative economic impact, both locally and over a broader geographical scope, and this is especially the case with regard to the cattle industry. Therefore, much interest exists in evaluating proteins that might be able to increase the fertility of sperm. Heparin binding proteins (HBPs), specifically the fertility associated antigen (FAA) and the Type-2 tissue inhibitor of metalloproteinase (TIMP-2), act to favor the capacitation and acrosome reaction and perhaps even modulate the immune system’s response toward the sperm. The objective of this research was to determine the effect on fertility of adding recombinant FAA (rFAA) and recombinant TIMP-2 (rTIMP-2) to bovine semen before cryopreservation for use in an artificial insemination (AI) program in a tropical environment. For this experiment, 100 crossbred (Bos taurus x Bos indicus) heifers were selected based on their estrus cycle, body condition score (BCS), of 4 to 6 on a scale of 1 to 9, and adequate anatomical conformation evaluated by pelvic and genital (normal) measurements. Heifers were synchronized using estradiol benzoate (EB), Celosil® (PGF2α) (Shering-Plough) and a controlled internal drug release (CIDR) device was inserted that contained progesterone. Inseminations were performed in two groups at random, 50 animals per group. The control group was inseminated with conventional semen. The treatment group was inseminated with semen containing rFAA (25 µg/mL) and rTIMP-2 (25 µg/mL). In the control group a 16% pregnancy rate was obtained versus a 40% pregnancy rate for the HBP treatment group, resulting in a significant difference (P = 0.0037). Given the results herein, one may conclude that the HBPs can increase fertility and could be an option for cattle in tropical conditions; however, one needs to consider the environment, nutrition, and the genetic interaction affecting the final result in whatever reproductive program that is implemented.

171 IN VITRO FERTILIZATION USING FROZEN - THAWED SEXED SEMEN TREATED WITH RECOMBINANT HEPARIN-BINDING PROTEINS

The decrease in the fertility of males can have a negative economic impact on the production of milk and meat in cattle. As a result, interest exists in evaluating diverse proteins that might be able to increase the fertility of sperm. This is the case of the heparin-binding proteins (HBP), specifically the fertility-associated antigen (FAA) and the Type-2 tissue inhibitor of metalloproteinase (TIMP-2), which act to favour capacitation and the acrosome reaction, perhaps by modulating the immune system response toward the sperm. The objective of this study was to evaluate the effect of adding recombinant FAA and recombinant TIMP-2 to Hereford sexed semen (X-chromosome-bearing sperm) for use in an IVF program. For IVF, X-sorted semen was prepared both with and without HBP. For this, 25 μg of FAA and 25 μg of TIMP-2 were added per milliliter of semen and were left to adsorb for 20 min. The semen was then diluted, packaged in straws and cryopreserved. Ovaries were obtained from a slaughterhouse and transported to the laboratory to obtain cumulus–oocyte complexes by follicular aspiration and were cultured in maturation medium (TCM-199 supplemented with FSH, LH and oestradiol) for 24 h. All incubations were performed at 38.5°C in a humidified atmosphere of 5% CO2 in air. For insemination, frozen-thawed semen was washed by centrifugation in 2 concentration gradients of a silica-based colloid solution. The sperm concentration used for IVF was 1 × 106 spermatozoa mL–1. Gametes were co-incubated for 22 h in fertilization medium (TALP added with BSA, heparin, penicillamine, hypotaurine and epinephrine). For IVD, presumptive zygotes were incubated for 7 days in a modified IVD medium (SOF) supplemented with FCS and BSA. A total of 363 in vitro-matured oocytes were used in the control vs 405 oocytes in the treatment group. A Pearson chi-square test was used to determine the statistical significance. The cleavage rates for the control (61.4%) and treatment (83.7%) groups were different (P < 0.001). However, the numbers of blastocysts, expanded blastocysts and hatched blastocysts produced in the control (10.2, 6.3 and 0%, respectively) and treatment (7.4, 7.4 and 0.7%, respectively) groups were similar (all P > 0.1). The fact that there was an increase in the early embryonic development rates and not in the development of the embryos to the blastocyst stage suggests that HBP could play a role in fertilization, but not in embryo development. The results also demonstrate that the addition of the recombinant proteins to the semen increased the fertilizing capacity of the sperm at a concentration of 1 × 106 sperm cells mL–1 of fertilization medium. To further elucidate the effect of the HBP, an evaluation of sperm penetration and pronuclei formation is proposed as a complement to these results. We thank Tod C. McCauley, Roy L. Ax and the staff at TMI Laboratories International Inc. for their assistance. We also thank Richard W. Lenz and the staff at Sexing Technologies for their assistance.

