Twin management in the mare: A review.

Twin gestation in the mare is undesirable and can have disastrous consequences. As in many cases, the key to success in twin management lies in a thorough follow-up and accurate recording of clinical findings in the pre-breeding examination. A pregnancy diagnosis in the mobility phase is imperative for a good outcome in the event of twin reduction. If a twin gestation is not diagnosed during this early pregnancy stage, several other procedures exist for managing post-fixation twins (>16 days) with varying degrees of success. Most twin pregnancies are the result of multiple ovulations (dizygotic twins). However, monozygotic twins are also sporadically diagnosed, due to the increasing number of transferred in vitro produced equine embryos. In these cases, the most optimal treatment strategy still needs to be determined. This review provides an overview of the various twin reduction techniques described with the expected prognosis as well as of some less reported techniques with their results. In addition, physiological events and the reduction techniques are demonstrated to the user in virtual 3-dimensional illustrations.

Automatic early detection of induced colic in horses using accelerometer devices.

BACKGROUND: To seek appropriate veterinary attention for horses with colic, owners must recognise early signs. Direct observation of horse behaviour has several drawbacks: it is time-consuming, hard to see subtle and common behavioural signs, and is based on intuition and subjective decisions. Due to recent advances in wearables and artificial intelligence, it may be possible to develop diagnostic software that can automatically detect colic signs. OBJECTIVES: To develop a software algorithm to aid in the detection of colic signs and levels of pain. STUDY DESIGN: In vivo experiments. METHODS: Transient colic was induced in eight experimental mares with luteolytic doses of prostaglandin. Veterinarians observed the horses before and throughout the interventions and assigned pain scores which were used to separate colic episodes into none (pain score ≤5), level 1 (pain score 6-10) or level 2 (pain score ≥11). Accelerometric data and videos were collected throughout the experiments and using accelerometric data, the horse's behaviour was classified into normal and 10 pain-related behaviours and an activity index was calculated. Models were designed that utilised behaviour and activity index characteristics both detecting the presence of colic and assessing its severity. To determine the accuracy of the model, the ground truth, that is the veterinarians' observation of colic signs and assessment of pain level, was compared with the automatic detection system. RESULTS: The cross-validation analysis demonstrated an accuracy of 91.2% for detecting colic and an accuracy of 93.8% in differentiating between level 1 colic and level 2 colic. The model was able to accurately classify 10 pain-related behaviours and distinguish them from normal behaviour with a high accuracy. MAIN LIMITATIONS: We included a limited number of horses with severe pain related behaviours in the dataset. This constraint affects the accuracy of categorising colic severity rather than limiting the algorithms' capacity to identify early colic signs. CONCLUSIONS: Our system for early detection of colic in horses is unique and innovative, and it can distinguish between colic of varying severity.

Anti-Müllerian Hormone and OPU-ICSI Outcome in the Mare.

Anti-Müllerian hormone (AMH) reflects the population of growing follicles and has been related to mammalian fertility. In the horse, clinical application of ovum pick-up and intracytoplasmic sperm injection (OPU-ICSI) is increasing, but results depend largely on the individuality of the mare. The aim of this study was to assess AMH as a predictor for the OPU-ICSI outcome in horses. Therefore, 103 mares with a total follicle count above 10 were included in a commercial OPU-ICSI session and serum AMH was determined using ELISA. Overall, the AMH level was significantly correlated with the number of aspirated follicles and the number of recovered oocytes (p < 0.001). Mares with a high AMH level (≥2.5 µg/L) yielded significantly greater numbers of follicles (22.9 ± 1.2), oocytes (13.5 ± 0.8), and blastocysts (2.1 ± 0.4) per OPU-ICSI session compared to mares with medium (1.5-2.5 µg/L) or low AMH levels (<1.5 µg/L), but no significant differences in blastocyst rates were observed. Yet, AMH levels were variable and 58% of the mares with low AMH also produced an embryo. In conclusion, measurement of serum AMH can be used to identify mares with higher chances of producing multiple in vitro embryos, but not as an independent predictor of successful OPU-ICSI in horses.

The Effects of Red Light on Mammalian Sperm Rely upon the Color of the Straw and the Medium Used.

