Proteomic analysis of sperm from fertile stallions and subfertile stallions due to impaired acrosomal exocytosis.

Thoroughbred stallions that carry a double-homozygous genotype A/A-A/A for SNPs rs397316122 and rs69101140 in exon 5 of the FKBP6 gene (chr13; EquCab3.0) are uniquely subfertile due to impaired acrosomal exocytosis (IAE). In this study, the sperm proteome in frozen/thawed semen from subfertile Thoroughbred stallions was studied and compared to that of frozen/thawed sperm from fertile Thoroughbred stallions. A total of 2,220 proteins was identified, of which 140 proteins were found to be differentially abundant in sperm from the subfertile stallions compared to that of fertile stallions (83 less and 57 more abundant). Proteins of differential abundance in sperm from the subfertile stallions were mainly overrepresented in the "metabolism" and the "metabolism of lipids" pathways. One of these proteins, arylsulfatase F (ARSF), was studied by immunofluorescence. A lower proportion of sperm displaying ARSF signal at the acrosome region was observed in sperm from subfertile Thoroughbred stallions. In addition, heterologous zona pellucida binding assays revealed that sperm from subfertile Thoroughbred stallions bound at a lower proportion to zonae pellucidae than sperm from fertile Thoroughbred stallions. In conclusion, a group of differential abundance proteins, including some of acrosome origin, were identified in sperm from subfertile stallions with acrosome dysfunction.

Treatment of mares with the non-steroidal anti-inflammatory drug (NSAID) phenylbutazone transiently affects in vitro maturation of equine oocytes and blastocyst development after Intracytoplasmic Sperm Injection (ICSI).

Mares enrolled in assisted reproductive technologies (ARTs) programs are often treated with non-steroidal anti-inflammatory drugs (NSAIDs), particularly phenylbutazone (Bute), due to chronic lameness. The current study was performed to determine the effect of Bute administration on the developmental competence of in vitro-matured equine oocytes subjected to Intracytoplasmic Sperm Injection (ICSI). In a Preliminary Study, immature cumulus-oocyte complexes (COCs) recovered by post-mortem ovary harvested from two healthy mares (n = 2) treated for 10 days with Bute (4.4 mg/kg, PO, BID), and four non-treated healthy mares (n = 4), were matured in vitro and subjected to Piezo-driven ICSI. Lower oocyte in vitro maturation [Bute: 25% (3/12) vs. Control: 61% (28/46)] and blastocyst rates [Bute: 0% (0/12) vs. Control: 18% (5/28)] were observed in the Bute-treated when compared to the Control mares (P < 0.05). In the Main Experiment, a group of healthy mares (n = 9) received a daily dose of Bute (4.4 mg/kg, orally, SID) for 10 days. A control group of mares (n = 10) was treated with an equal volume of placebo. Mares in both groups were subjected to ultrasound-guided transvaginal oocyte aspiration (TVA) on days 3, 33, and 77 following the last dose of Bute (PT). Recovered COCs from both mare groups were matured in vitro and subjected to Piezo-driven ICSI. By day-3 PT, oocyte in vitro maturation rate was similar between mare groups [Bute: 65% (36/55) vs. Control: 67% (78/116); P > 0.05], while oocyte recovery [Bute: 53% (55/103) vs. Control: 70% (116/166)], cleavage [Bute: 31% (11/36) vs. Control: 62% (48/78)] and blastocyst rates [Bute: [0%] (0/36) vs. Control: 28% (22/78)] were significantly different (P < 0.05). By day 33 PT and 77 PT, differences on oocyte recovery, in vitro maturation, cleavage, and blastocyst rates were not observed between mare groups. In summary, the administration of Bute for 10 consecutive days (4.4 mg/kg, PO, SID, or BID) is associated with a decrease in the ability of immature equine oocytes to undergo in vitro-maturation (Preliminary Study) and develop to the blastocyst stage following ICSI (Preliminary Study and Main Experiment). This negative effect appeared to be transient, as 30- and 77-days post-treatment, no differences on in vitro maturation, cleavage or blastocyst rates were observed.

Comparison of two processing techniques on the sperm quality of semen contaminated with urine.