Fertility-associated antigen on Nelore bull sperm and reproductive outcomes following first-service fixed-time AI of Nelore cows and heifers

The objective was to determine whether the presence of fertility-associated antigen (FAA) on sperm collected from Nelore ( Bos indicus) bulls can be used to assess potential fertility of sperm for use at first-service fixed-time AI (TAI). Six Nelore bulls were selected based on FAA status (FAA-negative: N = 3; FAA-positive: N = 3) and the ability to produce neat semen with ≥ 70% morphologically normal sperm and 60% estimated progressive motility before cryopreservation. In Experiment 1, suckled multiparous Nelore cows (N = 835) were evaluated for body condition score (BCS) and received an intravaginal progesterone device (CIDR) and 2.0 mg of estradiol benzoate (Day 0). On Day 9 the CIDR was removed, 12.5 mg of PGF 2α and 0.5 mg of estradiol cypionate were administered, and calves were removed for 48 h. All cows received TAI on Day 11 (48 h after CIDR removal). Pregnancy per TAI (P/TAI) was not different between FAA-positive and FAA-negative bulls (41.5% vs. 39.3%, respectively). There was an effect of AI technician on P/TAI (36.0% vs. 43.9%; P < 0.05) and BCS tended to affect P/TAI (P = 0.09), as cows with BCS ≥ 2.75 were 1.4 times more likely to become pregnant compared with cows with BCS < 2.75. In Experiment 2, nulliparous Nelore heifers (N = 617) were evaluated for BCS and received a CIDR and estradiol benzoate (2.0 mg) on Day 0. On Day 7, all heifers received PGF 2α (12.5 mg). On Day 9, CIDR inserts were removed and all heifers received estradiol cypionate (0.6 mg) and 200 IU eCG. All heifers received TAI on Day 11 (48 h after CIDR removal). Pregnancy/TAI was different (P = 0.04) between FAA-positive and FAA-negative bulls (33.7% vs. 40.7%, respectively). Presence of FAA on sperm was unsuccessful in assessing the potential fertility of sperm for use in TAI.

Recombinant Human Seminal DNase.

Seminal plasma deposited simultaneously with extended semen in the presence of neutrophils led to higher fertility in pigs and horses. The mechanism by which fertility was improved is not known, however, data suggest that seminal plasma conveys DNase activity which inhibits negative effects of neutrophils. Seminal protein extracts reduced sperm-neutrophil binding in vitro in a dose-dependent manner by hydrolyzing DNA extruded from activated neutrophils. DNase activity exists in semen from several mammalian species, and inhibits formation of sperm-neutrophil complexes. Seminal fertility-associated antigen (FAA) is a basic 31 kDa non-glycosylated heparin binding protein homologous to the family of DNase-I-like proteins (bovine FAA shares 88% identity to human DNase1L3). It is secreted by accessory sex glands and binds to sperm at ejaculation. FAA possesses two DNase-I-like signature motifs, and recombinant bovine FAA hydrolyzed DNA in vitro. FAA is a protein marker of fertility in bulls; sperm extracts containing detectable FAA indicated higher bull fertility compared to extracts lacking FAA. The objective of this study was to clone the human FAA homolog, determine whether it displayed biological activity previously described in seminal plasma, and investigate the hypothesis that human FAA modulates fertility. To that end, the human homolog to bovine FAA was cloned and expressed as a recombinant N-terminal his-tagged fusion protein. Gene-specific primers successfully amplified a 411 bp product of the human FAA gene spanning the DNase-I-like motifs. PCR product was cloned into a TOPO expression vector and transformed into BL21(DE3) cells. Expression of recombinant was induced by IPTG (4h, 37C). Optimal protein induction and solubility was determined by extraction in non-denaturing or denaturing buffers. Bacterial cell pellet was lysed and clarified extracts were purified by metal affinity chromatography. Purity was assessed by SDS-PAGE and verified by Western blotting. Blots were probed with anti-HisG-HRP conjugated antibody and with anti-bovine FAA monoclonal antibody to verify identity of the human homolog. Blots probed with anti-bovine FAA were incubated with goat anti-mouse HRP secondary antibody and developed by incubation in HRP substrate. Purified rFAA was renatured by multi-step dialysis with insoluble aggregates removed by centrifugation. DNA activity exhibited by human seminal plasma and purified recombinant human FAA was analyzed by degradation of calf thymus DNA using agarose gel electrophoresis. Seminal plasma from 12 patients displayed variation in amount of DNase activity detected. Marked degradation occurred in one-third, intermediate degradation in another third, with little to no activity in remaining samples. The expressed recombinant shared 90% nucleotide identity with bovine recombinant FAA (390 bp). Affinity-purified recombinant displayed a single band of 19.6 kDa using an anti-HisG and anti-FAA antibody. Human recombinant FAA displayed DNase activity in a dose response manner above 50 µg/ml. These data demonstrate high homology between bovine and human FAA and indicate that FAA is a primary source of intrinsic DNase activity in human semen. The recombinant human homolog to FAA can now be exploited (1) to evaluate if it modulates sperm/neutrophil binding, (2) as an antigen to develop antibodies for cytological studies, and (3) to use those antibodies to quantify FAA in semen in relation to fertility of patients.

