Effect of probiotics and prebiotics on the composition of the equine fecal and seminal microbiomes and sperm quality: A pilot study.

Probiotic and prebiotic effects on equine semen and gastrointestinal microbiome composition and sperm quality are unknown. This study aimed to evaluate the effects of pre-, pro- or synbiotic supplementation on fecal and semen microbiome composition and sperm quality parameters of stallions. This Latin square crossover trial involved four miniature pony stallions receiving control diet only, or addition of a pro-, pre- or synbiotic formulation. Full-length 16S rRNA gene amplicon sequencing was used to measure diversity of semen and fecal microbiomes. Total sperm count, total motility, progressive motility, DNA integrity, lipid peroxidation and mitochondrial oxidative stress, biomarkers of sperm quality, were measured after each intervention. A general linear model was employed to analyse and compare microbiome diversity measures and sperm quality data across four time points. Shannon's diversity index (alpha-diversity), and evenness of semen and gastrointestinal microbiomes were significantly different (p<0.001). A trend was observed for prebiotic effects on the diversity indices of the GI microbiome (p= 0.07). No effects of treatments were observed on either semen microbiome or sperm quality. Pre-, pro- and synbiotic supplements showed no negative effect on sperm quality parameters observed. This proof of concept provides preliminary data to inform future studies exploring the relationship between microbiomes and fertility.

The semen microbiome of miniature pony stallions.

CONTEXT: Little is known about the microbial composition of stallion semen. AIMS: To describe the microbiota detected in equine semen of healthy miniature pony stallions. METHODS: Semen specimens were collected using a Missouri artificial vagina at a single time point. PacBio (Pacific Biosciences) genomic DNA sequencing of the 16S rRNA gene was performed on these specimens, following which next-generation microbiome bioinformatics platform QIIME2 was used to process fastq files and analyse the amplicon data. The data were categorised into genus, family, class, order and phylum. KEY RESULTS: Firmicutes and Bacteroidetes phyla predominated (76%), followed by Proteobacteria (15%). Bacteroidales, Clostridiales and Cardiobacteriales predominated the microbial rank of order (86%). Class was mainly composed of Bacteroidia, Clostridia and Gammaproteobacteria (87%), while family was mainly composed of Porphyromonadaceae , Family_XI and Cardiobacteriaceae (62%). At the level of genus, 80% of the abundance was composed of seven genera, namely Porphyromonas, Suttonella, Peptoniphilus, Fastidiosipila, Ezakiella, Petrimonas and an unknown taxon. CONCLUSIONS: The findings indicate that specific microbiota may be characteristic of healthy miniature pony stallions' semen with some inter-individual variations observed. IMPLICATIONS: Larger equine studies involving fertile and infertile subjects could be informed by this study and could explore the relationship of the semen microbiome to male fertility.

Cloned Foal Born from Postmortem-Obtained Ear Sample Refrigerated for 5 Days Before Fibroblast Isolation and Decontamination of the Infected Monolayer Culture.

A 6-year-old mare, a valuable polo horse, died of complications following postcolic surgery. To preserve its genetics, ear skin samples were collected immediately after death and stored in an equine embryo transfer medium at 4°C for 5 days. After trypsin digestion, monolayer fibroblast cultures were established, but signs of massive bacterial infection were found in all of them. As an ultimate attempt for rescue, rigorously and repeatedly washed cells were individually cultured in all wells of four 96-well dishes. New monolayers were established from the few wells without contamination and used for somatic cell nuclear transfer. Four of the six Day 7 blastocysts derived from 14 reconstructed zygotes were transferred in four naturally cycling mares on Day 5 after ovulation. The embryo transfers resulted in 2 pregnancies, one from a fresh and one from a vitrified blastocyst. The vitrified embryo transfer resulted in a healthy offspring, now 21 months old, genetically and phenotypically identical to the somatic cell donor animal.

Vaginal and vestibular electrical resistance as an alternative marker for optimum timing of artificial insemination with liquid-stored and frozen-thawed spermatozoa in sows.

