Efficiency of Semen Cryopreservation in Stallions.

Differences in the cryotolerance of spermatozoa exist among stallions, but it remains to be determined to what extent such differences are affected by breed. In this study, post-thaw semen quality in stallions presented for semen cryopreservation was analysed retrospectively (1012 ejaculates from 134 stallions of 5 breeds). The percentage of frozen-thawed ejaculates acceptable for artificial insemination (AI) and the number of insemination doses per cryopreserved ejaculate was calculated. Logistic regression analysis revealed sperm motility in raw semen as the most important explanatory variable for the percentage of cryopreserved ejaculates with a post-thaw quality acceptable for AI. Of the other variables included into the model, stallion age was the most important parameter with more acceptable ejaculates in younger than in older stallions. Logistic regression also showed more acceptable frozen-thawed ejaculates in Arab stallions versus Warmbloods, Quarter Horses and Icelandic horses. The analysis thus demonstrates differences in the percentage of acceptable cryopreserved ejaculates among horse breeds. Season was a less relevant explanatory variable for percentage of acceptable cryopreserved ejaculates. Logistic regression revealed total sperm count as the most important variable determining the number of cryopreserved semen doses obtained per acceptable ejaculate. In conclusion, logistic regression analysis revealed stallion age and breed as explanatory variables for the percentage of cryopreserved ejaculates acceptable for AI.

Evaluation of a modular in vitro neurovascular procedure simulation for intracranial aneurysm embolization.

BACKGROUND: Rapid development in endovascular aneurysm therapy continuously drives demand for suitable neurointerventional training opportunities. OBJECTIVE: To investigate the value of an integrated modular neurovascular training environment for aneurysm embolization using additively manufactured vascular models. METHODS: A large portfolio of 30 patient-specific aneurysm models derived from different treatment settings (eg, coiling, flow diversion, flow disruption) was fabricated using additive manufacturing. Models were integrated into a customizable neurointerventional simulator with interchangeable intracranial and cervical vessel segments and physiological circuit conditions ('HANNES'; Hamburg ANatomic Neurointerventional Endovascular Simulator). Multiple training courses were performed and participant feedback was obtained using a questionnaire. RESULTS: Training for aneurysm embolization could be reliably performed using HANNES. Case-specific clinical difficulties, such as difficult aneurysm access or coil dislocation, could be reproduced. During a training session, models could be easily exchanged owing to standardized connectors in order to switch to a different treatment situation or to change from 'treated' back to 'untreated' condition. Among 23 participants evaluating hands-on courses using a five-point scale from 1 (strongly agree) to 5 (strongly disagree), HANNES was mostly rated as 'highly suitable for practicing aneurysm coil embolization' (1.78±0.79). CONCLUSION: HANNES offers a wide variability and flexibility for case-specific hands-on training of intracranial aneurysm treatment, providing equal training conditions for each situation. The high degree of standardization offered may be valuable for analysis of device behavior or assessment of physician skills. Moreover, it has the ability to reduce the need for animal experiments.

Effect of presence or absence of antibiotics and use of modified single layer centrifugation on bacteria in pony stallion semen.

Bacteria contaminate semen during collection and handling. The objective of this study was to identify the bacteria in pony stallion semen, the effects of antibiotics included in commercial semen extenders (lincomycin and spectinomycin) and the effect of modified single layer centrifugation (MSLC), on bacterial load. Ejaculates from six pony stallions, 3 ejaculates per animal, were extended in EquiPlus extender either with or without antibiotics. Aliquots were processed by MSLC to form four treatment groups: control and MSLC with antibiotics (CA and SA, respectively) and control and MSLC without antibiotics (CW and SW, respectively). Bacteriological examinations were carried out within 2 hr. Thirty-one species of bacteria were isolated from one or more ejaculates, with Corynebacterium spp. being the most frequently detected. Corynebacterium spp. were present in all ejaculates. The MSLC resulted in a significantly lower total bacterial count than controls (CA vs. SA, p < 0.001; CW vs. SW, p < 0.0001).

