F. Palm

Influence of different semen extenders and seminal plasma on PMN migration and on expression of IL-1β, IL-6, TNF-α and COX-2 mRNA in the equine endometrium

After artificial insemination or mating an inflammatory response is induced by spermatozoa and components of the inseminate or ejaculate. In order to investigate the inflammatory reaction of the endometrium to different semen extenders, phosphate buffered saline (PBS), seminal plasma (SP), skim milk-based extender (SM) or egg yolk semen extender (EY) was inoculated into the uterus of oestrous mares (n=8) during four consecutive cycles in alternating order. Twelve hours after treatment, a uterine lavage was performed and an endometrial biopsy was taken. An additional biopsy was taken in the oestrous cycle before experiments were started. No differences in volume, pH, specific density or cell count of lavage fluid were found between the treatments. A significantly (p<0.01) lower number of leukocytes in the endometrium was identified in pre-experiment biopsies (68+/-5 leukocytes per field) compared to PBS (154+/-32), SP (175+/-22), SM (193+/-29) and EY treatments (113+/-17). PMN numbers were lower (p<0.01) after infusion of EY (23+/-10) compared to PBS (59+/-21) and SM extender (69+/-21). The number of eosinophils increased after inoculation of SP (p<0.05 vs. PBS, SM and EY). All treatments increased expression of interleukins (IL)-1beta and 6, tumor necrosis factor-alpha (TNF-alpha) and cyclooxgygenase-2 (COX-2) in the endometrium compared to pre-experiment values. Expression of COX-2 mRNA was significantly higher after infusion of SM than after PBS treatment (p<0.04). In conclusion, extender alone as well as seminal plasma and PBS causes an inflammatory endometrial response with the least pronounced response induced by EY-based semen extender.

Embryo transfer induces a subclinical endometritis in recipient mares which can be prevented by treatment with non-steroid anti-inflammatory drugs

We tested the hypothesis that subclinical endometritis occurs after embryo transfer (ET) in the horse. Recipient mares were treated with meclofenamic acid (M) or flunixin meglumin (F) after ET or were left untreated (n=9 per group). Embryos were re-collected 4 days after transfer. Endometrial biopsies were taken for histology and analysis of cyclooxygenase-2 (COX-2) by immunohistochemistry and for PCR. Bacteriological swabs were collected from the uterus and lavage fluid of donor and recipient mares. Progesterone and prostaglandin F(2alpha) release was analysed in recipient mares after ET. Four days after ET, four embryos were recovered from group M and three from group F and untreated mares, each. The number of polymorph nuclear neutrophils was reduced in treated mares (p<0.05). Expression of mRNA for inflammatory cytokines did not differ between groups. In group M, expression of endometrial prostaglandin-E-synthase was higher than in group F (p<0.05). Three out of nine control mares underwent preterm luteolysis (p<0.05 vs. treatment groups), prostaglandin release (p<0.05) and the number of COX-2 positive cells (p<0.01) were significantly higher than in treated mares. Only few bacteriological swabs were positive. In conclusion, treatment of embryo recipient mares with non-steroid anti-inflammatory drugs inhibits the inflammatory response of the endometrium after ET. Meclofenamic acid may have advantages in comparison to flunixin meglumin due to a different influence on prostaglandin synthesis that may not result in inhibition of embryonic mobility.

Effect of a dietary antioxidant supplementation on semen quality in pony stallions

