The objective of this study was to establish a rapid and efficient assay that would assess acrosomal status and function of the stallion acrosome. Ejaculates from fertile and subfertile stallions were extended to 25x106/mL and divided into aliquots (1mL) treated with no ionophore (control) or 10µM A23187 and incubated at 37ºC for 0, 1, 2, and 3h. Following incubation, samples were fixed with 2% paraformaldehyde for 10 minutes at room temperature; then stored at 4°C in Dulbecco’s Phosphate-buffered saline (DPBS) for 0, 24, and 72 hours (i.e. post-fixation storage). After post-fixation storage samples were then permeabilized with 95% ethanol at -20ºC for 10 minutes. Samples were resuspended in 20% fetal bovine serum in DPBS, labeled with fluorescein isothiocyanate for 10 minutes, and analyzed by flow cytometry. Post-fixation storage produced fewer (P<0.05) acrosome intact (AI) spermatozoa and a higher (P<0.05) fluorescence intensity than respective fresh samples. Regardless of incubation time or treatment, cool-stored samples averaged ~6% lower (P<0.001) AI spermatozoa than the corresponding fresh semen; however, cooled storage did not alter (P>0.2) the overall fluorescence properties as compared to fresh semen (730±8.08 vs. 734±8.01 fluorescence intensity units, respectively). For fertile stallions, the percentage of AI spermatozoa was higher (p<0.01) in control samples than A23187 samples at incubation times 1, 2, and 3h (Control-59, 56, and 51% vs. A23187- 46, 29, and 23%, respectively), but not at Time 0. For subfertile stallions, the percentage of AI spermatozoa was not affected by ionophore treatment (P>0.05) or incubation period (P>0.05). The results suggest that post-fixation storage in DPBS for up to three days is still representative of the acrosomal competence of the original sample. In addition, spermatozoa stored for 24 hours in an Equitainer™ exhibited a small (~6%) but significant decrease in the percentage AI spermatozoa. Storage conditions may therefore, affect acrosomal integrity and contribute to reduced fertility when cooled-semen is used. Subfertile stallions exhibited little response [<11% acrosome reacted (AR)] after 3h of A23187 exposure, while the fertile stallions demonstrated a substantial response (≥ 36% AR) as soon as 1h after ionophore exposure. This assay diagnosed acrosomal dysfunction in stallions with unexplained subfertility.

Understanding the genes that regulate spermatogenesis and steroidogenesis in the testis is critical for enhancement of stallion fertility. Stallion testicular samples were used to identify candidate genes by cDNA microarrays that simultaneously assessed expression levels of 9132 genes. First, gene expression was compared between light (spermatogenically active) and dark (spermatogenically inactive) testis tissue of 1.5-year-old horses (n = 3). Ninety-three genes were differentially expressed (35 light specific, 58 dark specific) in matched paired samples. Second, gene expression was compared between testicular tissue of two mature stallions, one with normal quality semen (fertile) and one with poor quality semen (subfertile). A total of 233 genes were differentially expressed (122 in fertile tissue, 111 in subfertile tissue). Of these, phosphodiesterase 3B (PDE3B), steroidogenic acute regulatory (StAR) protein, and outer dense fiber of sperm tails 2 (ODF2) mRNAs, were localized and quantified by in situ hybridization (ISH) in mature stallions and/or in unilateral cryptorchids. ISH revealed differences (P < 0.05) among mature stallions (n = 10) for PDE3B (localized to seminiferous tubules) and StAR protein (localized to interstitial spaces) mRNAs. A positive correlation coefficient (r = .556, p = .025) was found between StAR protein mRNA and plasma concentration of testosterone. Additionally, both gene products were evaluated in 1-year-old (n = 3) and 3-year-old (n = 3) unilateral cryptorchid stallions. Expression of both PDE3B and StAR protein gene was significantly higher in mature, descended testes compared to mature, retained testes and the descended and retained testes of immature, cryptorchid stallions. StAR protein gene demonstrated significantly higher expression in immature retained testes compared to immature descended testes. A precision-cut tissue slice (PCTS) in vitro culture system was evaluated as a potential tool to study equine testes function. Testes from immature stallions (n = 3) were cut into slices (mean slice weight = 13.85 +/- 0.20 mg; mean slice thickness = 515.00 +/- 2.33 ?m) and exposed to medium containing ovine luteinizing hormone (oLH) at concentrations of 0, 5, 50 and 500 ng/ml for 6 h at 32 degrees C. Medium content of testosterone and estradiol was increased 500% and 120%, respectively, by addition of oLH versus that observed for the testis tissue slices treated with 0 ng oLH (control). An oLH concentration-dependent increase in StAR protein mRNA in tissue slices was detected by in situ hybridization; whereas, differences for PDE3B and ODF2 mRNAs were not observed. Collectively, these results demonstrate that the stallion is an excellent model for studying male fertility due to the initiation of spermatogenesis, frequency of cryptorchidism, and routine castration providing useful tissue to use for studying gene expression.