Development of a pen-side test to objectively determine the ideal time for artificial insemination (AI) in the sow would save producers time and money. Current processes rely on identification of oestrus via subjective behavioural and physiological markers that are indicative of high blood oestrogen concentrations. This study attempted to use measurements of electrical resistance (ER) in the cervical mucus to pinpoint timing of AI accurately enough to lead to equivalent pregnancy rates as a natural mating. Thirty-six sows were divided into 3 groups and observed for signs of oestrus. Seven sows did not show any oestrus behaviour and were excluded from the study. The remaining 29 sows were inseminated via natural mating and conventional oestrus detection (n = 14), or inseminated artificially with either liquid-stored semen (n = 8) or frozen-thawed semen (n = 7) according to timing indicated from electrical resistance measurements in the vagina and vestibule. Sows that were artificially inseminated on the basis of the electrical resistance readings had a lower pregnancy rate (P = 0.034) and less piglets born alive per litter (P < 0.05) than those that were naturally mated according to a conventional oestrus detection regime. However, the pregnancy rate and total piglets born alive did not differ between the two groups that underwent artificial insemination. Change in electrical resistance in the vagina has the potential to accurately predict ovulation timing, but more work is required to refine the timing of AI in relation to the readings before the technique can be adopted by industry.

Cloning horses by somatic cell nuclear transfer: Effects of oocyte source on development to foaling.

The cloning of horses is a commercial reality, yet the availability of oocytes for cloned embryo production remains a major limitation. Immature oocytes collected from abattoir-sourced ovaries or from live mares by ovum pick-up (OPU) have both been used to generate cloned foals. However, the reported cloning efficiencies are difficult to compare due to the different somatic cell nuclear transfer (SCNT) techniques and conditions used. The objective of this retrospective study was to compare the in vitro and in vivo development of equine SCNT embryos produced using oocytes recovered from abattoir-sourced ovaries and from live mares by OPU. A total of 1,128 oocytes were obtained, of which 668 were abattoir-derived and 460 were OPU-derived. The methods used for in vitro maturation and SCNT were identical for both oocyte groups, and the embryos were cultured in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham medium supplemented with 10% fetal calf serum. Embryo development in vitro was assessed, and Day 7 blastocysts were transferred to recipient mares. The embryos were transferred fresh when possible, and a cohort of vitrified-thawed OPU-derived blastocysts was also transferred. Pregnancy outcomes were recorded at Days 14, 42 and 90 of gestation and at foaling. The rates of cleavage (68.7 ± 3.9% vs 62.4 ± 4.7%) and development to the blastocyst stage (34.6 ± 3.3% vs 25.6 ± 2.0%) were superior for OPU-derived embryos compared with abattoir-derived embryos (P < 0.05). Following transfer of Day 7 blastocysts to a total of 77 recipient mares, the pregnancy rates at Days 14 and 42 of gestation were 37.7% and 27.3%, respectively. Beyond Day 42, the percentages of recipient mares that still had a viable conceptus at Day 90 (84.6% vs 37.5%) and gave birth to a healthy foal (61.5% vs 12.5%) were greater for the OPU group compared with the abattoir group (P < 0.05). Surprisingly, more favourable pregnancy outcomes were achieved when blastocysts were vitrified for later transfer, probably because the uterine receptivity of the recipient mares was more ideal. A total of 12 cloned foals were born, 9 of which were viable. Given the differences observed between the two oocyte groups, the use of OPU-harvested oocytes for generating cloned foals is clearly advantageous. Continued research is essential to better understand the oocyte deficiencies and increase the efficiency of equine cloning.

Establishment and characterization of oviductal organoids from farm and companion animals†.