Anti-Müllerian hormone profiling in prepubertal horses and its relationship with gonadal function.

Anti-Müllerian hormone (AMH) has gained increasing interest as a biomarker for assessment of gonadal activity. The ability to predict the ovarian follicular reserve of prepubertal female horses (fillies) or to identify stallions with testicular pathologies already during their prepubertal life has not been analyzed so far. Both would help to select fertile horses and reduce costs associated with keeping animals. The objectives of the present study were to (1) assess AMH, LH, FSH, progesterone (females) and testosterone (males) dynamics in prepubertal horses from birth onwards and (2) determine whether AMH concentrations detected in plasma of prepubertal female and male horses are correlated with postpubertal gonadal development. Warmblood foals (n = 30, 14 females, 10 normal males and 6 males with abnormal testicular development) born between February and May of two consecutive years (n = 28 in the first year and n = 2 the next year), were included in the study. Information on gestational length, parity of the dam and placental weight was collected for all foals. Blood samples for hormone analysis were collected from birth onwards every four weeks up to the age of one year. At two years, blood samples were collected on the day when antral follicle count (AFC) and total testicular volume (TTV) were assessed. AMH was detectable in the plasma of all animals from birth onwards and its concentration was significantly higher (P < .001) in males than in females, regardless of testicular development. In males, AMH and testosterone concentration were similar for all animals during the first year of life, regardless of testicular development. At two years, AMH concentration was higher (P < .05) in males with abnormal testicular development than in those with normal testes. In females, AMH concentration at two years was correlated with AMH concentration at birth (P < .05) and with AFC (P < .001). At birth, LH concentration was lower (P < .05) in stallions with abnormal testes (0.3 ±â€¯0.2 ng/ml) than in controls (0.6 ±â€¯0.2 ng/ml). A high negative correlation between AMH concentration and gestation length was observed in males during the first eight weeks of life (P < .01, r = -0.64 to -0.71). Elevated progesterone concentrations over 1 ng/ml were observed in several females starting with 20 weeks of age. This was paralleled by an increase in AMH concentration and was preceded by FSH and LH increases. In conclusion, AMH determination can be reliably used from two years onwards to identify stallions with abnormal testicular development, but it is inconclusive before puberty. In female horses, determination of AMH concentration at a prepubertal age allows for prediction of AMH and AFC after puberty. We suggest that premature luteinisation occurs before the onset of puberty in female horses and that LH secretion in the perinatal period is involved in testicular development and descent in the horse.

Reproductive stage and sex steroid hormone levels influence the expression of mesenchymal stromal cell (MSC) markers in the equine endometrium.

Mesenchymal stem or stromal cells (MSCs) play key roles in tissue homeostasis. In the cyclic equine endometrium, this may be regulated by changes in serum concentrations of sex steroid hormones. This study was designed to investigate the changes in endometrial expression of MSC markers during reproductive cycles in mares and the influence of sex steroid hormones on endometrial MSC proliferation in vitro. Endometrial biopsies were collected from pony mares at different reproductive stages (estrus; day 5 and 13 after ovulation; seasonal anestrus; 20 h and 7days post-partum; n = 5 per stage) and were analyzed by RT-qPCR. MSC (CD29, CD44, CD73, CD90, CD105) and perivascular (CD146, NG2) markers were present in all samples irrespective of reproductive stage. Transcript levels of most markers were present at lowest levels on day 5 after ovulation and at 20 h post-partum. MSCs isolated from endometrial tissue (n = 6 mares) were cultured in the presence of progesterone (0.01-100 µM) and estradiol 17ß (0.1-1 µM), and cell proliferation was analyzed using alamarBlue® assay. Relative to cells incubated in steroid-depleted media, both progesterone and estradiol 17ß moderately increased cell proliferation (1.1- and 1.2-fold, respectively) independently of the concentration used. In conclusion, our results suggest that levels of MSC markers in equine endometrium dynamically change across reproductive cycles and that MSC populations are in part regulated by sex steroids.

Sperm Quality during Storage Is Not Affected by the Presence of Antibiotics in EquiPlus Semen Extender but Is Improved by Single Layer Centrifugation.