Urospermia in stallions can occur intermittently, consistently, or as an isolated event, and may result in reduced sperm quality which is often assumed to reduce fertility. Although sperm quality declines in urospermic ejaculates, fertility has not been assessed in mares bred with urine contaminated semen. The aims of this study were to compare sperm quality after simple dilution (SD), cushioned centrifugation (CC) alone, or cushioned centrifugation combined with a 40 % silane-coated silica solution (SC) in semen contaminated with 0, 20, or 40 % (v/v) urine. Sperm quality values tended to decrease as the percent urine increased within all treatments (SD, CC, SC) after 24 h of cooled storage. However, SC treated groups had higher sperm quality compared to SD and CC when exposed to 20 or 40 % (v/v) urine. Differences in pregnancy rates among treatment groups (SD with 0 or 40 % (v/v) urine, or 40 % (v/v) urine followed by SC) were unable to be detected.

Factors affecting the analysis and interpretation of sperm quality in frozen/thawed stallion semen.

In the current study, we examined: 1) the agreement (bias) between fluorescence-based methods (NucleoCounter-SP100 [NC] vs. flow cytometry [FC]) for determining the viability (VIAB) of frozen/thawed stallion sperm; 2) the agreement between post-thaw sperm total motility (TMOT) and VIAB; 3) whether a difference between TMOT and VIAB [VIAB - TMOT] in frozen/thawed stallion sperm could be explained by the level of lipid peroxidation in viable sperm (VLPP); 4) the repeatability of post-thaw analysis of sperm quality; and 5) the effect of final post-thaw semen dilution (10, 30, or 50 x 106 sperm/mL) on sperm motion characteristics. Post-thaw VIAB was similar between NC and FC (P > 0.05), and the agreement between these two methods was high (bias: 1 to -3). The agreement between post-thaw TMOT and VIAB decreased as the pre-freeze percentages of morphologically normal sperm and DNA quality decreased: bias - 4 to - 25. The bias between [VIAB - TMOT] and VLPP ranged from - 5 to 7. Differences in post-thaw sperm quality (TMOT, PMOT, VIAB, and sperm concentration) were not observed when analyzing one or three straws per ejaculate (P > 0.05). There was no effect of post-thaw sperm concentration (i.e., 10 vs. 30 vs. 50 x 106 sperm/mL) on sperm motion characteristics (P > 0.05). This study reports factors other than post-thaw sperm motility that warrant further consideration when analyzing frozen/thawed stallion sperm.

Lactate as the sole energy substrate induces spontaneous acrosome reaction in viable stallion spermatozoa.

BACKGROUND: Equine spermatozoa appear to differ from spermatozoa of other species in using oxidative phosphorylation preferentially over glycolysis. However, there is little information regarding effects of different energy sources on measured parameters in equine spermatozoa. OBJECTIVE: To determine the effect of three individual energy substrates, glucose, pyruvate, and lactate, on motion characteristics, membrane integrity, and acrosomal status of stallion spermatozoa. MATERIALS AND METHODS: Freshly ejaculated stallion spermatozoa were incubated with combinations of glucose (5 mm), pyruvate (10 mm), and lactate (10 mm) for 0.5 to 4 h. Response to calcium ionophore A23187 (5 µm) was used to evaluate capacitation status. Motility was evaluated using computer-assisted sperm analysis, and plasma membrane and acrosomal integrity were evaluated by flow cytometry. RESULTS: Incubation with lactate alone for 2 h increased acrosomal sensitivity to A23187. Notably, incubation with lactate alone for 4 h induced a significant spontaneous increase in acrosome-reacted, membrane-intact (viable) spermatozoa, to approximately 50% of the live population, whereas no increase was seen with incubation in glucose or pyruvate alone. This acrosomal effect was observed in spermatozoa incubated at physiological pH as well as under alkaline conditions (medium pH approximately 8.5). Sperm motility declined concomitantly with the increase in acrosome-reacted spermatozoa. Sperm motility was significantly higher in pyruvate-only medium than in glucose or lactate. The addition of pyruvate to lactate-containing medium increased sperm motility but reduced the proportion of live acrosome-reacted spermatozoa in a dose-dependent fashion. DISCUSSION: This is the first study to demonstrate that incubation with a specific energy substrate, lactate, is associated with spontaneous acrosome reaction in spermatozoa. The proportion of live, acrosome-reacted spermatozoa obtained is among the highest reported for equine spermatozoa. CONCLUSION: These findings highlight the delicate control of key sperm functions, and may serve as a basis to increase our understanding of stallion sperm physiology.