Organoid technology has provided a unique opportunity to study early human development and decipher various steps involved in the pathogenesis of disease. The technology is already used in clinics to improve human patient outcomes. However, limited knowledge of the methodologies required to establish organoid culture systems in domestic animals has slowed the advancement and application of organoid technology in veterinary medicine. This is particularly true for the field of reproduction and the application of assisted reproductive technologies (ART). Here, we have developed a platform to grow oviductal organoids from five domestic species-bovine, porcine, equine, feline, and canine. The organoids were grown progressively from single cells derived from the enzymatic digestion of freshly collected infundibular/fimbrial samples. The addition of WNT, TGFß, BMP, ROCK, and Notch signaling pathway activators or inhibitors to the organoid culture medium suggested remarkable conservation of the molecular signals involved in oviductal epithelial development and differentiation across species. The gross morphology of organoids from all the domestic species was initially similar. However, some differences in size, complexity, and growth rate were subsequently observed and described. After 21 days, well-defined and synchronized motile ciliated cells were observed in organoids. Histopathologically, oviductal organoids mimicked their respective native tissue. In summary, we have carried out a detailed cross-species comparison of oviductal organoids, which would be valuable in advancing our knowledge of oviduct physiology and, potentially, help in increasing the success of ART.

NAD+, Sirtuins and PARPs: enhancing oocyte developmental competence.

Oocyte quality is the limiting factor in female fertility. It is well known that maternal nutrition plays a role in reproductive function, and manipulating nutrition to improve fertility in livestock has been common practice in the past, particularly with respect to negative energy balance in cattle. A deficiency in nicotinamide adenine dinucleotide (NAD+) production has been associated with increased incidences of miscarriage and congenital defects in humans and mice, while elevating NAD+ through dietary supplements in aged subjects improved oocyte quality and embryo development. NAD+ is consumed by Sirtuins and poly-ADP-ribose polymerases (PARPs) within the cell and thus need constant replenishment in order to maintain various cellular functions. Sirtuins and PARPs play important roles in oocyte maturation and embryo development, and their activation may prove beneficial to in vitro embryo production and livestock breeding programs. This review examines the roles of NAD+, Sirtuins and PARPs in aspects of fertility, providing insights into the potential use of NAD+-elevating treatments in livestock breeding and embryo production programs.

Supplemental Nicotinic Acid Elevates NAD+ Precursors in the Follicular Fluid of Mares.

A deficiency in NAD+ has previously been linked with increased occurrences of congenital abnormalities and embryonic death in humans and mice. Early embryonic death is a major factor involved in pregnancy loss in mares, and very little is known regarding the NAD+ requirements for optimum reproductive function in horses. The aim of this study was to determine the effect of supplementing the diet of mares with nicotinic acid (NA) on the composition of NAD+ metabolites in the blood and follicular fluid. Vehicle alone or NA (3 g per os) were administered to seven mares over a minimum of 3 consecutive days during the follicular phase of the oestrous cycle. Blood samples were collected immediately prior to supplemental feeding and follicular fluid aspiration. Follicular fluid was collected from the dominant follicle through transvaginal ultrasound-guided aspiration. Blood and follicular fluid samples were processed and analysed by mass spectrometry. The concentration of nicotinamide mononucleotide (NMN) in the follicular fluid of NA-fed mares was 4-fold greater than that in the corresponding plasma and 10-fold greater than that in the follicular fluid of vehicle-fed mares. The concentrations of NA, nicotinamide (NAM) and nicotinuric acid (NUR) tended to be greater in the follicular fluid of NA-supplemented mares than in the corresponding plasma. The results show that NA supplementation increased the bioavailability of NAD+ precursors in the follicular fluid of the dominant follicle, which is proposed to better promote the maturation of good quality oocytes, especially in older mares.

Conception and early pregnancy in the mare: lipidomics the unexplored frontier.

Lipids are dynamic biological molecules that play key roles in metabolism, inflammation, cell signalling and structure. They are biologically significant in the physiology of conception and reproduction. Many of the mechanisms surrounding equine conception and the early feto-maternal dialogue are yet to be understood at a biochemical level. Recently, lipidomic technologies have advanced considerably and analytical strategies have been enhanced and diversified. Consequently, in-depth lipidomic exploration now has the potential to reveal new lipid biomarkers and biochemical relationships that improve our understanding of the processes leading to efficient and successful reproduction. This review considers the role of lipids in conception and establishment of pregnancy, providing new insights into the enigmatic pathways governing early reproductive physiology of the mare. Lay summary: This paper discusses the role that lipids play in the very early stages of pregnancy in the mare. Lipids are microscopic non-soluble molecules that are important components of living cells. The manuscript discusses how lipids influence the reproductive cycle of mares, including ovulation and the detailed biological process of becoming pregnant. It explains how lipids are identified in a laboratory setting with a newly developing technology known as 'lipodomics'. The technology may lead to a more detailed understanding of how mares become pregnant. The focus of the paper is on mare reproduction, but it also draws on similarities with reproduction in other mammals. Remarkably there are gaps in much of our knowledge about the finer details of pregnancy in the horse, and the paper summarises what we already know about lipids, highlighting areas for further research.