Lipid peroxidation contributes to the damage of the sperm plasma membrane. In different species, dietary supplementation with antioxidants has been shown to improve semen quality. Therefore, we tested effects of dietary supplementation with antioxidants and l-carnitin on semen quality in Shetland pony stallions (n=6). Semen was collected twice a week over a time period of 16 weeks. From weeks 5 to 12, a special diet for stallions containing a variety of antioxidants (STALLION, Pavo Pferdenahrung GmbH, Goch, Germany; tocopherol 300 mg/day; ascorbic acid 300 mg/day; l-carnitin 4000 mg/day; folic acid 12 mg/day) was added to the basal diet (hay, mineral supplements, water). Ejaculates were evaluated for total sperm count, semen motility (percentage of totally and progressively motile spermatozoa, longevity for 24 h at 5 degrees C) and membrane integrity (SYBR-14/PI staining): All values given are means+/-S.E.M. No changes in motility, progressive motility and membrane integrity or semen longevity for 24 h were detected. A slight but significant reduction of morphologically abnormal spermatozoa was found (weeks 1-4: 43.7+/-7.1%; weeks 13-16: 39.4+/-7.2%, p<0.05). Results show that a supplementary diet with antioxidants in the given concentration and duration does not result in pronounced effects on semen quality of stallions. It is therefore questionable to support stallions with dietary antioxidants as long as they receive an adequately balanced basal diet.

Uterine involution and endometrial function in postpartum pony mares

OBJECTIVE To determine endometrial regeneration in postpartum mares by analysis of histologic features, apoptosis and cell proliferation markers, lectin binding, cytokines, and progesterone and estrogen receptors in endometrial biopsy specimens. ANIMALS 9 postpartum mares. PROCEDURES Mares were examined on postpartum days 1, 9, and 16, and uterine biopsy specimens were obtained for histologic examination. Lectin binding was analyzed histochemically, and expressions of Ki-67 antigen (proliferation marker), lysozyme, and caspase 3 (apoptosis marker) were studied immunohistochemically. Gene expressions for cytokines (interleukin-1beta, -6 and -8 and tumor necrosis factor-alpha), cyclooxygenase 2, prostaglandin-E-synthase, and estrogen and progesterone receptors were determined by use of quantitative real-time PCR assay. RESULTS On day 1, neutrophils predominated but by day 9 had largely been replaced by lymphocytes and macrophages. High numbers of cells with staining for caspase 3 were found on day 1, but numbers decreased by day 9. In contrast, the number of cells with staining for Kiel 67 antigen increased between days 1 and 9. Lectin binding to the endometrium changed over time. Relative mRNA expressions for cytokines and prostaglandin-E-synthase did not differ among days. Expressions of progesterone and estrogen receptors were minimal on day 1 and increased by day 9. CONCLUSIONS AND CLINICAL RELEVANCE Early postpartum endometrial cells underwent apoptosis, but during the second week, postpartum proliferation of cells predominated. Lectin binding reflected changes in endometrial glycocalyx patterns. Increased expression of estrogen receptors allowed the endometrium to respond to estrogen during foal heat, and in subsequent diestrus, the endometrium was able to respond to progesterone.

Effects of Altrenogest Treatment of Mares in Late Pregnancy on Parturition and on Neonatal Viability of their Foals

In this study, effects of altrenogest treatment (0.088 mg/kg daily) given to mares during late gestation until parturition on the time and the process of foaling, neonatal adaptation and postnatal development were analysed. The number of animals was 6 in the treatment group and 7 in the control group. Gestational length tended to be shorter in mares given altrenogest. Birth weight of the foals and weight of the placenta did not differ between groups. The second stage of parturition was prolonged in the altrenogest-treated mares (p<0.05). Foals born to altrenogest-treated mares had a significantly lower respiratory rate than control foals during the first 30 minutes of life (p<0.05). At no time differences in heart rate and body temperature were found between groups. In foals of altrenogest treated mares, venous plasma pH was significantly higher than in control foals at 15 and 30 minutes after birth (p<0.05). Base excess in foals of altrenogest treated mares was significantly higher than in control foals at 45 minutes and up to 12 hours after birth (p<0.05). There were significantly more problems in the perinatal period (3/6) in foals born after altrenogest treatment to their dams than in control foals (0/7; p<0.05). In conclusion, treatment with altrenogest did not prevent parturition and its effectiveness to prevent abortion or preterm foalings in mares with disturbed pregnancies should be doubted. In addition, altrenogest treatment of mares affected adaptation of the foals to the extrauterine environment.

