Occurrence of ultrasonographic assessed placental abnormalities, treatments, pregnancy outcome, and subsequent fertility on a large warmblood stud farm: A retrospective field study.

Little is known about the incidence and outcome of high-risk pregnancies in equine practice and clinical studies on spontaneous occurring placentitis cases and treatments are missing. Therefore, the aims of this retrospective field study were to (1) describe the incidence and severity of ultrasonographic assessed placental abnormalities (UPA) in 4,192 pregnancies on a large commercial warmblood stud farm in 2017 - 2019 and (2) characterize these UPA cases and their pregnancy outcome. UPA severity (Placental abnormality score (PSc) 1-3; low to high), nine treatment regimens (TM1-9) used in UPA cases and treatment duration as well as subsequent fertility were analyzed in the group of UPA mares. The proportion of pregnancies affected by UPA was 4.2 % (n=177/4192). Placental abnormality severity was scored as PSc1 (51.4 %), PSc2 (32.8 %) and PSc3 (15.8 %). The generalized mixed model revealed PSc was affected by mare age and mare status (own pregnancy (OP) or embryo transfer recipient (ER)) (P=0.035) with ER mares having increased PSc compared with mares having their own pregnancy. Abortion occurred in 17/177 (9.6 %) UPA pregnancies. Overall, at the end of the next season, 61.1 % of UPA mares were pregnant, 32.0 % barren, and 6.9 % open (n=175). Pregnancy was established in 62/91(68.1 %) of mares with PSc1, 31/58 (53.4 %) with PSc2 and 14/26 (53.8 %) with PSc3. Most pregnancies were achieved in the first 81/107 (75.7 %) or second 18/107 (16.8 %) inseminated cycle. In conclusion, early detection and treatment of ultrasonographic assessed placental abnormalities can save high-risk pregnancies in > 90 % of cases with a satisfying subsequent fertility.

No increase in pregnancy rate of mares after preovulatory deep uterine horn application of misoprostol.

A potential source of fertility loss in mares is oviductal dysfunction, potentially caused by masses or debris in the lumen, that may prevent either sperm from reaching the fertilization site or the embryo from reaching the uterus. Recently a novel therapeutic method leading to increased pregnancy results was described by infusing misoprostol, a synthetic prostaglandin E1, in the uterus of mares with unexplained fertility problems. In this study, we aimed, after examining the compatibility of misoprostol with semen, to evaluate the pregnancy rate after routine preovulatory deep uterine horn application of misoprostol in clinically normal oestrous mares, which were inseminated in the same cycle. In experiment 1, ejaculates of 10 stallions diluted with INRA 96™ were mixed with different concentrations of misoprostol (0.01 mg/mL, 0.001 mg/mL, 0.0001 mg/mL, and 0.00001 mg/mL) and total semen motility was evaluated immediately, 12, 24, 48, and 72 h later, and compared with a control sample (mixed with NaCl 0.9%). In experiments 2 and 3, 33 privately-owned clinically normal oestrous mares were each allocated to a treatment or control group. Ovulation was then induced with intramuscularly 2.25 mg deslorelin acetate. At the moment of ovulation induction (experiment 2) and 24 h earlier (experiment 3), 0.2 mg misoprostol diluted in 2 mL NaCl 0.9% were applied deep in the uterine horn (treatment groups) and pure 2 mL NaCl 0.9% in the mares of the control groups. Mares were then inseminated 24 h after deslorelin administration and prior to ovulation with commercial chilled-warmed or frozen-thawed semen, as well as immediately after ovulation detection (both types of semen) maximally 48 h after ovulation induction. In experiment 1, regardless of time and compared with the control groups, all solutions with different concentrations of misoprostol had a negative effect on total motility of semen, which was significant for the highest concentrations (0.01 mg/mL: 18.0% reduction, CI = 22-13%, p = < 0.01). We found no beneficial effect of preovulatory uterine treatment with misoprostol on pregnancy rate (OR = 0.45, CI = 0.15-1.31, p = 0.14): in experiment 2, 2/11 (18.2%) mares of the treatment group became pregnant vs. 12/22 (54.5%) mares in the control group (OR = 0.19, CI = 0.03-1.06, p = 0.07), in experiment 3, 5/14 (35.7%) mares in the treatment group vs. 7/19 (36.8%) mares in the control group (OR = 0.95, CI = 0.23-4.02, p = 0.95), respectively. In conclusion, pregnancy rate was not increased in reproductively normal mares with routine preovulatory deep uterine horn application of misoprostol.