Previous research has determined that irradiation of mammalian sperm with red light increases motility, mitochondrial activity, and fertilization capacity. In spite of this, no study has considered the potential influence of the color of the straw and the extender used. Therefore, this study tests the hypothesis that the response of mammalian sperm to red light is influenced by the color of the straw and the turbidity/composition of the extender. Using the horse as a model, 13 ejaculates from 13 stallions were split into two equal fractions, diluted with Kenney or Equiplus extender, and stored at 4 °C for 24 h. Thereafter, each diluted fraction was split into five equal aliquots and subsequently packed into 0.5-mL straws of red, blue, yellow, white, or transparent color. Straws were either nonirradiated (control) or irradiated with a light-dark-light pattern of 3-3-3 (i.e., light: 3 min, dark: 3 min; light: 3 min) prior to evaluating sperm motility, acrosome and plasma membrane integrity, mitochondrial membrane potential, and intracellular ROS and calcium levels. Our results showed that irradiation increased some motion variables, mitochondrial membrane potential, and intracellular ROS without affecting the integrities of the plasma membrane and acrosome. Remarkably, the extent of those changes varied with the color of the straw and the extender used; the effects of irradiation were more apparent when sperm were diluted with Equiplus extender and packed into red-colored straws or when samples were diluted with Kenney extender and packed into transparent straws. As the increase in sperm motility and intracellular ROS levels was parallel to that of mitochondrial activity, we suggest that the impact of red light on sperm function relies upon the specific rates of energy provided to the mitochondria, which, in turn, vary with the color of the straw and the turbidity/composition of the extender.

Red LED Light Acts on the Mitochondrial Electron Chain of Donkey Sperm and Its Effects Depend on the Time of Exposure to Light.

This work aimed to investigate how stimulation of donkey sperm with red LED light affects mitochondrial function. For this purpose, freshly diluted donkey semen was stimulated with red light for 1, 5, and 10 min, in the presence or absence of oligomycin A (Omy A), a specific inhibitor of mitochondrial ATP synthase, or FCCP, a specific disruptor of mitochondrial electron chain. The results obtained in the present study indicated that the effects of red LED light on fresh donkey sperm function are related to changes in mitochondria function. In effect, irradiation of donkey sperm resulted in an increase in mitochondrial membrane potential (MMP), the activity of cytochrome C oxidase and the rate of oxygen consumption. In addition, in the absence of oligomycin A and FCCP, light-stimulation augmented the average path velocity (VAP) and modified the structure of motile sperm subpopulations, increasing the fastest and most linear subpopulation. In contrast, the presence of either Omy A or FCCP abolished the aforementioned effects. Interestingly, our results also showed that the effects of red light depend on the exposure time applied, as indicated by the observed differences between irradiation protocols. In conclusion, our results suggest that exposing fresh donkey sperm to red light modulates the function of their mitochondria through affecting the activity of the electron chain. However, the extent of this effect depends on the irradiation pattern and does not exclude the existence of other mechanisms, such as those related to thermotaxis.

Single Layer Centrifugation Improves the Quality of Fresh Donkey Semen and Modifies the Sperm Ability to Interact with Polymorphonuclear Neutrophils.

This study sought to determine whether single layer centrifugation (SLC) of fresh donkey semen with Equicoll has any impact on sperm quality parameters and on the modulation of endometrial reaction following semen deposition using an in vitro model. Seventeen ejaculates from five jackasses were obtained using an artificial vagina and diluted in a skim-milk extender. Samples were either selected through SLC (Equicoll) or non-treated (control). Two experiments were performed. The first one consisted of incubating selected or non-selected spermatozoa at 38 °C for 180 min. Integrity and lipid disorder of sperm plasma membrane, mitochondrial membrane potential, and intracellular levels of calcium and reactive oxygen species were evaluated at 0, 60, 120, and 180 min. In the second experiment, polymorphonuclear neutrophils (PMN) isolated from jennies blood were mixed with selected and unselected spermatozoa. Interaction between spermatozoa and PMN was evaluated after 0, 60, 120, and 180 min of co-incubation at 38 °C. SLC-selection increased the proportions of spermatozoa with an intact plasma membrane and low lipid disorder, of spermatozoa with high mitochondrial membrane potential and with high calcium levels, and of progressively motile spermatozoa. In addition, selection through SLC augmented the proportion of phagocytosed spermatozoa, which supported the modulating role of seminal plasma proteins on sperm-PMN interaction. In conclusion, SLC of fresh donkey semen increases the proportions of functionally intact and motile spermatozoa, and appears to remove the seminal plasma proteins that inhibit sperm-PMN binding.