"Just the Facts Ma'am": Moral and Ethical Considerations for Artificial Intelligence in Medicine and its Potential to Impact Patient Autonomy and Hope.

Applying machine-based learning and synthetic cognition, commonly referred to as artificial intelligence (AI), to medicine intimates prescient knowledge. The ability of these algorithms to potentially unlock secrets held within vast data sets makes them invaluable to healthcare. Complex computer algorithms are routinely used to enhance diagnoses in fields like oncology, cardiology, and neurology. These algorithms have found utility in making healthcare decisions that are often complicated by seemingly endless relationships between exogenous and endogenous variables. They have also found utility in the allocation of limited healthcare resources and the management of end-of-life issues. With the increase in computing power and the ability to test a virtually unlimited number of relationships, scientists and engineers have the unprecedented ability to increase the prognostic confidence that comes from complex data analysis. While these systems present exciting opportunities for the democratization and precision of healthcare, their use raises important moral and ethical considerations around Christian concepts of autonomy and hope. The purpose of this essay is to explore some of the practical limitations associated with AI in medicine and discuss some of the potential theological implications that machine-generated diagnoses may present. Specifically, this article examines how these systems may disrupt the patient and healthcare provider relationship emblematic of Christ's healing mission. Finally, this article seeks to offer insights that might help in the development of a more robust ethical framework for the application of these systems in the future.

Lactate-induced spontaneous acrosomal exocytosis as a method to study acrosome function in stallion sperm.

Evaluation of acrosome function in stallion sperm is mostly based on the use of inducers of acrosomal exocytosis (AE), such as the calcium ionophore A23187 or progesterone. Recently, it has been reported that incubation of stallion sperm under presumed capacitating conditions (i.e., medium formulated with calcium, bicarbonate, and bovine serum albumin) using a lactate-only containing medium (Lac-MW) results in a high rate of spontaneous AE in viable sperm (AE/Viable). In the current study, we developed an alternative assay of acrosome function for stallion sperm following the incubation of sperm in a medium formulated only with lactate as an energy substrate (Lac-MW). In Experiment 1, freshly ejaculated stallion sperm was incubated with 10 µM A23187, Lac-MW, or Control, for up to 6 h under capacitating conditions. The percentages of motile sperm, viable sperm, total AE (Total AE), and AE in viable sperm (AE/Viable) were compared among treatment groups. Incubation in Lac-MW, but not with Control or A23187, resulted in a time-dependent increase in the percentage of AE/Viable, as determined by flow cytometry, particularly at 4 and 6 h of incubation (P < 0.05). In Experiment 2, freshly ejaculated sperm was incubated in Lac-MW for up to 6 h, and the occurrence of protein tyrosine phosphorylation and AE/Viable were determined. At 4h and 6h of incubation in Lac-MW, ∼40% of the sperm displayed a protein tyrosine phosphorylation immunofluorescence pattern that coincides with that recently associated with stallion sperm capacitation (i.e., immunofluorescence signal at the acrosome and midpiece). In Experiment 3, the rate of AE/Viable sperm was compared among freshly ejaculated, cool-stored, and frozen/thawed stallion sperm. Except at 2h incubation in Lac-MW, differences in mean AE/Viable among fresh, cool-stored, and frozen/thawed sperm were not observed (P > 0.05). In Experiment 4, the relationship between Total AE (A23187), or AE/Viable (Lac-MW), and in vivo fertility of 5 stallions was determined. A linear relationship was observed between mean AE/Viable and the per-cycle (r = 0.93; P < 0.05) and seasonal (r = 0.66; P < 0.05) pregnancy rates of five stallions used for artificial insemination with cool-stored semen. In Experiment 5, frozen/thawed sperm from subfertile Thoroughbred (TB) stallions, known to carry the susceptibility genotype for Impaired Acrosomal Exocytosis (IAE; FKBP6 A/A-A/A) was evaluated following incubation in Lac-MW. Sperm from subfertile TB stallions with IAE had lower mean AE/Viable, at both 4h and 6h incubation in Lac-MW, when compared to that of fertile control stallions (P < 0.05). Overall, the Lac-MW model validated in the current study may be a useful complementary assay to evaluate the ability of stallion sperm to physiologically undergo AE and to study stallion fertility potential. This acrosome function assay can be used to evaluate fresh, cool-stored, or frozen/thawed stallion sperm, and describes a strong linear relationship with in vivo-fertility of stallions used in artificial insemination programs.