Assessment of anti-Müllerian hormone in mares' transitional period and in relation to fertility in elderly mares.

Anti-Müllerian Hormone (AMH) has been linked to reproductive tract abnormalities in mares and stallions. This study aimed at evaluating AMH as a biomarker for two reproductive conditions in mares. In the first part of this study, plasma AMH was evaluated as an early indicator of the onset of cyclicity in mares in the transitional period from the anovulatory phase during winter anoestrus to the cyclic phase during the breeding season. Ten mares between 8 and 17 years old were included in the experiment which lasted from mid-February until the end of April. Ovarian activity was monitored with ultrasonography three times per week, the detection of a corpus haemorrhagicum/luteum was documented and antral follicle counts (AFC) were recorded. Blood samples were collected weekly by jugular venipuncture during the whole study period to compare AMH concentrations before and after the first ovulation of the year. The second objective was to evaluate if plasma AMH concentrations in middle-aged mares are linked to fertility and could serve as a prognostic marker in that age group. A total of 41 privately-owned clinically sound mares aged between 12 and 21 years of various breeds were inseminated with fresh or frozen semen. Mares were scanned between day 14 and 20 and the "early pregnancy rate" included only positive pregnancy examinations after the first observed cycle in the season of each mare. Potential associations between the early pregnancy rate in the first cycle and the explanatory factors AMH concentrations, age, status of the mare, stud, development of post-breeding endometritis, number of inseminations and semen type were analysed using logistic regression models. In the first part of the study, correlation between AMH and AFC for the whole study period (P = 0.0002, ρ = 0.55) as well as prior to (P = 0.008, ρ = 0.58) and after the first ovulation (P = 0.0007, ρ = 0.69) were observed. However, AMH concentrations before and after the first ovulation of the year were not statistically different. The second part of the study revealed no association between early pregnancy rate and AMH concentrations or any of the other mentioned factors. In conclusion, this study showed no evidence of a difference between AMH concentrations before and after the first ovulation of the year thus not supporting the use of AMH as a biomarker to predict the onset of cyclicity in mares. We could furthermore not show a relationship between plasma AMH concentrations and early pregnancy rates in this cohort of animals.

Embryo survival in the oviduct not significantly influenced by major histocompatibility complex social signaling in the horse.

The major histocompatibility complex (MHC) influences sexual selection in various vertebrates. Recently, MHC-linked social signaling was also shown to influence female fertility in horses (Equus caballus) diagnosed 17 days after fertilization. However, it remained unclear at which stage the pregnancy was terminated. Here we test if MHC-linked cryptic female choice in horses happens during the first days of pregnancy, i.e., until shortly after embryonic entrance into the uterus and before fixation in the endometrium. We exposed estrous mares to one of several unrelated stallions, instrumentally inseminated them with semen of another stallion, and flushed the uterus 8 days later to test for the presence of embryos. In total 68 embryos could be collected from 97 experimental trials. This success rate of 70.1% was significantly different from the mean pregnancy rate of 45.7% observed 17 days after fertilization using the same experimental protocol but without embryo flushing. Embryo recovery rate was not significantly dependent on whether the mares had been socially exposed to an MHC-dissimilar or an MHC-similar stallion. These observations suggest that MHC-linked maternal strategies affect embryo survival mainly (or only) during the time of fixation in the uterus.

Genetic parameters and estimated breeding values for traits of raw and frozen-thawed semen in German Warmblood stallions.