Contamination of semen with bacteria arises during semen collection and handling. This bacterial contamination is typically controlled by adding antibiotics to semen extenders but intensive usage of antibiotics can lead to the development of bacterial resistance and may be detrimental to sperm quality. The objective of this study was to determine the effects of antibiotics in a semen extender on sperm quality and to investigate the effects of removal of bacteria by modified Single Layer Centrifugation (MSLC) through a colloid. Semen was collected from six adult pony stallions (three ejaculates per male). Aliquots of extended semen were used for MSLC with Equicoll, resulting in four treatment groups: control and MSLC in extender with antibiotics (CA and SA, respectively); control and MSLC in extender without antibiotics (CW and SW, respectively). Sperm motility, membrane integrity, mitochondrial membrane potential and chromatin integrity were evaluated daily by computer-assisted sperm analysis (CASA) and flow cytometry. There were no differences in sperm quality between CA and CW, or between SA and SW, although progressive motility was negatively correlated to total bacterial counts at 0 h. However, MSLC groups showed higher mean total motility (P < 0.001), progressive motility (P < 0.05), membrane integrity (P < 0.0001) and mitochondrial membrane potential (P < 0.05), as well as better chromatin integrity (P < 0.05), than controls. Sperm quality remained higher in the MSLC groups than controls throughout storage. These results indicate that sperm quality was not adversely affected by the presence of antibiotics but was improved considerably by MSLC.

The PGF2α agonists luprostiol and d-cloprostenol reliably induce luteolysis in luteal phase mares without evoking clinical side effects or a stress response.

In the present study we have evaluated a possible stress reaction in response to two different PGF2α analogs-luprostiol and D-cloprostenol--and their effects on estrous cycle characteristics. In a cross-over-design eight mares received in alternating order either luprostiol (Treatment LUP; 3.75 mg im), D-cloprostenol (Treatment CLO; 22.5µg im) or saline (Treatment CON; NaCl 0.9% 0.5ml im) on day 8 after ovulation. Injection of either LUP or CLO, but not of CON resulted in a significant decline of progesterone concentration in plasma to baseline concentrations within two days (time: p<0.001, treatment: p<0.01, time × treatment: p<0.05). The treatment to ovulation interval was significantly shorter in LUP and CLO than in CON cycles (LUP: 9.4 ± 0.4 d; CLO: 9.4 ± 1.3 d; CON: 16.1 ± 0.8 d; p<0.001). Injection of either LUP or CLO, but not of CON significantly increased salivary cortisol concentration (immediately before injection: CON 1.3 ± 0.2, LUP 1.4 ± 0.3, CLO 1.4 ± 0.3 ng/ml; 60 min after injection: CON 1.0 ± 0.3, LUP 8.0 ± 1.4, CLO 4.2 ± 0.7 ng/ml; time: p<0.01, treatment: p<0.001, time × treatment: p<0.001). Heart rate decreased over time (p<0.05) independent of treatment and no changes in heart rate variability were detected. Injection of the PGF2α analogs CLO and LUP reliably induced luteolysis and apart from a transient increase in salivary cortisol concentration no signs of a physiological stress response or apparent side effects occurred.

Heart rate and heart rate variability in pregnant dairy cows and their fetuses determined by fetomaternal electrocardiography.