Influence of Seminal, Physical, and Mating Behavior Traits of Bulls on Number of Calves Sired per Bull in a Multisire Herd

A study was conducted to quantify the influence of behavior, physical characteristics, and seminal traits of bulls on reproductive performance in a multisire herd. Braunvieh (n = 6) and Bonsmara (n = 6) bulls (18 to 24 mo) were evaluated for BCS, social dominance rank (SDR), serving capacity, sperm motility and morphology, and fertility-associated antigen classification of an ejaculate. Bulls (n = 12) were mated with multiparous cows (n = 305) for 90 d. Paternity was verified by DNA typing. All bulls were fertility-associated antigen-positive. Bull breed, sperm motility and morphology, and SDR were associated with number of calves sired per bull (P < 0.05, R2= 0.75). More calves born early in the calving period (P < 0.025) were sired by bulls with ≥ 80% normal sperm. Results indicate that assessment of SDR in conjunction with a breeding soundness exam can determine number of calves sired per bull. Furthermore, selection of bulls that produce at least 80% morphologically normal sperm may enhance the percentage of cows impregnated early in the breeding season.

Immunolocalization of Fertility-Associated Antigen (FAA) on bovine, equine, and human sperm

Immunolocalization of Fertility-Associated Antigen (FAA) on bovine, equine, and human sperm

Artificial insemination outcomes in beef females using bovine sperm with a detectable fertility-associated antigen.

In this study, semen samples from 25 bulls that had passed a breeding soundness evaluation were analyzed for the presence or absence of a 31-kDa protein, known as fertility-associated antigen (FAA), on spermatozoal membranes. Eighteen bulls had FAA on sperm (FAA-positive) and seven were devoid of FAA on sperm (FAA-negative). A single ejaculate from each bull was extended and frozen with 25 to 30 x 10(6) sperm in .5-mL straws. Crossbred replacement heifers (n = 865) were estrus-synchronized and artificially inseminated either at timed AI or 12 h after they were detected in estrus. Mature cows (n = 285) were inseminated 12 h after they were detected in estrus during a 45-d AI period. Pregnancy rates (pooled) to first AI service for females (n = 764) inseminated with FAA-positive sperm were 65.6% and were 49.7% for females (n = 386) inseminated with FAA-negative sperm (P < .005). Among the estrus-synchronized replacement heifers, pregnancy rates to synchronized AI service for heifers (n = 550) inseminated with FAA-positive sperm were 62% and were 45.7% for heifers (n = 315) inseminated with FAA-negative sperm (P < .005). These data indicate that pregnancy rates to first AI service at spontaneous and synchronized estrus are higher when using semen from bulls with detectable FAA on spermatozoal membranes compared to semen from bulls devoid of FAA on membranes. Fertility-associated antigen is an important determinant for fertility potential of sperm from bulls to be used in AI breeding programs.