Objectives of the present study were to estimate genetic parameters for frozen-thawed semen traits of 271 fertile German Warmblood stallions and genetic correlations with raw semen quality traits. Semen samples were collected from stallions utilized for semen collection and artificial insemination (AI) on the Lower Saxon National Stud Celle and the North Rhine-Westphalian National Stud Warendorf. Semen quality variables were analyzed in 63,972 raw (gel-free volume, concentration, progressive motility, number of sperm) and 3681 frozen-thawed samples (motility, DNA fragmentation index (DFI), non-viable sperm). A multivariate linear animal model was used to estimate additive genetic and permanent environmental variances among stallions as well as estimated breeding values (EBVs) for all semen traits. Heritability estimates were greatest for DFI (h2 = 0.45) and least for non-viable sperm counts ( h2 = 0.11). Additive genetic correlations between progressive sperm motility in raw semen and DFI (rg = -0.79) as well as non-viable sperm (rg = -0.45) were negative. The EBVs for frozen-thawed semen traits ranged from 49 to 181 with mean reliabilities of 0.28 to 0.43. The EBVs for progressively motile sperm post-thawing and DFI were the most highly correlated traits with EBVs for stallion fertility (r = 0.38 and r = -0.17). Stallions with relatively greater EBVs for progressive motility in raw semen may be most suitable when freezing semen for storage and subsequently thawing it for AI. Using EBVs for semen traits in selection of stallions to AI mares appears as an option for genetic improvement to enhance fertility after AI.

Stallion semen quality depends on major histocompatibility complex matching to teaser mare.

The major histocompatibility complex (MHC) has repeatedly been found to influence mate choice of vertebrates, with MHC-dissimilar mates typically being preferred over MHC-similar mates. We used horses (Equus caballus) to test whether MHC matching also affects male investment into ejaculates after short exposure to a female. Semen characteristics varied much among stallions. Controlling for this variance with a full-factorial within-subject experimental design, we found that a short exposure to an MHC-dissimilar mare enhanced male plasma testosterone and led to ejaculates with elevated sperm numbers as compared to exposure to an MHC-similar mare. Sperm velocity seemed not affected by the treatment. Overall genetic similarity between stallions and mares (determined from polymorphic microsatellites on 20 different chromosomes) played no significant role here. The MHC type of the teaser mare also affected characteristics of cold-stored sperm after 24 and 48 hr. As expected from ejaculate economics, sperm viability was elevated after exposure to an MHC-dissimilar mare. However, oxidative stress and the percentage of sperm with a high DNA fragmentation were mostly increased after exposure to an MHC-dissimilar mare, depending also on whether the teaser mare was in oestrous or not. We conclude that males can quickly adjust ejaculate quality relative to a female's MHC, and that this male reaction to the social environment can also affect important characteristics of cold-stored semen.

Major histocompatibility complex-linked social signalling affects female fertility.

Genes of the major histocompatibility complex (MHC) have been shown to influence social signalling and mate preferences in many species, including humans. First observations suggest that MHC signalling may also affect female fertility. To test this hypothesis, we exposed 191 female horses (Equus caballus) to either an MHC-similar or an MHC-dissimilar stimulus male around the time of ovulation and conception. A within-subject experimental design controlled for non-MHC-linked male characteristics, and instrumental insemination with semen of other males (n = 106) controlled for potential confounding effects of semen or embryo characteristics. We found that females were more likely to become pregnant if exposed to an MHC-dissimilar than to an MHC-similar male, while overall genetic distance to the stimulus males (based on microsatellite markers on 20 chromosomes) had no effect. Our results demonstrate that early pregnancy failures can be due to maternal life-history decisions (cryptic female choice) influenced by MHC-linked social signalling.

Testicular volumetry and prediction of daily sperm output in stallions by orchidometry and two- and three-dimensional sonography.

