The use of sex-sorted semen in horses.

The application of sex-sorted semen in horses has historically lagged the bovine industry due to differences in the reproductive physiology and grater variability in equine semen quality. Recent advancements, including SuperGen™ sorters and Ultraplus™ semen processing methods developed by STgenetics® (Navasota, Texas), have significantly improved the efficiency of the sex-sorting process and reduced the sperm damage previously reported. To facilitate the broader use sex-sorted semen in the equine industry, it is essential to address challenges such as shipping cooled semen to a central sorting facility, minimizing oxidative stress and DNA fragmentation, and developing effective methods for short-term cooling or long-term cryopreservation of the sex-sorted sperm.

Spermatozoal acrosome dysfunction and its role in stallion subfertility.

Cases of stallion subfertility due to acrosome dysfunction have been recognized since the 1990s. While some of these were observed in stallions with reduced sperm motility and morphology, a more severe form has been reported in stallions with normal-to-excellent sperm quality parameters, which is also uniquely observed in individuals of the Thoroughbred registry. These stallions carry a susceptibility genotype (A/A-A A in the gene FKBP6, exon 5) for Impaired Acrosomal Exocytosis (IAE).

A study on methods for preimplantation genetic testing (PGT) on in vivo- and in vitro-produced equine embryos, with emphasis on embryonic sex determination.

Two methods for preimplantation genetic testing (PGT) have been described for equine embryos: trophoblast cell biopsy (TCB) or blastocoele fluid aspiration (BFA). While TCB is widely applied for both in vivo- and in vitro-produced embryos, BFA has been mostly utilized for in vivo-produced embryos. Alternative methods for PGT, including analysis of cell-free DNA (CFD) in the medium where in vitro-produced embryos are cultured, have been reported in humans but not for equine embryos.

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.

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.

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 10 sperm/mL) on sperm motion characteristics. Post-thaw VIAB was similar between NC and FC (P > 0.

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.

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.

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.

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).

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.

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.

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.

Flow-cytometric analysis of membrane integrity of stallion sperm in the face of agglutination: the "zombie sperm" dilemma.

Purpose: To define the effect of sperm agglutination, associated with incubation under capacitating conditions, on accuracy of membrane assessment via flow cytometry and to develop methods to mitigate that effect.

Methods: Sperm motility was measured by CASA. Sperm were stained with PI-PSA or a novel method, LD-PSA, using fixable live/dead stain and cell dissociation treatment, before flow-cytometric analysis.

The effects of metabolic substrates glucose, pyruvate, and lactate added to a skim milk-based semen extender for cooled storage of stallion sperm.

Under in vitro conditions, stallion sperm might preferentially use energy substrates that primarily undergo mitochondrial metabolism. The present study sought to determine the effects of glucose, pyruvate, lactate, or their combinations on the quality of stallion sperm subjected to cooled storage at different temperatures, when using a skim milk-based semen extender. In Experiment 1, no substrate (Control), glucose (40 mM; Glu-40), pyruvate (2 mM, 19.

Inclusion of supplemental antibiotics (amikacin - penicillin) in a commercial extender for stallion semen: Effects on sperm quality, bacterial growth, and fertility following cooled storage.

In this study, the effectiveness of supplementing INRA-96® extender (INRA-Control; original antibiotic formulation: potassium penicillin G = 38 μg/mL; gentamicin sulfate = 105 μg/mL; amphotericin B = 0.315 μg/mL) with amikacin sulfate and potassium penicillin G (AP) was determined. In Exp.

Effects of glucose concentration in semen extender and storage temperature on stallion sperm quality following long-term cooled storage.

In Experiment 1, the effects of glucose concentration in extender (0 mM, 67 mM, 147 mM, 270 mM; G0, G67, G147, and G270, respectively) and storage temperature of extended semen (5, 10, 15 and 20 °C) were evaluated after storage for up to 5 days (T0h to T120h). For all time points tested, mean total (TMOT) and progressive (PMOT) sperm motility were lower in G0 than all other treatment groups (P < 0.05).