Red-Light Irradiation of Horse Spermatozoa Increases Mitochondrial Activity and Motility through Changes in the Motile Sperm Subpopulation Structure.

Previous studies in other mammalian species have shown that stimulation of semen with red-light increases sperm motility, mitochondrial activity, and fertilizing capacity. This study sought to determine whether red-light stimulation using a light emitting diode (LED) at 620-630 nm affects sperm motility and structure of motile subpopulations, sperm viability, mitochondrial activity, intracellular ATP levels, rate of O2 consumption and DNA integrity of horse spermatozoa. For this purpose, nine ejaculates were collected from nine different adult stallions. Upon collection, semen was diluted in Kenney extender, analyzed, its concentration was adjusted, and finally it was stimulated with red-light. In all cases, semen was packaged in 0.5-mL transparent straws, which were randomly divided into controls and 19 light-stimulation treatments; 6 consisted of a single exposure to red-light, and the other 13 involved irradiation with intervals of irradiation and darkness (light-dark-light). After irradiation, sperm motility was assessed using a Computerized Semen Analysis System (CASA). Flow cytometry was used to evaluate sperm viability, mitochondrial membrane potential and DNA fragmentation. Intracellular levels of ATP and O2 consumption rate were also determined. Specific red-light patterns were found to modify kinetics parameters (patterns: 4, 2-2-2, 3-3-3, 4-4-4, 5-1-5, and 5-5-5 min), the structure of motile sperm subpopulations (patterns: 2, 2-2-2, 3-3-3, and 4-1-4 min), mitochondrial membrane potential (patterns: 4, 3-3-3, 4-4-4, 5-1-5, 5-5-5, 15-5-15, and 15-15-15 min), intracellular ATP levels and the rate of O2 consumption (pattern: 4 min), without affecting sperm viability or DNA integrity. Since the increase in some kinematic parameters was concomitant with that of mitochondrial activity, intracellular ATP levels and O2 consumption rate, we suggest that the positive effect of light-irradiation on sperm motility is related to its impact upon mitochondrial activity. In conclusion, this study shows that red LED light stimulates motility and mitochondrial activity of horse sperm. Additional research is needed to address the impact of red-light irradiation on fertilizing ability and the mechanisms through which light exerts its effects.

Irradiating frozen-thawed stallion sperm with red-light increases their resilience to withstand post-thaw incubation at 38 °C.

The aim of this study was to evaluate whether red-light stimulation increases the longevity and resilience of cryopreserved stallion sperm to withstand post-thaw incubation for 120 min. Sixteen frozen straws of 0.5 mL from eight stallions were used. Samples were cryopreserved, thawed through incubation at 38 °C for 30 s and divided into the control and samples exposed to red-light using a triple LED photo-activation system (wavelength: 620-630 nm). Three irradiation protocols consisting of different light-dark-light intervals (1-1-1, 2-2-2 and 3-3-3 min) were tested. Sperm quality parameters were analyzed immediately after light-stimulation (0 min) and after 120 min of incubation at 38 °C. Sperm motility was evaluated using a Computerized Semen Analysis System (CASA), and flow cytometry and different fluorochromes were used to evaluate the integrity and lipid disorder of plasma membrane, mitochondrial membrane potential and intracellular levels of peroxides and superoxides. Irradiation significantly increased the percentages of spermatozoa with high mitochondrial membrane potential (1-1-1 pattern) and the intracellular levels of peroxides (2-2-2 pattern) at 0 min. In addition, sperm kinematic parameters (2-2-2 and 3-3-3 patterns) and percentages of viable spermatozoa with low membrane lipid disorder (3-3-3 pattern) were significantly higher in irradiated samples than in the control at 120 min. Our results indicate that red-light stimulation could help increase the resilience of frozen-thawed stallion sperm to withstand post-thaw incubation at 38 °C for 120 min and that these effects rely on the irradiation pattern. Further research should evaluate whether light-stimulation could also have a positive on fertility rates after artificial insemination.