Accurate determination of the testes volume and prediction of the daily sperm output (DSO) is valuable information for reproductive management of a stallion. The aim of this study was to compare different methods for measuring the testes volume, including caliper, 2D and 3D ultrasound. Special emphasis was on feasibility of 3D volume analysis. First, 22 castrated testes were measured and derived volumes were compared with volumes determined via volume displacement in a graded cylinder with saline solution. Then, during the breeding season, testes sizes of 52 stallions were measured in vivo and analyzed. With the derived volumes, predicted DSO (pDSO) values were calculated which were compared with actual values (aDSO) determined from semen evaluation. Analyses of castrated testes revealed a discrepancy between volume assessments via the caliper and ultrasound methods and actual volumes as found via volume displacement. The smallest difference was found for 3D volume analysis, followed by caliper and 2D ultrasound. Testicular volumes of breeding stallions were highest if determined via 3D ultrasound, followed by measurements using 2D ultrasound and caliper. Correlation between the total testicular volume (TTV) and aDSO was high with volume assessment via ultrasound (2D: r = 0.639, p < 0.001, and 3D: r = 0.604, p < 0.001), and moderate for using caliper (r = 0.46, p < 0.01). Linear regression analyses of TTV and aDSO values revealed that changes in aDSO in part could be explained by differences in testes volume: 32% and 27% in case of 3D and 2D ultrasound, and 12% with caliper. pDSO values that were predicted from testicular measurements correlated best with aDSO values from semen collection protocols in case of using 3D ultrasound (r = 0.56, p < 0.001), followed by 2D ultrasound (r = 0.52; p < 0.001) and caliper (r = 0.34, p = 0.01). In conclusion, 3D ultrasound can be performed on equine testes for more accurate volume predictions, which in turn may increase precision when determining the breeding potential of a stallion.

Birth of healthy calves after intra-follicular transfer (IFOT) of slaughterhouse derived immature bovine oocytes.

To circumvent the negative impacts of in vitro culture on bovine embryos, we have recently established a new method, the so called intra-follicular oocyte transfer (IFOT), enabling in vivo fertilization and in vivo development of in vitro matured oocytes up to the blastocyst stage as well as to term. In this study, we raised the question whether immature bovine oocytes could also be transferred into a pre-ovulatory follicle to support in vivo maturation prior to subsequent in vivo fertilization, in vivo development as well as to term. To unravel that question, a total of 791 immature oocytes were transferred in groups of ∼50 into pre-ovulatory follicles of 16 recipient heifers. Consequently, we were able to recollect a total of 306 structures 8 days thereafter (38.5%). All in all, 12 heifers (75%) gave embryos developed to the morula or blastocyst stage in addition to the expected native embryos. Among all recollected structures, 40.1% had developed to the morula and/or blastocyst stage, meaning a total efficiency of 17.3% based on all transferred oocytes. Of impact, IFOT-embryos reached significantly higher developmental rates to the Morula and/or blastocyst stage until day 7 compared to in vitro cultured control embryos, despite being derived from the same charge of slaughterhouse ovaries (40.1 vs. 29.3%). This implicates a beneficial effect of the follicular environment for the intrinsic quality of the fertilized embryos during maturation and for subsequent developmental rates up to the blastocyst stage. Finally, the birth of two healthy calves after transfer of frozen-thawed IFOT-derived blastocysts to final recipients established the first proof of principle that IFOT of immature bovine oocytes generates bovine blastocysts bearing developmental capacity to term. Likewise, to the best of our knowledge, these calves are the first calves derived from full in vivo development of immature slaughterhouse derived oocytes. Thus, the results of the present study clearly demonstrate that IFOT of immature slaughterhouse-derived oocytes is now a feasible technique. Since efficiencies following IFOT achieved within the present study were improved compared to previous studies, IFOT now offers an attractive option for designing new scientific experiments.

Quality of seminal fluids varies with type of stimulus at ejaculation.