A matter of agreement: The effect of the technique and evaluator on the analysis of morphologic defects in stallion sperm.

Analysis of sperm morphology is an important part of the stallion breeding soundness evaluation since it provides an objective measure of a stallion's sperm quality and is one of many factors that estimate a stallion's fertility potential. To describe the effect of sperm quality level on the technique (Differential Interference Contrast - DIC; Phase-contrast - PH; Dip-Quick staining - DQ; and eosin-nigrosin staining - EN; semen samples fixed in buffered-formal saline) and evaluator (three evaluators; using only DIC), stallions were categorized based on sperm quality into three categories: High: >57% normal sperm, Moderate: 23-56% normal sperm, or Low: <23% normal sperm (four stallions per category). The data were analyzed using three different statistical methods: Analysis of Variance (ANOVA), correlative analysis, and Bland-Altman method (agreement). A higher level of agreement among techniques was observed between DIC and PH for morphologically normal sperm. The agreement between the alternative methods (EN, DQ, or PH) and the standard method (DIC) varied, depending on the sperm quality level (High, Moderate, or Low). Some morphological defects (e.g., AH, AMP) were constantly underestimated with the staining methods (DQ, EN) compared to DIC and PH, particularly in ejaculates with low sperm morphology. Underestimation of some abnormalities, due to the technique or the evaluator, has the potential to alter the clinical interpretation of stallion fertility.

Preclinical evaluation of a third-generation absorbable antibacterial envelope.

BACKGROUND: The TYRX (Medtronic) absorbable antibacterial envelope has been shown to stabilize implantable cardiac devices and reduce infection. A third-generation envelope was developed to reduce surface roughness with a redesigned multifilament mesh and enhanced form factor but identical polymer coating and antibiotic concentrations as the currently available second-generation envelope. OBJECTIVE: The purpose of this study was to compare drug elution, bacterial challenge efficacy, stabilization, and absorption of second- vs third-generation envelopes. METHODS: Antibiotic elution was assessed in vitro and in vivo. For efficacy against gram-positive/gram-negative bacteria, 40 rabbits underwent device insertions with or without third-generation envelopes. For stabilization (migration, rotation), 5 sheep were implanted with 6 devices each in second- or third-generation envelopes. Prespecified acceptance criteria were <83-mm migration and <90° rotation. Absorption was assessed via gross pathology. RESULTS: Elution curves were equivalent (similarity factors ≥50 per Food and Drug Administration guidance). Third-generation envelopes eluted antibiotics above minimal inhibitory concentration (MIC) in vivo at 2 hours postimplant through 7 days, consistent with second-generation envelopes. Bacterial challenge showed reductions (P <.05) in infection with second- and third-generation envelopes. Device migration was 5.5 ± 3.5 mm (third-generation) vs 9. 9 ±7.9 mm (second-generation) (P <.05). Device rotation was 18.9° ± 11.4° (third-generation) vs 17.6° ± 15.1° (second-generation) and did not differ (P = .79). Gross pathology confirmed the absence of luminal mesh remainders and no differences in peridevice fibrosis at 9 or 12 weeks. CONCLUSION: The third-generation TYRX absorbable antibacterial envelope demonstrated equivalent preclinical performance to the second-generation envelope. Antibiotic elution curves were similar, elution was above MIC for 7 days, infections were reduced compared to no envelope, and acceptance criteria for migration, rotation, and absorption were met.

The stallion sperm acrosome: Considerations from a research and clinical perspective.