Seminal Plasma, Sperm Concentration, and Sperm-PMN Interaction in the Donkey: An In Vitro Model to Study Endometrial Inflammation at Post-Insemination.

In the donkey, artificial insemination (AI) with frozen-thawed semen is associated with low fertility rates, which could be partially augmented through adding seminal plasma (SP) and increasing sperm concentration. On the other hand, post-AI endometrial inflammation in the jenny is significantly higher than in the mare. While previous studies analyzed this response through recovering Polymorphonuclear Neutrophils (PMN) from uterine washings, successive lavages can detrimentally impact the endometrium, leading to fertility issues. For this reason, the first set of experiments in this work intended to set an in vitro model through harvesting PMN from the peripheral blood of jennies. Thereafter, how PMN, which require a triggering agent like formyl-methionyl-leucyl-phenylalanine (FMLP) to be activated, are affected by donkey semen was interrogated. Finally, we tested how four concentrations of spermatozoa (100 × 106, 200 × 106, 500 × 106 and 1000 × 106 spermatozoa/mL) affected their interaction with PMN. We observed that semen, which consists of sperm and SP, is able to activate PMN. Whereas there was a reduced percentage of spermatozoa phagocytosed by PMN, most remained attached on the PMN surface or into a surrounding halo. Spermatozoa not attached to PMN were viable, and most of those bound to PMN were also viable and showed high tail beating. Finally, only sperm concentrations higher than 500 × 106 spermatozoa/mL showed free sperm cells after 3 h of incubation, and percentages of spermatozoa not attached to PMN were higher at 3 h than at 1 h, exhibiting high motility. We can thus conclude that semen activates PMN in the donkey, and that the percentage of spermatozoa phagocytosed by PMN is low. Furthermore, because percentages of spermatozoa not attached to PMN were higher after 3 h than after 1 h of incubation, we suggest that PMN-sperm interaction plays an instrumental role in the reproductive strategy of the donkey.

Effects of red-light irradiation on the function and survival of fresh and liquid-stored donkey semen.

This study sought to determine whether sperm irradiation using a light emission diode (LED) at 620-630 nm affects the motility, membrane integrity (viability), mitochondrial activity and intracellular levels of reactive oxygen species (ROS) in fresh diluted and liquid-stored donkey semen. With this purpose, sixteen ejaculates (eight fresh diluted and eight cooled-stored) were collected from eight adult jackasses. Fresh semen samples were diluted in Kenney extender and stimulated with red-light after collection, whereas cooled semen was stored at 4 °C for 24 h after dilution and then irradiated. In all cases, semen samples were packed into 0.5-mL transparent straws, which were then randomly divided into control and 19 treatments: six consisted of single red-light exposure, and the other 13 involved irradiation at light-dark-light intervals. Upon irradiation, sperm motility, membrane integrity mitochondrial membrane potential (MMP) and intracellular levels of superoxide anion (·O2-) and hydrogen peroxide (H2O2) were evaluated. While specific light-patterns increased both sperm motility and mitochondrial activity, they did not affect sperm membrane integrity and had no clear impact on intracellular ROS levels. The effects of irradiation patterns differed between fresh and cooled semen since, whereas 1 and 4 min patterns induced the greatest increments in the total and progressive motility of fresh semen, 4 min, 4-1-4 and 4-4-4 were the most suitable for cooled-stored samples. In both fresh diluted and cooled-stored semen, the motility increase observed after light-stimulation for 4 min was concomitant with changes in the percentages of spermatozoa with high mitochondrial membrane potential. In summary, this study shows, for the first time, that specific irradiation patterns increase sperm motility and mitochondrial activity in the donkey. Furthermore, the precise effect of red-light appears to depend on the specific functional status of cells, with separate effects on fresh and cooled samples.

Seminal plasma has limited counteracting effects following induction of oxidative stress in donkey spermatozoa.