The theory of ejaculate economics was mainly built around different sperm competition scenarios but also predicts that investments into ejaculates depend on female fecundity. Previous tests of this prediction focused on invertebrates and lower vertebrate, and on species with high female reproductive potential. It remains unclear whether the prediction also holds for polygynous mammals with low female reproductive potential (due to low litter size and long inter-birth intervals). We used horses (Equus caballus) to experimentally test whether semen characteristics are adjusted to the oestrous cycle of the mare a stallion is exposed to during few moments before ejaculation. We analysed 122 weekly semen samples collected from 16 stallions during exposure to either an oestrous or a dioestrous mare. Semen volume and the rate of motile sperm were higher when stallions were exposed to an oestrous than to a diestrous mare, while total sperm counts and sperm velocity remained unchanged. Sperm collected after exposure to an oestrous mare also showed reduced oxidative degeneration of cell membranes over a period of 48 hours. We conclude that stallions invest more into their seminal fluids when the chance of fertilization is elevated, and that this adjustment of ejaculate quality can happen very quickly.

MHC-correlated preferences in diestrous female horses (Equus caballus).

Genes of the major histocompatibility complex (MHC) have been shown to influence communication in many vertebrates, possibly with context-specific MHC-correlated reactions. Here we test for MHC-linked female preferences in the polygynous horse (Equus caballus) by repeatedly exposing 19 mares to a group of seven sexually experienced stallions. Each mare was tested four times during two consecutive reproductive cycles, twice during estrus and twice during diestrus. Male plasma testosterone concentrations were determined from weekly blood samples, and equine leukocyte antigen (ELA) class I and II alleles were determined serologically at the end of the experiments. Perception of male attractiveness was strongly dependent on estrous cycle: mean preference scores did not correlate for mares in diestrus and estrus and varied more during estrus than during diestrus. We found elevated female interests for MHC-dissimilar stallions, but only during diestrus, not during estrus. Female preferences were not significantly predicted by mean male testosterone plasma concentrations. However, testosterone concentrations changed during the 11 weeks of the experiment. By the end of the experiment, average testosterone concentration was significantly correlated to the average number of MHC alleles the stallions shared with the mares. We conclude that the MHC affects female preferences for stallions, but non-MHC linked male characteristics can overshadow effects of the MHC during estrus.

High interindividual and intraindividual variation of oxytocin secretion in estrous mares exposed to stallions, but no significant link to mate preferences.

Oxytocin is a hormone that may not only influence reproductive mechanisms in mammals but also their social behavior, including pair bonding. We therefore tested if the concentrations of oxytocin and other hormones reveal mate preferences of 13 mares in estrus. Each mare was first exposed to two stallions (haphazardly selected out of seven) and her behavior recorded. The mare was then returned to her box (i.e., no contact to stallions during that time). Approximately 4.5 hours later, venous blood samples were collected every minute during 30 minutes preceding exposure to one of the two previously used stallions, 6 minutes during exposure, and 30 minutes after exposure back in the mare's box. The procedure was repeated in the consecutive estrus cycle, with the difference that the mare was each exposed to the other of the two stallions during oxytocin measurements. In 20 of the 26 trials, oxytocin concentrations were significantly elevated during exposure to the stallion, without significant associations to cortisol and estradiol concentrations. We found no significant association between oxytocin secretion and preferences in the previous choice situation. While estradiol concentration showed a high repeatability over the two cycles, we found considerable intraindividual differences in oxytocin and cortisol plasma concentration among the two cycles. Partially, the variation in oxytocin concentrations could be linked to the time of ovulation, with lower oxytocin plasma concentrations in mares which ovulated later than expected. In conclusion, when teasing under experimental conditions, we found high interindividual and intraindividual variation among mares in the increase of oxytocin plasma concentrations, depending on the timing of ovulation. However, oxytocin levels seemed to be no predictor of mare preference.

Heritability of semen traits in German Warmblood stallions.