During the fertilization process, the interaction between the sperm and the oocyte is mediated by a process known as acrosomal exocytosis (AE). Although the role of the sperm acrosome on fertilization has been studied extensively over the last 70 years, little is known about the molecular mechanisms that govern acrosomal function, particularly in species other than mice or humans. Even though subfertility due to acrosomal dysfunction is less common in large animals than in humans, the evaluation of sperm acrosomal function should be considered not only as a complementary but a routine test when individuals are selected for breeding potential. This certainly holds true for stallions, which might display lower levels of fertility in the face of "acceptable" sperm quality parameters determined by conventional sperm assays. Nowadays, the use of high throughput technologies such as flow cytometry or mass spectrometry-based proteomic analysis is commonplace in the research arena. Such techniques can also be implemented in clinical scenarios of males with "idiopathic" subfertility. The current review focuses on the sperm acrosome, with particular emphasis on the stallion. We aim to describe the physiological events that lead to the acrosome formation within the testis, the role of very specific acrosomal proteins during AE, the methods to study the occurrence of AE under in vitro conditions, and the potential use of molecular biology techniques to discover new markers of acrosomal function and subfertility associated with acrosomal dysfunction in stallions.

Effects of egg yolk level, penetrating cryoprotectant, and pre-freeze cooling rate, on the post-thaw quality of stallion sperm.

The current study determined the effect of the egg-yolk (phospholipid source) level (egg yolk [20% EY] vs. skim-milk + egg yolk [SM + 2% EY]), cryoprotectant (glycerol [Gly] vs. glycerol + methylformamide [Gly + MF]), and pre-freeze cooling rate (-0.1 vs. -1 vs. -5 °C/min) on post-thaw stallion sperm quality. In Experiment 1, ejaculates (n = 27) from 9 stallions (3 ejaculates each) with varied sperm quality (High, Average, or Low) were frozen in EY-Gly, SMEY-Gly, EY-Gly + MF, or SMEY-Gly + MF extenders. Sperm in each group were cooled from 22° to 5°C using either -0.1 °C/min or -1 °C/min linear cooling rates prior to freezing. In Experiment 2, ejaculates (n = 24) from 12 stallions (2 ejaculates each) with High or Average sperm quality were frozen in EY-Gly, EY-Gly + MF, or in BotuCrio (BC) extenders. Sperm in each group were cooled from 22° to 5°C using either -1 or -5 °C/min linear cooling rates prior to freezing. In Experiment 1, for stallions with High or Average sperm quality, either cooling rate generally resulted in lower sperm quality for the SMEY-based extenders than for the EY-based extenders (P < 0.05). Stallions with Low sperm quality were unaffected by any experimental treatment (P > 0.05). In Experiment 2, a -5 °C/min cooling rate yielded lower sperm quality in BC than in EY-Gly or EY-Gly + MF groups (P < 0.05); however, a -1 °C/min cooling rate yielded similar sperm quality among these treatments (P > 0.05). In summary, the phospholipid level in the freezing extender and the pre-freeze cooling rate, but not the penetrating cryoprotectant, affected the post-thaw quality of stallion sperm.

Sperm factors associated with the production of equine blastocysts by intracytoplasmic sperm injection (ICSI) using frozen/thawed semen.