Apoptosis in testicular germ cells has been demonstrated in many species. Features of apoptosis reported in other species were used to confirm use of the TUNEL assay in stallion testes. Eight stallions with normal testicular size and semen quality were evaluated to determine the germ cell types and stages where apoptosis most commonly occurs. Mean numbers of TUNEL-positive germ cells per 100 Sertoli cell nuclei were highest in stages IV and V of the seminiferous epithelial cycle corresponding to meiotic divisions of primary spermatocytes and mitotic proliferation of B1 and B2 spermatogonia. Round and elongated spermatids were labeled less frequently by the TUNEL assay. To examine the relationships between germ cell apoptotic rate and spermatogenic efficiency, seminal traits were assessed to classify stallions into normal or reduced quality semen groups. Apoptotic rates were higher for stages IV-VI and stage VIII seminiferous tubules in stallions with reduced semen quality. Daily sperm production (DSP) per gram and per testis were lower for stallions with reduced semen quality. Regression analyses revealed negative linear relationships for germ cell apoptotic rate with DSP/g, DSP/testis, daily sperm output, progressively motile sperm and morphologically normal sperm in ejaculates. Mean circulating concentrations of inhibin were lower for stallions ejaculating reduced quality semen. Apoptotic rate was negatively correlated with concentrations of inhibin and estradiol-17b and positively correlated with concentrations of LH and FSH. To study germ cell apoptosis and formation of the Sertoli cell barrier during the initiation of spermatogenesis, tubule development was classified based on lumen score. Formation of a seminiferous tubule lumen was consistent with events leading to development of a Sertoli cell barrier. A primary wave of apoptosis removed early differentiating germ cell types prior to the formation of a tubule lumen facilitating both the formation of a tubule lumen and a Sertoli cell barrier. A second wave of apoptosis occurred after the formation of a lumen but before seminiferous tubule cross-sections contained a full complement of germ cells. In conclusion, apoptosis is an essential mechanism during normal spermatogenesis. Apoptosis also accounts for low numbers of normal sperm seen in the ejaculates of some stallions.

Current equine sperm cryopreservation methods fail to reliably prevent damages to important cellular structures such as the cell membrane and DNA. The objective of this study was to determine the effects of supplementing a stallion semen extender with 1 or 10 mM resveratrol on post-thaw sperm characteristics. Results showed that sperm death was increased with 10 mM compared to both the control and 1 mM (P < 0.05). DNA fragmentation was increased in the 1 mM treatment compared to the control (P < 0.05). ROS activity was reduced the most in the 10 mM with differences between all groups (P < 0.05). Membrane integrity was not different between groups (P > 0.05). Motility of the control was higher than the treatment groups (P < 0.05). Resveratrol was able to reduce ROS but was unable to preserve motility or viability at the concentrations tested.