The objectives of the present study were to evaluate genetic parameters for semen quality traits of 241 fertile German Warmblood stallions regularly employed in artificial insemination (AI). Stallions were owned by the National Studs Celle and Warendorf in Germany. Semen traits analyzed were gel-free volume, sperm concentration, total number of sperm, progressive motility and total number of progressively motile sperm. Semen protocols from a total of 63,972 ejaculates were collected between the years 2001 and 2014 for the present analysis. A multivariate linear animal model was employed for estimation of additive genetic and permanent environmental variances among stallions and breeding values (EBVs) for semen traits. Heritabilities estimated for all German Warmblood stallions were highest for gel-free volume (h(2)=0.28) and lowest for total number of progressively motile sperm (h(2)=0.13). The additive genetic correlation among gel-free volume and sperm concentration was highly negative (rg=-0.76). Average reliabilities of EBVs were at 0.37-0.68 for the 241 stallions with own records. The inter-stallion variance explained between 33 and 61% of the trait variance, underlining the major impact of the individual stallion on semen quality traits analyzed here. Recording of semen traits from stallions employed in AI may be recommended because EBVs achieve sufficient accuracies to improve semen quality in future generations. Due to favorable genetic correlations, sperm concentration, total number of sperm and total number of progressively motile sperm may be increased simultaneously.

Fourier transform infrared spectroscopic analysis of sperm chromatin structure and DNA stability.

Sperm chromatin structure and condensation determine accessibility for damage, and hence success of fertilization and development. The aim of this study was to reveal characteristic spectral features coinciding with abnormal sperm chromatin packing (i.e., DNA-protein interactions) and decreased fertility, using Fourier transform infrared spectroscopy. Chromatin structure in spermatozoa obtained from different stallions was investigated. Furthermore, spermatozoa were exposed to oxidative stress, or treated with thiol-oxidizing and disulfide-reducing agents, to alter chromatin structure and packing. Spectroscopic studies were corroborated with flow cytometric analyses using the DNA-intercalating fluorescent dye acridine orange. Decreased fertility of individuals correlated with increased abnormal sperm morphology and decreased stability toward induced DNA damage. Treatment with the disulfide reducing agent dithiothreitol resulted in increased sperm chromatin decondensation and DNA accessibility, similar as found for less mature epididymal spermatozoa. In situ infrared spectroscopic analysis revealed that characteristic bands arising from the DNA backbone (ν1230, ν1086, ν1051 cm(-1) ) changed in response to induced oxidative damage, water removal, and decondensation. This coincided with changes in the amide-I region (intensity at ν1620 vs. ν1640 cm(-1) ) denoting concomitant changes in protein secondary structure. Reduction in protein disulfide bonds resulted in a decreased value of the asymmetric to symmetric phosphate band intensity (ν1230/ν1086 cm(-1) ), suggesting that this band ratio is sensitive for the degree of chromatin condensation. Moreover, when analyzing spermatozoa from different individuals, it was found that the asymmetric/symmetric phosphate band ratio negatively correlated with the percentage of morphologically abnormal spermatozoa.

Sperm Membrane Behaviour during Cooling and Cryopreservation.

Native sperm is only marginally stable after collection. Cryopreservation of semen facilitates transport and storage for later use in artificial reproduction technologies, but cryopreservation processing may result in cellular damage compromising sperm function. Membranes are thought to be the primary site of cryopreservation injury. Therefore, insights into the effects of cooling, ice formation and protective agents on sperm membranes may help to rationally design cryopreservation protocols. In this review, we describe membrane phase behaviour of sperm at supra- and subzero temperatures. In addition, factors affecting membrane phase transitions and stability, sperm osmotic tolerance limits and mode of action of cryoprotective agents are discussed. It is shown how cooling only results in minor thermotropic non-cooperative phase transitions, whereas freezing causes sharp lyotropic fluid-to-gel phase transitions. Membrane cholesterol content affects suprazero membrane phase behaviour and osmotic tolerance. The rate and extent of cellular dehydration coinciding with freezing-induced membrane phase transitions are affected by the cooling rate and ice nucleation temperature and can be modulated by cryoprotective agents. Permeating agents such as glycerol can move across cellular membranes, whereas non-permeating agents such as sucrose cannot. Both, permeating and non-permeating protectants preserve biomolecular and cellular structures by forming a protective glassy state during freezing.

Characterization of the equine blood-testis barrier during tubular development in normal and cryptorchid stallions.