Insights into the NAD+ biosynthesis pathways involved during meiotic maturation and spindle formation in porcine oocytes.

Treatments that elevate NAD+ levels have been found to improve oocyte quality in mice, cattle, and pigs, suggesting that NAD+ is vital during oocyte maturation. This study aimed to examine the influence of different NAD+ biosynthetic pathways on oocyte quality by inhibiting key enzymes. Porcine oocytes from small antral follicles were matured for 44 h in a defined maturation system supplemented with 2-hydroxynicotinic acid [2-HNA, nicotinic acid phosphoribosyltransferase (NAPRT) inhibitor], FK866 [nicotinamide phosphoribosyltransferase (NAMPT) inhibitor], or gallotannin [nicotinamide mononucleotide adenylyltransferase (NMNAT) inhibitor] and their respective NAD+ pathway modulators (nicotinic acid, nicotinamide, and nicotinamide mononucleotide, respectively). Cumulus expansion was assessed after 22 h of maturation. At 44 h, maturation rates were determined and mature oocytes were fixed and stained to assess spindle formation. Each enzyme inhibitor reduced oocyte maturation rate and adversely affected spindle formation, indicating that NAD+ is required for meiotic spindle assembly. Furthermore, NAMPT and NMNAT inhibition reduced cumulus expansion, whereas NAPRT inhibition affected chromosomal segregation. Treating oocytes with gallotannin and nicotinamide mononucleotide together showed improvements in spindle width, while treating oocytes with 2-HNA and nicotinic acid combined showed an improvement in both spindle length and width. These results indicate that the salvage pathway plays a vital role in promoting oocyte meiotic progression, while the Preiss-Handler pathway is essential for spindle assembly.

Supplementing media with NAD+ precursors enhances the in vitro maturation of porcine oocytes.

In vitro maturation (IVM) is an important reproductive technology used to produce embryos in vitro. However, the developmental potential of oocytes sourced for IVM is markedly lower than those matured in vivo. Previously, NAD+-elevating treatments have improved oocyte quality and embryo development in cattle and mice, suggesting that NAD+ is important during oocyte maturation. The aim of this study was to examine the effects of nicotinic acid (NA), nicotinamide (NAM) and nicotinamide mononucleotide (NMN) on oocyte maturation and subsequent embryo development. Porcine oocytes from small antral follicles were matured for 44 h in a defined maturation medium supplemented with NA, NAM and resveratrol or NMN. Mature oocytes were artificially activated and presumptive zygotes cultured for 7 days. Additionally, oocytes were matured without treatment then cultured for 7 days with NMN. Supplementing the IVM medium with NA improved maturation and blastocyst formation while NAM supplementation improved cleavage rates compared with untreated controls. Supplementing the IVM or embryo culture media with NMN had no effect on maturation or embryo development. The results show that supplementing the maturation medium with NA and NAM improved maturation and developmental potential of porcine oocytes.

Nicotinic acid supplementation at a supraphysiological dose increases the bioavailability of NAD+ precursors in mares.