Purification and characterization of fertility‐associated antigen (FAA) in bovine seminal fluid

Heparin-binding proteins (HBP) recognized by a monoclonal antibody (M1) are produced by male accessory sex glands and bind to distinct regions of ejaculated bull sperm. Immunoblots of sperm proteins probed with M1 identified HBP variants of approximately 31-, 24-, and 21.5-kDa that were associated with increased fertility of bulls. The purpose of this study was to identify the 31-kDa HBP known as fertility-associated antigen (FAA). FAA was isolated by heparin-affinity chromatography and reversed-phase high performance liquid chromatography near homogeneity. Biochemical characterization indicated that FAA was an unglycosylated, basic protein. FAA protein was detected in seminal vesicle and prostate gland homogenates, and FAA extracted from sperm membranes by treatment with hypertonic media was identical biochemically to seminal fluid-derived FAA. N-terminal sequence analysis of purified FAA yielded a 26 amino acid sequence (L K I X S F N V R S F G E S K K A G F N A M R V I V) with 73% identity to a recently identified human deoxyribonuclease (DNase) I-like protein. Two internal amino acid sequences generated from lys-C digested FAA were 85% and 92% identical to the same DNase I-like protein. In conclusion, we have identified a bovine seminal heparin-binding protein that binds to sperm and is indicative of bull fertility as being similar to the family of DNase I-like proteins. These data demonstrate the presence of a novel DNase I-like protein in bull accessory sex glands and form the groundwork for the identification of a candidate genetic marker for fertility of bulls. Mol. Reprod. Dev. 54:145–153, 1999. © 1999 Wiley-Liss, Inc.

Purification and characterization of fertility-associated antigen (FAA) in bovine seminal fluid.

Heparin-binding proteins (HBP) recognized by a monoclonal antibody (M1) are produced by male accessory sex glands and bind to distinct regions of ejaculated bull sperm. Immunoblots of sperm proteins probed with M1 identified HBP variants of approximately 31-, 24-, and 21.5-kDa that were associated with increased fertility of bulls. The purpose of this study was to identify the 31-kDa HBP known as fertility-associated antigen (FAA). FAA was isolated by heparin-affinity chromatography and reversed-phase high performance liquid chromatography near homogeneity. Biochemical characterization indicated that FAA was an unglycosylated, basic protein. FAA protein was detected in seminal vesicle and prostate gland homogenates, and FAA extracted from sperm membranes by treatment with hypertonic media was identical biochemically to seminal fluid-derived FAA. N-terminal sequence analysis of purified FAA yielded a 26 amino acid sequence (L K I X S F N V R S F G E S K K A G F N A M R V I V) with 73% identity to a recently identified human deoxyribonuclease (DNase) I-like protein. Two internal amino acid sequences generated from lys-C digested FAA were 85% and 92% identical to the same DNase I-like protein. In conclusion, we have identified a bovine seminal heparin-binding protein that binds to sperm and is indicative of bull fertility as being similar to the family of DNase I-like proteins. These data demonstrate the presence of a novel DNase I-like protein in bull accessory sex glands and form the groundwork for the identification of a candidate genetic marker for fertility of bulls.

Fertility-associated antigen on bull sperm indicates fertility potential.

A 30-kDa heparin-binding protein named fertility-associated antigen (FAA) was identified in sperm membranes of beef bulls with greater fertility potential. In a survey of 2,191 beef bulls, 88% had FAA present in sperm membranes (FAA-positive), and 12% were FAA-negative. In the first study, 54 Santa Gertrudis and 51 Santa Cruz bulls were grouped (1 to 14 bulls per group) according to FAA profiles and were bred to 2,403 cows at ratios of 1 bull: 25 cows. Fertility for 14 groups of FAA-positive bulls averaged 88%, whereas three groups of FAA-negative bulls impregnated 79% of the cows. Thus, FAA-positive bulls were nine percentage points more (P < .01) fertile than FAA-negative bulls. In the second study, 2-yr-old Santa Cruz bulls (n = 26) were grouped according to FAA profiles and serving capacity. The fertility of the group of 12 high-serving-capacity, FAA-positive bulls was 87% of 270 cows. The group of six FAA-negative bulls with high serving capacity impregnated 78% of 143 cows. Among the groups of bulls with high serving capacity, FAA-positive bulls were nine percentage points more (P < .05) fertile than FAA-negative bulls. The group of eight FAA-positive bulls with low serving capacity impregnated the least (P < .01) percentage (69%) of 238 cows. Serving capacity of bulls should be considered when optimizing fertility potential. Among bulls with acceptable physical characteristics and serving capacity, determination of FAA profiles in sperm can be used as a tool to identify subfertile bulls.