The formation of the blood-testis barrier (BTB) is defined as occurring with the first appearance of spermatocytes at around puberty and is vital for normal spermatogenesis. This barrier between two adjacent Sertoli cells (SCs) consists of a cell junctional protein complex, which includes tight junctions (TJs), adherens junctions, and gap junctions. In many mammalian species, BTB composition has already been investigated, whereas little is known about the equine BTB. In the present study, immunohistochemistry and qualitative Western Blot analysis were used to assess the expression and distribution patterns of the junctional proteins claudin-11 (TJ), zonula occludens-1 (TJ associated), N-cadherin (adherens junctions), and connexin 43 (gap junctions) in equine testes during tubular development and in testes of stallions exhibiting unilateral cryptorchidism. Therefore, testes of 21 warmblood stallions (aged 12 months-11 years) were obtained during routine surgical castration. In the normal adult equine testis, the junctional proteins are localized at the basolateral region of the seminiferous tubules forming a circumferential seal corresponding to the known BTB localization. N-cadherin is additionally expressed along the lateral SC surface. In immature seminiferous cords still lacking a lumen, a diffuse distribution pattern of the junctional proteins throughout the SC cytoplasm is visible. As lumen formation advances, the immunolocalization shifts progressively toward the basolateral SC membranes. Additionally, apoptotic germ cells were detected and quantified in prepubertal stallions using terminal deoxynucleotidyl transferase dUTP nick end labeling assay and correlated with junctional protein localization. In the retained testis of cryptorchid stallions, which exhibit an aberrant testicular morphology, a deviating expression of the junctional proteins is visible. The present data show for the first time that (1) the equine SC junctional complex contains claudin-11, zonula occludens-1, N-Cadherin, and connexin 43, as already described for men or mice, and that (2) different distribution patterns of these proteins exist during testicular development in the context of lumen formation (lumen scores: 1-7) and in retained testes of unilateral cryptorchid stallions.

Effects of ground semen collection on weight bearing on hindquarters, libido, and semen parameters in stallions.

Collection of semen on the ground from the standing stallion represents an alternative method to dummy mount semen collection and is of increasing popularity for sport stallions, males suffering from health problems, or in studs without a dummy or suitable mare at disposal. Our aim was to collect and compare spermatological and physiological data associated with traditional and ground semen collection. Twelve of 23 Franches-Montagnes stallions were selected to carry out semen collection on a dummy and while standing in a crossed experimental protocol. Semen quantity and quality parameters, weight bearing on hindquarters, and behavioral and libido data were recorded. Ground versus dummy mount semen collection was accompanied by lower seminal volume (15.9 ± 14.6 vs. 22.0 ± 13.3 mL; P < 0.01) and lower total sperm count (4.913 ± 2.721 × 10(9) vs. 6.544 ± 2.856 × 10(9) sperm; P < 0.001). No significant differences were found concerning sperm motility and viability. Time to ejaculation was longer, and the number of attempts to ejaculation was higher (P = 0.053) in the standing position compared with the mount on the dummy. A higher (P < 0.01) amount of tail flagging was manifested by the stallions during ejaculation on the dummy compared to when standing. There was no difference in weight bearing on hindquarters when comparing dummy collection (51.2 ± 2.5%) and standing collection (48.9 ± 5.5%). Ground semen collection can be considered as a viable option for stallions that cannot mount a dummy or a mare. However, it requires training and may be not easily accepted by all stallions. Owners should be advised that ground semen collection is associated with significantly lower sperm numbers than with dummy mount semen collection.

Effects of an anabolic steroid (Durateston) on testicular angiogenesis in peripubertal stallions.