NAD+ deficiency has recently been linked with increased occurrences of congenital abnormalities and embryonic death in human and animal subjects. Early embryonic death is a major component of pregnancy loss in mares and very little is known regarding the requirement for NAD+ in horses. The aim of this study was to quantify NAD+ and its metabolites in the plasma and urine of mares after orally administering an acute dose of nicotinic acid and determine the absorption, metabolism and excretion of this essential precursor for NAD+ biosynthesis. Nicotinic acid (5 g per os) was administered to four mares via a dosing syringe. Blood samples were collected at 0, 0.25, 0.5, 1, 2, 4, 6 and 22 h, and urine samples were collected at 0, 3, 6 and 22 h. The samples were processed and analysed by mass spectrometry. A general additive model was applied to all metabolite concentration values followed by a post-hoc multiple comparisons test. Nicotinic acid was rapidly absorbed into peripheral blood within 15 min of administration and the concentrations of nicotinic acid, nicotinamide (NAM), nicotinuric acid, nicotinic acid mononucleotide and nicotinic acid adenine dinucleotide (NaAD) increased significantly in plasma at 30 min. The concentrations of NAM, nicotinic acid riboside and NaAD increased significantly in urine at 3 h. The levels of NAM and NaAD remained significantly elevated in plasma at 22 h, sixfold and ninefold greater, respectively, than the basal levels at 0 h. While the extracellular levels of NAD+ in the samples remained undetected, the large, sustained elevation of NaAD levels in plasma indicates that the NAD+ levels were boosted within the cellular compartments. The results show that nicotinic acid supplementation increases the bioavailability of NAD+ precursors in mares, which is proposed to be beneficial during periods of peak NAD+ demand, such as during early embryo development.

Serum Concentrations of AMH and E2 and Ovarian and Uterine Traits in Gilts.

Poor sow retention due to reproductive failure is a common reproductive inefficiency amongst piggeries. This shows that traditional methods of gilt selection are inadequate and a marker of reproductive success is needed. The aim of this study was to determine whether circulating levels of AMH and E2 at D80 and D160 are associated with uterine and ovarian traits at D160. Uterine weight, horn length and horn diameter were measured, and ovarian follicle counts were determined histologically. There was a negative relationship between both D80 and D160 AMH levels and D160 ovarian follicle populations. There was also a positive relationship between D80 E2 levels and uterine capacity in gilts that were pubertal at D160. The findings indicate that D80 and D160 AMH could be used to predict ovarian reserve and that D80 E2 levels may be indicative of uterine capacity in precocial gilts.

Effect of carbohydrates on lipid metabolism during porcine oocyte IVM.

Porcine oocytes contain a large amount of endogenous lipid, which is thought to function as an intracellular source of energy. The aim of this study was to determine the effects of stimulating or inhibiting lipid metabolism using l-carnitine or etomoxir respectively on the IVM of porcine oocytes cultured in media of varying carbohydrate composition. In the presence of pyruvate and lactate, exclusion of glucose inhibited oocyte nuclear and cytoplasmic maturation compared with oocytes matured in media containing low (1.5mM) and high (4.0mM) concentrations of glucose. In the absence of pyruvate and lactate in low-glucose medium only, a greater proportion of l-carnitine-treated oocytes progressed to the MII stage compared with untreated oocytes. The inclusion of pyruvate and lactate significantly altered the distribution of cytoplasmic lipid droplets and elevated the ATP content of oocytes, whereas the l-carnitine treatment did not. Further, the inhibitory effect of etomoxir on nuclear maturation was decreased in high- compared with low-glucose medium. The results indicate that carbohydrate substrates are absolutely necessary for effective porcine oocyte maturation, and that l-carnitine supplementation can only partially compensate for deficiencies in carbohydrate provision.

Anti-Müllerian hormone and Oestradiol as markers of future reproductive success in juvenile gilts.

There is a need for an early marker for reproductive success in gilts as the traditional process for selecting breeding females is inefficient. There is evidence that circulating anti-Müllerian hormone (AMH) is indicative of ovarian reserve, antral follicle populations, gonadotropin responsiveness and fertility in various species other than the pig. Additionally, oestradiol (E2) has been shown to mark antral follicle populations in cattle and pregnancy outcomes in women, after gonadotropin treatment. The aims of this study were to determine whether 1) serum levels of AMH or E2, prior to or after gonadotropin injection at 60, 80 or 100 days of age, and 2) hormonal changes in response to gonadotropin stimulation (i.e. declining, plateauing or increasing hormone levels), are associated with future reproductive success in juvenile gilts. Serum samples were obtained at 0, 2 and 4 days after injection and mating and litter data were collected until parity three. Results showed that, regardless of age group and parity, Day 0 E2 levels were positively associated with the probability of stillbirth (P = 0.035) and E2 levels on Day 0 (P = 0.032), Day 2 (P = 0.045) and Day 4 (P = 0.019) were negatively associated with the number of piglets born alive. Further, both a single measurement of serum AMH levels at Day 2 (P = 0.048) and the AMH response type were associated with gestation length (P = 0.012). These findings suggest that serum AMH and E2 levels can be used to inform the selection of gilts for the breeding herd.