Study objectives were to; 1) determine if supplementing of n-3 fatty acids improves membrane integrity, and hence viability and motility of stallion spermatozoa following cold storage, and following cryopreservation, and 2) determine if n-3 supplementation alters the fatty acid composition of stallion spermatozoa. Data indicate that following 90 d of n-3 supplementation daily sperm output and the percentage of morphologically normal sperm in neat semen are increased. Omega-3 supplementation for 90 d did not improve spermatozoal motility or viability following short-term preservation (0, 24 h, 48 h), or following cryopreservation. Although motility was unchanged in this study, individual stallion responses did indicate that n-3 supplementation in stallions with marginal to poor semen quality may benefit from n-3 supplementation. Finally, n-3 fatty acid supplementation does alter plasmalemma fatty acid composition. Spermatozoa from supplemented stallions had increased docosahexaenoic acid (DHA) concentrations as compared to non-supplemented stallions. It is postulated that an increase in long chain n-3 fatty acids, specifically DHA in spermatozoa membrane improves membrane integrity, and thus enhances spermatozoa recovery following the stresses of cold storage and cryopreservation. This phenomenon appears to be beneficial to stallions with marginal to poor quality ejaculates.

One of the requirements for a successful artificial insemination is the quality of stallion´s ejaculate. This thesis deals with influence of sperm velocity fraction on fertility. Semen samples in this thesis were evaluated by using an objective computerized method CASA, system SCA. Sperm motility in monitoring was performed in 75 samples from 12 stallions involved in artificial insemination fresh sperm in ZH Písek. Total and progressive motility, rapid, medium and slow motility were monitored. Stallion´s age was from 3-21 years. The average of fertility was 56,19 %. The average of total motility was 78,07 %. The average of progressive motility was 38,35 %. Furthermore, the spermatozoa were divided into 3 velocity fractions and the average percentage of the individual fractions in the samples was as follows: 27,30 % rapid spermatozoa, 21,27 % medium spermatozoa and 29,50% slow spermatozoa. Then the average percentage of rapid progressive spermatozoa (13,43 %), medium progressive spermatozoa (24,92 %) and non-progressive spermatozoa (39,72 %) were found. There was a positive correlation between fertility and the rate of rapid motile spermatozoa (r=0,34). The difference between individual stallions (p-value < 0,01) in the mean percentage of rapid spermatozoa in the ejaculate was demonstrated. The stallion with the highest rate of rapid motile spermatozoa and the best pregnancy was welsh part-bred stallion. On the other hand, the worst pregnancy results and one of the lowest percentages of rapid motile spermatozoa had noriker stallion. The highest success rate of pregnancy was found after the second insemination.The results of this work have shown that the quality of the ejaculate has a very significant effect on good pregnancy but it is just one of many acting factors.

The use of cooled and frozen/thawed semen offers many advantages to breeders. However, many stallions produce spermatozoa that are unable to endure the stresses of cooling/storage and freezing/thawing. Improving the quality and viability of equine spermatozoa via appropriate dietary manipulation could make these stallions commercially viable for cooling or cryopreservation. To evaluate whether spermatozoa quality and viability can be improved by supplementation of omega-3 fatty acids, and if improvements can be made by altering the sources of these fats, nine miniature stallions were placed into 1 of 2 treatment groups and fed either a fish- or algae/flaxseed-based supplement which was added to the basal concentrate. Motion characteristics, membrane integrity and morphology of spermatozoa in fresh, cooled/stored (24 and 48 h), and frozen/thawed semen samples were analyzed. When comparing spermatozoa obtained from stallions in each treatment, no differences were found (P > 0.05) in motility, percentage of membrane intact spermatozoa, and percentage of morphologically normal spermatozoa of stallions. Overall, omega-3 supplementation did not appear to have a beneficial effect on offsetting the harmful effects of the cooling and freezing processes. However, when analyzing the data of one stallion that had < 40% progressive motility (PMOT) after 24 h of cooling and storage, a significant increase was observed in total motility, and progressive motility of fresh and 24 h cooled/stored spermatozoa was observed when supplemented with the fish-based supplement. Thus, omega-3 fatty acid supplementation may be most beneficial for stallions that produce lower quality ejaculates. However, further studies should be conducted, with a larger sample size, in order to substantiate these findings.