Intracytoplasmic Sperm Injection (ICSI) using frozen/thawed sperm is a common procedure to obtain embryos from fertile or subfertile mares and stallions. Stallion-associated factors that impact the efficiency of ICSI have been studied less than those associated with the mare. Three experiments were conducted: Experiment 1: the effect of freezing extender composition and cryoprotectant; Experiment 2: the effect of sperm exposure to seminal plasma prior to freezing (ejaculated vs. epididymal sperm; two-freeze/thaw cycles each); and Experiment 3: the effect of sperm morphologic feature used for fertilization (normal vs. cytoplasmic droplet vs. bent tail); on the blastocyst rate after ICSI. In Experiment 1, stallion sperm was cryopreserved using commercially available extenders containing: a) 2% egg-yolk + milk + 4% glycerol (MFR5); b) 2% egg-yolk + milk + 2% glycerol + 3% methyl formamide (CMMFR5); c) 20% egg-yolk + 4.75% glycerol (LE); or d) 20% egg-yolk + 2% glycerol + 3% methyl formamide (CMLE). Sperm from each of the treatment groups were used for Piezo-driven ICSI on in vitro-matured equine oocytes (n = 321). Extender CMLE resulted in a lower cleavage rate (35%) than the other treatment groups (MFR5: 74%, CMMFR5: 62%, LE: 68%; P < 0.05). Extender MFR5 yielded a higher blastocyst rate per injected oocyte (21/82 [26%]) than the Groups LE (8/77 [10%]), CMLE (4/80 [5%]) or CMMFR5 (4/82 [5%]; P < 0.05). Extender MFR5 also yielded a higher blastocyst rate per cleaved oocyte (34%) than Groups LE, CMLE or CMMFR5 (15%, 14%, 8%; respectively P < 0.05). In Experiment 2, ejaculated (EJ) and epididymal (EPD) sperm from a fertile stallion which was initially cryopreserved in the CMLE extender, was thawed and re-cryopreserved in MFR5 extender for use in ICSI. Sperm from both groups (EJ vs. EPD) were used for ICSI on in vitro matured oocytes (n = 127). Differences were not detected for cleavage rate (EJ: 36/63 [57%] vs. EPD: 49/64 [77%]), blastocyst rate per injected oocyte (EJ: 11/63 [17%] vs. EPD: 11/64 [17%]), or blastocyst rate per cleaved oocyte (EJ: 31% vs. EPD: 22%) between treatment groups (P > 0.05). In Experiment 3, morphologically normal sperm (N), or sperm with proximal droplets (PD) or bent tails (BT), were obtained from a single fertile stallion and were used for ICSI on in vitro matured oocytes (n = 75). No differences were detected among treatment groups for cleavage rate (N: 19/25 [77%] vs. PD: 20/25 [88%] vs. BT: 18/25 [72%]), blastocyst rate per injected oocyte (N: 6/25 [24%] vs. PD: 5/25 [20%] vs. BT: 2/25 [8%]), and blastocyst rate per cleaved oocyte (N: 32% vs. PD: 23% vs. BT: 11%; P > 0.05). In conclusion, the current study indicates that freezing extender composition used for stallion sperm cryopreservation has an impact on the developmental competence of in vitro-matured equine oocytes after ICSI and in vitro culture. Furthermore, we were unable to detect differences on cleavage and blastocyst rates when performing ICSI when using: 1) ejaculated or epididymal sperm; or 2) sperm with different morphologic features. The results from the current study provide additional insight regarding stallion-related factors that should be considered when performing ICSI in horses.

Radiographic Identification of Cardiac Implantable Electronic Device Manufacturer: Smartphone Pacemaker-ID Application Versus X-ray Logo.

Radiographic identification of the cardiac implantable electronic device (CIED) manufacturer facilitates urgent interrogation of an unknown CIED. In the past, we relied on visualizing a manufacturer-specific X-ray logo. Recently, a free smartphone application ("Pacemaker-ID") was made available. A photograph of a chest X-ray was subjected to an artificial intelligence (AI) algorithm that uses manufacturer characteristics (canister shape, battery design) for identification. We sought to externally validate the accuracy of this smartphone application as a point-of-care (POC) diagnostic tool, compare on-axis to off-axis photo accuracy, and compare it to X-ray logo visualization for manufacturer identification. We reviewed operative reports and chest X-rays in 156 pacemaker and 144 defibrillator patients to visualize X-ray logos and to test the application with 3 standard (on-axis) and 4 non-standard (off-axis) photos (20° cranial; caudal, leftward, and rightward). Contingency tables were created and chi-squared analyses (P < .05) were completed for manufacturer and CIED type. The accuracy of the application was 91.7% and 86.3% with single and serial application(s), respectively; 80.7% with off-axis photos; and helpful for all manufacturers (range, 85.4%-96.6%). Overall, the application proved superior to the X-ray logo, visualized in 56% overall (P < .0001) but varied significantly by manufacturer (range, 7.7%-94.8%; P < .00001). The accuracy of the Pacemaker-ID application is consistent with reports from its creators and superior to X-ray logo visualization. The accuracy of the application as a POC tool can be enhanced and maintained with further AI training using recent CIED models. Some manufacturers can enhance their X-ray logos by improving placement and design.