In this study, fetomaternal electrocardiograms were recorded once weekly in cattle during the last 14 weeks of gestation. From the recorded beat-to-beat (RR) intervals, heart rate and heart rate variability (HRV) variables standard deviation of the RR interval (SDRR) and root mean square of successive RR differences (RMSSD) were calculated. To differentiate between effects of lactation and gestation, pregnant lactating (PL) cows (n = 7) and pregnant nonlactating (PNL) heifers (n = 8) were included. We hypothesized that lactation is associated with stress detectable by HRV analysis. We also followed the hypothesis that heart rate and HRV are influenced by growth and maturation of the fetus toward term. Maternal heart rate changed over time in both groups, and in PL cows, it decreased with drying-off. During the last 5 weeks of gestation, maternal heart rate increased in both groups but was lower in PL cows than in PNL heifers. Maternal HRV did not change over time, but SDRR was significantly higher in PL cows than in PNL heifers, and significant interactions of group × time existed. On the basis of HRV, undisturbed pregnancies are thus no stressor for the dam in cattle. Fetal heart rate decreased from week 14 to week 1 before birth with no difference between groups. Gestational age thus determines heart rate in the bovine fetus. The HRV variables SDRR and RMSSD increased toward the end of gestation in fetuses carried by cows but not in those carried by heifers. The increase in HRV indicates maturation of fetal cardiac regulation which may be overrun by high sympathoadrenal activity in fetuses carried by heifers as suggested by their low HRV.

Treatment with human chorionic gonadotrophin before ovulation increases progestin concentration in early equine pregnancies.

For prevention of early conceptus loss in the horse, treatment with progestins has become common practice. In cattle, treatment with human chorionic gonadotrophin (hCG) during the early postovulatory phase stimulates endogenous progesterone synthesis, which is an important factor for maintenance of early pregnancy via stimulation of endometrial function and conceptus development. In the present study we have therefore investigated the influence of treatment with hCG either for induction of ovulation or during the early luteal phase on plasma progestin concentrations, size of the corpus luteum and size of the conceptus in early pregnant mares. We hypothesized that hCG treatment stimulates progestin secretion and conceptus development. In Experiment 1, induction of ovulation with hCG (1500 IU i.v.; n=14) significantly increased progestin concentration between days 5 and 15 after ovulation compared to untreated controls (n=28; p<0.05; e.g. day 5 hCG i.v.: 17.2 ± 1.9, control: 13.9 ± 0.8 ng/ml). A significant interaction (p<0.05) of hCG treatment with size of the conceptus between days 30 and 40 of pregnancy was detected. In Experiment 2, treatment of mares with hCG (5000 IU) on day 5 after ovulation (n=12) did neither affect progestin secretion (e.g. day 8 hCG: 15.4 ± 1.6, control: 17.6 ± 1.2 ng/ml) nor luteal tissue area (e.g. day 8 hCG: 9.0 ± 0.7, control: 7.6 ± 1.4 cm(2)) compared to untreated mares (n=9). In conclusion, treatment of mares with hCG for induction of ovulation within 48 h before ovulation but not on day 5 of the luteal phase stimulates progestin secretion and may enhance conceptus development via stimulation of endometrial function during early pregnancy.

Changes in faecal bacteria and metabolic parameters in foals during the first six weeks of life.

Many foals develop diarrhoea within the first two weeks of life which has been suggested to coincide with postpartum oestrus in their dams. To analyse the pathogenesis of this diarrhoea we have determined faecal bacteria in foals and their dams (n=30 each), and serum IGF-1 and γ-globulins for 6 weeks after birth. In addition, effects of ß-carotene supplementation to mares (group 1: 1000 mg/day, n=15, group 2: control, n=15) on diarrhoea in foals were studied. Diarrhoea occurred in 92 and 79% of foals in groups 1 and 2, respectively, but was not correlated with oestrus in mares. Beta-carotene supplementation was without effect on foal diarrhoea. In mares, bacterial flora remained stable. The percentage of foals with cultures positive for E. coli was low at birth but increased within one day, the percentage positive for Enterococcus sp. was low for 10 days and for Streptococcus sp. and Staphylococcus sp. was low for 2-4 weeks. By 4 weeks of age, bacterial flora in foals resembled an adult pattern. Concentration of serum IGF-1 was low at birth (group 1: 149 ± 11, group 2: 166 ± 17ng/ml), increased after day 1 (day 7 group 1: 384 ± 30, group 2: 372 ± 36) but at no time differed between groups. Serum γ-globulin concentration in foals was low before colostrum intake and highest on day 1 (p<0.001 over time). In conclusion, neonatal diarrhoea in foals does not coincide with postpartum oestrus in their dams but with changes in intestinal bacteria and is not influenced by ß-carotene supplementation given to mares.