The aim of this study was to determine the effect of the anabolic steroid testosterone on the testicular vascularization, angiogenesis and expression of angiogenic factors, and their receptors in testes of peripubertal stallions. Seven peripubertal stallions were treated with Durateston and castrated either 4 (treatment group 1 [TG1]) or 12 weeks (TG2) after the last injection. The castration of seven untreated control stallions (control group [CG]) took place within the same time. In the testicular specimens, volume density (VD), numerical density (ND), and area of vessels were determined morphometrically. Immunohistochemically, the expression of vascular endothelial growth factor (VEGF) and its receptor VEGF-R2; angiopoietin 2 (Ang2) and its receptor Tie2 as well as of transforming growth factor α (TGF-α) was investigated. Morphometrically, the VD of TG1 (P = 0.000) and TG2 (P = 0.001) vessels and the ND of arterioles and venules and capillaries were higher (TG1, TG2: P < 0.05), and the area of capillary cross sections was smaller (TG1, TG2: P < 0.05) than that in the CG. Compared to TG2 horses, TG1 animals showed a higher (P < 0.05) VD and ND of vascular structures and a smaller (P < 0.05) area of capillary cross sections. In numerous vascular structures, especially of TG1, the TGF-α and, to a less extent, the Ang2 and VEGF-R2 expression was significantly higher (P < 0.05) than that in the CG. Sertoli cells in TG2 were characterized by a significantly higher expression (P < 0.05) of VEGF-R2 than in the CG. In summary, the most and smallest vessels could be detected in the testes of TG1. Most likely this is explainable by the highest expression of some angiogenic factors (TGF-α, Ang2) and receptors (VEGF-R2) investigated. This expression behavior may be stimulated by testosterone. As a significant decrease of morphometric parameters could be detected in TG2 compared to TG1, the stimulatory effect of testosterone seems to be temporary.

Female major histocompatibility complex type affects male testosterone levels and sperm number in the horse (Equus caballus).

Odours of vertebrates often contain information about the major histocompatibility complex (MHC), and are used in kin recognition, mate choice or female investment in pregnancy. It is, however, still unclear whether MHC-linked signals can also affect male reproductive strategies. We used horses (Equus caballus) to study this question under experimental conditions. Twelve stallions were individually exposed either to an unfamiliar MHC-similar mare and then to an unfamiliar MHC-dissimilar mare, or vice versa. Each exposure lasted over a period of four weeks. Peripheral blood testosterone levels were determined weekly. Three ejaculates each were collected in the week after exposure to both mares (i.e. in the ninth week) to determine mean sperm number and sperm velocity. We found high testosterone levels when stallions were kept close to MHC-dissimilar mares and significantly lower ones when kept close to MHC-similar mares. Mean sperm number per ejaculate (but not sperm velocity) was positively correlated to mean testosterone levels and also affected by the order of presentation of mares: sperm numbers were higher if MHC-dissimilar mares were presented last than if MHC-similar mares were presented last. We conclude that MHC-linked signals influence testosterone secretion and semen characteristics, two indicators of male reproductive strategies.

Use of density centrifugation for delayed cryopreservation of stallion sperm: perform sperm selection directly after collection or after storage?

Equipment for cryopreservation of stallion sperm is not always available. In such cases, diluted semen can be shipped to a facility for later cryopreservation. The aim of this study was to evaluate if selection of sperm via density centrifugation yields higher survival rates when cryopreservation is to be delayed (i.e. carried out after 1 day of storage at 5°C). Two-layer iodixanol as well as single-layer Androcoll density centrifugation were tested and compared with samples prepared with standard centrifugation. Special emphasis was placed on comparing centrifugation on the day of semen collection with centrifugation after 1-day refrigerated storage. Sperm morphology and motility as well as membrane and chromatin integrity were evaluated before and after centrifugation. Sperm motility and membrane integrity were also assessed after cryopreservation. It was found that both two- and single-layer density centrifugation processing resulted in higher percentages of morphologically normal and motile sperm with higher membrane and chromatin integrity, as compared to standard centrifugation or diluted samples. Differences were only in the order of magnitude of 5%. Recovery rates after density centrifugation were only approximately 30-40%. When cryopreservation was carried out after 1-day refrigerated storage, centrifugation processing of sperm directly after semen collection resulted in higher percentages of plasma membrane intact sperm post-thaw as compared to performing centrifugation processing of stored sperm just prior to cryopreservation. No significant differences in progressively motile sperm post-thaw were seen. Taken together, for delayed cryopreservation, it is best to perform density centrifugation directly after collection rather than immediately prior to cryopreservation.