The role of impaired acrosomal exocytosis (IAE) in stallion subfertility: A retrospective analysis of the clinical condition, and an update on its diagnosis by high throughput technologies.

Acrosomal dysfunction has been considered as a cause of subfertility in males of different species, including stallions. A subset of subfertile stallions with acrosomal dysfunction is unique because they have normal sperm quality (motility, morphology, viability, and DNA quality). The current work aims to describe the clinical characteristics of subfertile stallions that were diagnosed with Impaired Acrosomal Exocytosis (IAE) by using two high throughput methods: flow cytometry and molecular genetic analysis, and to identify the prevalence of subfertility due to IAE in stallions evaluated at Texas A&M University. Clinical data from 1,128 stallions evaluated during 17 years at a Veterinary Teaching Hospital was retrospectively analyzed. Only stallions with a history of subfertility not explained following a breeding soundness examination and/or conventional semen analysis, were included. For those stallions, the acrosomal exocytosis test (AE test), in which sperm is incubated at 37 °C for up to 2 h in the presence of the calcium ionophore A23187, was used to determine IAE. The difference in AE-Rate (AE-Diff) between each pair of fertile control stallion and subfertile stallion was categorized as: Normal: AE-Diff < 14%; Questionable: AE-Diff 15-29%; Abnormal: AE-Diff > 30%. In selected cases, blood or hair was procured for identification of the susceptibility genotype for IAE, A/A-A/A, in the FKBP6 gene, exon 5. Twenty-one (21) stallions (1.86% total population analyzed) had reduced fertility despite having acceptable sperm quality. Sperm from these stallions were subjected to the AE Test. Of these, 8 stallions had reduced sperm AE-rate, based on the AE Test (8/21; 38.1%). Subsequently, blood or hair samples from these 8 stallions which had either questionable (AE-Diff 15 - 29%; n = 5) or abnormal (AE-Diff > 30%; n = 3) responses to the AE Test were analyzed for the susceptibility genotype for IAE, A/A-A/A (FKBP6 gene, exon 5). Seven out of the eight (7/8) stallions carried this susceptibility genotype. All of these were Thoroughbreds. After 2 h of incubation, the viability in fertile stallion sperm was lower than in A/A-A/A stallions (4% vs. 25%, respectively; P < 0.05), while the AE-rate was higher for fertile than for A/A-A/A stallions (85% vs. 56%, respectively; P < 0.05). The use of two high throughput tests (i.e., flow cytometry and molecular genetic analysis) may complement each other in the diagnosis of IAE in breeding stallions. In this study, 5/7 subfertile stallions diagnosed with the IAE susceptibility genotype would have been diagnosed as normal with the AE Test. This study introduces a subset of stallions with the IAE genotype with fertility higher than has been previously reported (i.e., <15% per-cycle pregnancy rate), suggesting that IAE manifests as a broader range of subfertility.

Evaluation of Stallion Testicular Cell Types by Flow Cytometry.

Flow cytometry procedures can be used for evaluation of both spermatogenic efficiency and diagnose disorders of stallion spermatogenesis. Aims of this study were to compare two testicular sample acquisition techniques (needle aspirate-N and tissue wedge-T) and results when using flow cytometry and histology procedures. Testicular cell types were stained with acridine orange, and nine regions (R2 to R10) were identified and enumerated following acquisition by either N or T. Testes were also grouped and analyzed by size and sexual maturity (Small [immature] compared with Large [mature]) and used to determine if flow cytometry procedures could be used to detect differences. For both N and T, percentages of 2n cell types were greater in the Small than Large testes, whereas percentages of 1n cell types in N were greater in the Large than Small testes (P < .05). Testicular cell types in N regions were correlated to similar T regions (r between 0.51 and 0.99; P < .05) in both groups. Flow cytometry and histology scores were correlated in both groups (r between -0.95 and 0.93, P < .05). There were small differences in number of testicular cell types from N and T. With both sample acquisition methods, there was discrimination between the Small and Large testes, therefore, evaluation of testicular cell types using flow cytometry procedures might have clinical applications. Results with comparison of flow cytometry to histology procedures indicate that flow cytometry can be applied clinically to identify changes in testicular cell types of stallions using a needle aspirate.