Vitrification, not cryoprotectant exposure, alters the expression of developmentally important genes in in vitro produced porcine blastocysts.

The vitrification of embryos is common practice in advanced livestock breeding programs and in human fertility clinics. Recent studies have revealed that vitrification results in aberrant expression of a number of stress related genes. However, few studies have examined the effect that vitrification has on developmentally important genes, and none have been conducted in porcine embryos. The aim of this study was to determine the effects that different vitrification procedures and cryoprotectant combinations have on the expression of imprinted genes in in vitro produced (IVP) porcine blastocysts. The transcript levels of insulin-like growth factor 2 (IGF2) were lower in all groups of vitrified blastocysts compared to that in non-vitrified control blastocysts (P < 0.05). Expression levels of IGF2 and IGF2 receptor (IGF2R) in blastocysts that had been exposed to cryoprotectants without being vitrified were similar to that in non-vitrified control blastocysts (P > 0.05). Furthermore, blastocysts vitrified using ethylene glycol and propanediol combined, and those vitrified in a closed device, had IGF2R transcript levels similar to that in non-vitrified control blastocysts (P > 0.05). In conclusion, vitrification, but not exposure to cryoprotectants, caused aberrant expression of the imprinted genes IGF2 and IGF2R. Vitrification protocols that incorporated propanediol or a closed device were found to be least disruptive of gene expression in IVP porcine blastocysts.

From Peptide Masses to Pregnancy Maintenance: A Comprehensive Proteomic Analysis of The Early Equine Embryo Secretome, Blastocoel Fluid, and Capsule.

Early pregnancy in the mare is a poorly understood, high risk period during which the embryo communicates its presence to the maternal endometrium. Remarkably, the maternal recognition of pregnancy signal is unknown in the horse. This study aimed to profile the proteins secreted by equine blastocysts into their immediate environment, along with proteins contained in the blastocoel and within the acellular embryo capsule. Embryos were recovered on day 8 after ovulation and cultured for 48 hours. Secretomes of day 9 and day 10 embryos were analyzed by LC-MS/MS and supported by analysis of blastocoel fluid and embryo capsule. Analyses revealed 72 (24 h) and 97 (48 h) unique protein IDs in the embryo secretome, 732 protein IDs in blastocoel fluid, and 11 proteins IDs in the embryo capsule. Novel findings of interest include secretion of a pregnancy specific proteinase (PAG) by the equine embryo at day 10, along with detection of a prostaglandin receptor inhibiting protein (PTGFRN) and a progesterone potentiating factor (FKBP4) in blastocoel fluid. This is the first comprehensive proteomic analysis of the equine embryo secretome, and provides new insights into the unique physiology of early pregnancy in this species.

Cryotolerance of porcine blastocysts is improved by treating in vitro matured oocytes with L-carnitine prior to fertilization.