The aim of this study was to evaluate the response of donkey spermatozoa to oxidative stress induced by hydrogen peroxide, and to determine whether the presence of seminal plasma modulates the sperm response to that stress. Nine ejaculates were collected, extended in skim milk extender and split into two aliquots. Seminal plasma was removed from the first but not second aliquot. Samples were subsequently split into four aliquots supplemented with different concentrations of commercial hydrogen peroxide (0, 100 and 250µM and 50mM). Aliquots were incubated at 37°C under aerobic conditions and several sperm parameters, namely motility, viability, intracellular levels of peroxides and superoxides and mitochondrial membrane potential, were evaluated at 0, 1 and 3h. Exposure to hydrogen peroxide markedly decreased sperm motility but had much less of an effect on sperm viability, mitochondrial membrane potential and intracellular reactive oxygen species levels. A protective effect of seminal plasma against the loss of sperm motility was not apparent, but some kinetic parameters and relative levels of superoxides were better maintained when seminal plasma was present together with high concentration of hydrogen peroxide. In conclusion, oxidative stress induced by hydrogen peroxide reduces donkey sperm motility and has a less apparent effect on other sperm parameters. Finally, seminal plasma is only able to partially ameliorate the detrimental effect of this induced stress.

Examination of jackass (Equus asinus) accessory sex glands by B-mode ultrasound and of testicular artery blood flow by colour pulsed-wave Doppler ultrasound: Correlations with semen production.

The accessory sex glands play a major role in the production of seminal plasma, and testicular artery blood flow seems to strongly influence testicular function. However, very little ultrasound imaging of these organs has been undertaken in donkeys. The present work reports the results of such examinations in five jackasses along the year. The accessory glands were inspected by B-mode ultrasound while the testicular artery blood flow was assessed by colour pulsed-wave Doppler ultrasound. The testicular artery was examined at pampiniform plexus (PPT), supratesticular area (ST) and capsular artery (CA). Values were recorded for the total arterial blood flow (TABF), peak systolic velocity (PSV), end-diastolic velocity (EDV), resistive index (RI), pulsatility index (PI) and time average maximum velocity (TAMV). Semen was obtained and assessed for sperm concentration, viability, abnormalities and motility using a CASA system. The bulbourethral glands, prostate and ductus deferens ampullae were relatively larger than in the stallion. Bulbourethral glands and ampullae sizes were inversely correlated with sperm motility. An reduction in blood flow between the level the PPP and the CA was observed, helping to reduce testis temperature and oxygen pressure. Blood flow at the CA showed the strongest correlation with semen production. PI and RI were positively correlated with the CASA motility variable STR (p = .02, p = .06) and sperm viability (p = .01), while sperm concentration (p = .05) correlated inversely with PSV, EDV, TAMV and TABF. EDV also correlated negatively with the CASA variables VSL, LIN, STR and VAP (p ≤ .05). PI and RI were also negatively correlated with testis length (p = .0093, p = -.0438).

Total and specific activities of superoxide dismutase (SOD) in seminal plasma are related with the cryotolerance of jackass spermatozoa.

This study investigated whether the activities of four antioxidant enzymes present in jackass seminal plasma (SP), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) and glutathione reductase (GSR), are related to the sperm ability to withstand cryopreservation. Eighteen ejaculates from 16 healthy jackasses were collected and split into two aliquots. The first one was centrifuged (3,000×g, 4 °C for 10 min) and used to determine the activities of these four enzymes in SP, whereas the other was diluted in a skim-milk extender and then cryopreserved. Assessment of sperm motility and membrane integrity was performed before and after cryopreservation. Based on the percentages of total motile and viable spermatozoa at post-thaw, samples were classified as good (GFE) or poor (PFE) freezability ejaculates through cluster analyses. Total and specific activities of SOD in seminal plasma were higher (P < 0.05) in GFE than in PFE, whereas no significant differences between GFE and PFE were observed regarding total and specific activities of CAT, GPX and GSR. However, post-thaw sperm parameters were positively correlated with total and specific activities of CAT and negatively correlated with those of GSR. In conclusion, determination of total and specific activities of SOD in the seminal plasma of a given jackass ejaculate may predict the sperm ability to withstand cryopreservation. In addition, our results warrant further research on addressing whether SOD activity in seminal plasma does not only allow predicting the sperm cryotolerance of a given ejaculate but also that of all ejaculates from a given jackass.

Cryotolerance of Stallion Spermatozoa Relies on Aquaglyceroporins rather than Orthodox Aquaporins.