The chromosomal location of endocrine genes was established, and relationships between expression of specific endocrine genes and measures of testis function in normal and poor semen quality stallions was assessed. Consensus primer sequences for glucocorticoid receptor (GR) and luteinizing hormone receptor (LHR) were used to screen the CHORI-241 equine bacterial artificial chromosome (BAC) library. The identity of PCR-positive BAC clones was confirmed by sequencing. Verified BACs were mapped to horse metaphase chromosome spreads by fluorescence in situ hybridization (FISH). The BACs containing the GR and LHR were localized by FISH to ECA 14q16-q21 and ECA15q22-q23, respectively. In addition to FISH mapping, the 5000rad horse x hamster radiation hybrid (RH) panel was screened in duplicate. Two-point linkage analysis placed GR 0 cR from LEX047, while LHR was 36.67 cR from TKY011 on ECA14 and ECA15, respectively. Total testicular parenchymal weight, mean daily sperm production (DSP) per gram parenchyma and mean apoptotic rate (406.05 ± 24.33g vs. 180.01 ± 34.41g, 15.29 ± 0.87 vs. 10.24 ± 1.10, 6.70 ± 0.88 vs. 14.25 ± 1.11, respectively) differed (P<0.05) between normal (n=8) and poor semen quality (n=5) stallions. Also, plasma estradiol and inhibin concentrations were higher (P<0.05) in normal stallions than in poor semen quality stallions. Testicular expression of estrogen receptor beta (ER beta), &#946;B inhibin, prolactin receptor (PRLR), growth hormone receptor (GHR) and insulin-like growth factor I receptor (IGF-IR) mRNAs were all lower (P<0.05) in poor semen quality stallions than in normal stallions. The BACs and primers developed in this study will facilitate future investigations of GR and LHR gene structure in the horse as well as providing a resource for physiological investigation of these two genes that are primary regulators of stress responsiveness and fertility. These data add important endocrine genes to the horse cytogenetic map. Also, important hormonal and gene expression changes have been identified in poor semen quality stallions for further investigation.

Objective markers to identify higher fertility individuals are needed to maximize livestock breeding success. Two heparin-binding proteins, which are reflective of fertility in bulls, have been biochemically identified as fertility-associated antigen (FAA) and tissue inhibitor of metalloproteinases-2 (TIMP-2). These four studies were designed to examine the importance of those proteins in relation to reproduction in bulls and other livestock species. In the first study, indirect immuno-fluorescent microscopy was performed to localize FAA and TIMP-2 to livestock sperm. FAA was localized on spermatozoal acrosomes of bulls and rams, but no cross-reactivity was observed for stallions. TIMP-2 labeling was observed on acrosomes and posterior heads, which was species dependent. Localization patterns for FAA and TIMP-2 were further investigated during heparin-induced capacitation and acrosome reactions of bovine sperm. In study two, an enzyme-linked immunosorbent assay (ELISA) was developed to determine concentrations of FAA in bovine seminal plasma (SP). A commercially available TIMP-2 ELISA was utilized to quantify TIMP-2. Respective mean concentrations of FAA and TIMP-2 in SP were 6.661.487 ug/ml and 1.180.045 mg/ml. Concentrations of FAA in SP did not correspond to bull fertility potential, however, older bulls with higher concentrations of TIMP-2 in SP sired more calves. The third study evaluated utility of an amplified fragment length polymorphism with bovine TIMP-2 gene specific primers to amplify a 700 bp genomic DNA (gDNA) product from sperm. From 53 bulls screened, 22.6% were negative for the 700 bp amplicon. There was a three-fold likelihood for 700 bp negative bulls to not sire a calf compared to 700 bp positive bulls. The product was cloned and sequenced, but no homology to TIMP-2 was detected. Therefore, the product represented novel bovine gDNA sequence. The fourth study identified an equine homologue to the bovine FAA gene. Immuno-based diagnostics had not detected FAA in stallion semen. The equine DNA homologue was 88.5% identical in nucleotide and 86% in amino acid sequences to bovine FAA. Subtle differences in the amino acid sequence are likely responsible for the inability to detect FAA in stallion semen with FAA antibodies to bovine FAA.