Sufficient generation of adenosine triphosphate (ATP) by oocytes is critical for fertilization and embryo development. The objective of this study was to determine the effects of supplementing media with L-carnitine, a co-factor required for the metabolism of fatty acids, during the peri-fertilization period on embryo development and energy generation. Firstly, in vitro matured (IVM) porcine oocytes were co-incubated with sperm in IVF medium supplemented with 0‒24 mM L-carnitine. The blastocyst formation rate of the control group was greater than those of the L-carnitine groups (P < 0.05), except for the 3 mM L-carnitine group. Subsequently, oocytes and/or sperm were treated without or with 3 mM L-carnitine for either the 1 h pre-IVF oocyte incubation; the pre-IVF sperm preparation; the first 30 min of IVF; or the entire 5.5 h of IVF. Despite similar fertilization rates among the groups, the cleavage rate of the pre-IVF oocyte group was significantly greater than those of the other groups, except for the pre-IVF sperm group. Additionally, the oocyte ATP content and the cryotolerance of the resulting blastocysts were examined following the pre-IVF oocyte treatment. Oocyte ATP content was also similar among the groups (P > 0.05). Following vitrification, the post-warming survival rate of blastocysts derived from L-carnitine-treated oocytes was greater than that of blastocysts derived from untreated oocytes (42.4% vs. 24.9%; P < 0.05). In conclusion, a 1 h oocyte exposure to 3 mM L-carnitine immediately prior to insemination enhanced cleavage and improved the cryotolerance of resulting blastocysts. While the findings are suggestive of a lipolytic action, further studies are required to clarify the contributions of lipid metabolism and oxidative mechanisms to the observed effects of the L-carnitine treatment.

Characterization of an L-amino acid oxidase in equine spermatozoa.

This study demonstrates for the first time the presence of an L-amino acid oxidase (LAAO) enzyme in equine spermatozoa that is able to generate significant amounts of reactive oxygen species (ROS) and create a state of oxidative stress. RT-PCR analysis revealed that the mRNA for this enzyme was present in the equine testis and spermatozoa, while immunocytochemical studies demonstrated that the mature LAAO protein was located in the sperm head, particularly in the acrosomal and postacrosomal domains. Experimental studies demonstrated that the aromatic amino acids (L-phenylalanine > L-tryptophan > L-tyrosine) were substrates for this enzyme, eliciting the dose- and time-dependent generation of ROS via mechanisms that were enhanced by cell death. This unexpected result was confirmed by analyses of ROS generation in subcellular sperm fractions, which again located a majority of LAAO activity to the sperm head. Equine cryopreservation medium was shown to contain sufficient quantities of aromatic amino acids to activate the LAAO system and generate ROS. The biological significance of this activity was established in an experiment in which physiological concentrations of aromatic amino acids were found to suppress sperm motility but only if dead spermatozoa were present in the same suspension. The combination of aromatic amino acids and nonviable cells was also found to enhance the levels of lipid peroxidation in live spermatozoa. These results suggest the potential significance of LAAO activity in generating the oxidative stress associated with the cryopreservation of equine spermatozoa. It is possible that inhibitors of this enzyme system may facilitate the development of modified cryostorage regimes for clinical validation in vivo.

Reproductive physiology and ovarian folliculogenesis examined via 1H-NMR metabolomics signatures: a comparative study of large and small follicles in three mammalian species (Bos taurus, Sus scrofa domesticus and Equus ferus caballus).

The aim of this study was to characterize the composition of follicular fluid (FF) collected from the small and large follicles of three mammalian species, Bos taurus, Sus scrofa domesticus, and Equus ferus caballus, that display distinct ovulatory properties. For each species, five large FF samples and five small FF samples were analyzed using 1H-NMR spectroscopy. The FF metabolic profiles of the three species were very distinct. In cows and mares, the metabolic profiles of large FF and small FF were also very distinct. The concentrations of seventeen identified metabolites differed significantly between the sample groups. In mares, fourteen metabolites were found at much greater concentrations in large FF than in small FF (p<0.05). In cows, four metabolites differed in concentration between the large FF and small FF samples (p<0.05). A common feature of the monovulatory species was that the concentrations of α- and ß-glucose were much greater in large FF compared with small FF (p<0.05). Sow FF was characterized by the apparent absence of citrate (detected in cow and mare FF), and the presence of succinate (not detected in cow and mare FF). Another obvious difference between species was the concentration of lactate, which was minimal in mare FF compared with cow and sow FF (p<0.05). The findings provide valuable insights into reproductive physiology broadly, and indicate that the activities of central metabolic enzymes differ enormously between these species. Future investigations into species-specific differences in follicle metabolism would increase our understanding of the processes critical to folliculogenesis and the acquisition of oocyte developmental competence.