Aquaporins (AQPs), a family of ubiquitous water channels divided into orthodox AQPs, aquaglyceroporins (GLPs), and superAQPs, are present in stallion spermatozoa. The aim of this study was to elucidate the functional relevance of each group of AQPs during stallion sperm cryopreservation through the use of three different inhibitors: acetazolamide (AC), phloretin (PHL) and propanediol (PDO). Sperm quality and function parameters were evaluated in the presence or absence of each inhibitor in fresh and frozen-thawed samples. In the presence of AC, different parameters were altered (p < 0.05), but not in a concentration- or time-depending manner. PHL was found to decrease sperm motility, viability, acrosome integrity, and the percentages of spermatozoa with low membrane lipid disorder, high mitochondrial membrane potential (MMP) and high intracellular levels of calcium and superoxides (p < 0.05). Finally, the sperm motility, viability, acrosome integrity, the percentages of spermatozoa with low membrane lipid disorder, high MMP and high intracellular calcium levels were higher (p < 0.05) in PDO treatments than in the control. The sperm response to AC, PHL and PDO indicates that GLPs, rather than orthodox AQPs, play a crucial role during stallion sperm cryopreservation. Furthermore, post-thaw sperm quality was higher in PDO treatments than in the control, suggesting that this molecule is a potential permeable cryoprotectant.

Specific Activity of Superoxide Dismutase in Stallion Seminal Plasma Is Related to Sperm Cryotolerance.

While the removal of seminal plasma is a routine practice prior to equine sperm cryopreservation, this fluid contains the main source of antioxidant enzymes able to scavenge these reactive oxygen species. Therefore, stallion seminal plasma components may have an impact on ejaculate freezability. Against this background, this study was designed to investigate whether the activities of the main stallion seminal plasma antioxidant enzymes are related to sperm cryotolerance. With this purpose, 16 ejaculates were collected from 14 healthy stallions, and each ejaculate was split into two aliquots. The first one was used to evaluate the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione reductase (GSR) in seminal plasma. The second aliquot was extended and then processed for cryopreservation. Sperm motility and viability were evaluated before and after cryopreservation, and ejaculates were classified as of good (GFE) or poor freezability (PFE) based on total motile and viable spermatozoa at post-thaw. We observed that, while the specific activities of CAT, GPX, and GSR were similar between GFE and PFE, that of SOD was significantly (p < 0.05) higher in GFE than in PFE. We can thus conclude that, in stallions, the specific activity of SOD in the seminal plasma of a given ejaculate might be related to its freezability.

Activities of antioxidant seminal plasma enzymes (SOD, CAT, GPX and GSR) are higher in jackasses than in stallions and are correlated with sperm motility in jackasses.

This study compared the activities of four antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT; glutathione peroxidase, GPX; and glutathione reductase, GSR) in the seminal plasma of stallions and jackasses. Eighteen stallion ejaculates and 24 jack ejaculates were collected through an artificial vagina. Seminal plasma was obtained by several centrifugations at 3000×g and 4 °C for 10 min, and activities of SOD, CAT, GPX and GSR were subsequently determined. We also evaluated whether the collecting season had any influence on the activities of these four enzymes in both stallions and jackasses. Antioxidant capacity of seminal plasma was significantly higher in jackasses than in stallions (mean ±â€¯SEM, SOD: 1707.7 ±â€¯195.9 U/mL vs. 231.9 ±â€¯29.6 U/mL; CAT: 9094.7 ±â€¯1292.9 U/L vs.1682.7 ±â€¯525.9 U/L; GPX 845.4 ±â€¯106.0 U/L vs. 469.7 ±â€¯60.3 U/L; GSR: 50.3 ±â€¯5.1 U/L vs. 20.7 ±â€¯4.6 U/L). Furthermore, whereas season had no effect on the activity of these four enzymes in stallions, the activities of CAT and GPX in jack seminal plasma were significantly higher in the summer than in the other seasons. In addition, the activities of SOD and CAT were found to be significantly correlated with the percentages of progressively motile spermatozoa, and with the percentages of linearity and straightness, respectively, in jackasses. In contrast, the activities of these four enzymes were not correlated with sperm quality parameters in stallions. Finally, while SOD, CAT, and GPX activities but not those of GSR were correlated in jackasses, the activities of all four enzymes were correlated each other in stallions. We can thus conclude that the activities of SOD, CAT, GPX and GSR differ between the seminal plasma of stallions and donkeys, and vary between seasons in jackasses.