A inseminação artificial utlizando sêmen criopreservado de garanhões apresenta uma baixa utilização, principalmente pela variabilidade dos resultados e aumento dos custos relacionados à inseminação. Os lipídeos presentes nas membranas plasmáticas dos espermatozóides participam de diversos eventos associados à fertilização e por sofrerem alterações químicas e estruturais durantes os processos necessários à congelação podem estar relacionados a congelabilidade dos ejaculados. Este trabalho foi composto por dois experimentos. O experimento I utilizou amostras de espermatozóides de seis garanhões e seis touros e objetivou avaliar a possibilidade de análise direta das amostras de espermatozóides e o efeito do armazenamento em metanol por um período de 45 dias a -80°C. As amostras foram coletadas, \"lavadas\" por centrifugações sucessivas e armazenadas nos seguintes meios: PBS Ca++ Mg++ Free, Metanol padrão-HPLC e solução 1:1 PBS Ca++ Mg++ Free e Metanol padrão-HPLC. As amostras em PBS foram analisadas após extração lipídica ou adição de metanol padrão-HPLC previamente à análise. Foi confirmada a possibilidade de análise direta da composição lipídica em MALDITOF, sem necessidade de extração. O armazenamento em metanol ou solução contendo o mesmo não resultou em degradação no período observado, no entanto o armazenamento de amostras previamente extraídas por longos períodos resultaram em resultados alterados. O experimento II utilizou amostras de 16 garanhões, armazenadas em solução 1:1 PBS Ca++ Mg++ Free e Metanol padrão-HPLC. Os ejaculados de origem destas amostras foram submetidos à congelação e os resultados de análise in vitro de motilidade e morfologia espermática foram relacionados com os dados de composição lipídica através da análise dos componentes principais (PCA). Não foi observada nenhuma relação entre os resultados de análise seminal in vitro e a composição lipídica, apesar de alguns autores encontrarem tal relação, principalmente quanto à relação de determinados ácidos graxos e conteúdo total de fosfolipídeos. Estes autores utilizaram técnicas complexas, impossibilitando a aplicação na rotina veterinária a campo. A facilidade de preparo das amostras para MALDI-TOF torna esta técnica viável para análise de composição lipídica, no entanto novos testes devem ser realizados incluindo um maior número de amostas e dados de fertilidade a campo. / Artificial insemination with stallions\' frozen semen have not been widely used, mainly due to the high variability of field results and the increased cost associated with it. Sperm cell membrane lipids play an important role on several events associated with fertilization. These cells go through structural and chemicals changes during freezing and might participate in the freezeability of ejaculates. This work was composed by two experiments. In Experiment I sperm samples from six bulls and six stallions were collected, \"washed\" through centrifugation and stored at -80° in the following mediums: PBS Ca++ Mg++ Free, Methanol HPLC-standard and a solution 1:1 PBS Ca++ Mg++ Free and Methanol HPLC-standard. The samples on PBS were analyzed immediately after lipid extraction or after the addition of methanol. These samples were evaluated onthe day after collection and 45 days later for evaluation of the possibility of a direct analyses with methanol and the effect of long periods of storage with methanol on lipid degradation. The possibility of an extraction-free direct MALDI-TOF analysis was confirmed. The storage with methanol did not resulted in lipid degradation for the period studied, however the storage of previously extract samples resulted on irregular spectrums. In Experiment II 16 sperm samples from stallions were collected and frozen. An aliquot of 500&micro;l from each sample was washed through centrifugation and stored with a 1:1 PBS Ca++ Mg++ Free and Methanol HPLC-standard solution. In vitro sperm analysis of motility and morphology were studied for relations with the lipid profile through a principal components analysis (PCA). It was not observed relation between lipid profile and sperm motility and morphology, as previously reported by others authors. However, these authors methodology were highly complex , making it difficult for field application. MALDI-TOF sample preparation its easy and fast and new test with a larger number of samples and field fertility results may prove this is a viable tool for improving field results with frozen semen artificial insemination.