EcPV-2 is transcriptionally active in equine SCC but only rarely detectable in swabs and semen from healthy horses

Squamous cell carcinomas (SCC) are malignant tumours arising from keratinocytes. In horses, there is increasing evidence for Equus caballus papillomavirus type 2 (EcPV-2) being causally involved in SCC development. However, only little is known regarding intralesional transcription of the virus, and sparse information on the incidence of EcPV-2 infection in healthy equids is available so far. Using RT-PCR, total mRNA from 8 EcPV-2 DNA-positive and 1 EcPV-2 negative SCC/SCC precursor lesions was screened for the presence of EcPV-2 E6 and E1 transcripts. Using PCR, we tested 193 sample specimens (30 ocular swabs, 94 genital swabs, 54 semen and 15 milk samples) from a total of 161 apparently healthy horses for the presence of EcPV-2 genes E7 and E6 or E2. Positive results were confirmed by repeating the PCR reactions, and by amplicon sequencing. E6 mRNA was detectable in 8/8 EcPV-2 DNA-positive lesions, whereas only 3/8 scored positive for E1 mRNA. EcPV-2 PCR scored positive for DNA from 1/30 ocular swabs, 4/94 genital swabs, 0/54 semen and 0/15 milk samples, thus resulting in an overall detection rate of 5/193, i.e. 2.6%. The demonstrated presence of viral mRNA in all EcPV-2 DNA-positive lesions is suggestive for an active pathogenic role of the virus in SCC development. This finding and the low incidence of EcPV-2 DNA in healthy equids further strengthen the concept of an aetiologic association of EcPV-2 with equine SCC disease.

Effect of altrenogest‐treatment of mares in late gestation on adrenocortical function, blood count and plasma electrolytes in their foals

REASONS FOR PERFORMING STUDY Mares with compromised pregnancies are often treated with altrenogest to prevent abortion. However, there is only limited information about effects on the foal when altrenogest treatment is continued during final maturation of the fetus. OBJECTIVES To determine effects of altrenogest treatment during late gestation in mares on maturity, haematology changes, adrenocortical function and serum electrolytes in their newborn foals. METHODS Six mares were treated with altrenogest (0.088 mg/kg bwt) once daily from Day 280 of pregnancy until foaling and 7 mares served as controls. RESULTS Foals born to altrenogest-treated mares had a significantly lower neutrophil/lymphocyte ratio on the first day after birth than control foals (P<0.05). Basal plasma cortisol concentrations immediately after birth were higher in foals of altrenogest-treated mares than in control foals (P<0.05). Cortisol release in response to exogenous adrenocorticotropic hormone (ACTH)--except for higher values 15 min after ACTH injection in foals of altrenogest-treated mares on Day 1--revealed no differences in adrenocortical function between the groups of foals. Plasma potassium concentration in foals from altrenogest-treated mares compared to control foals was significantly lower immediately after birth (P<0.05) and plasma ionised calcium concentration was significantly lower 3 h after birth (P = 0.01). CONCLUSIONS AND POTENTIAL RELEVANCE Altrenogest treatment of pregnant mares prolonged labour had no major effects on adrenocortical function in foals. A reduced neutrophil/lymphocyte ratio in these foals may suggest either immunomodulatory effects of altrenogest or dysmaturity of the foals.

Heart rate and heart rate variability in pregnant warmblood and Shetland mares as well as their fetuses

Heart rate (HR) is an important parameter of fetal well-being. In horses, HR and heart rate variability (HRV) can be determined by fetomaternal electrocardiography (ECG) from mid-pregnancy to foaling. Normal values for physiological parameters in larger breeds are often used as reference values in ponies. However, HR increases with decreasing size of the animal and in ponies is higher than in warmblood horses. It is not known if fetal HR is affected by breed and if values obtained in larger breeds can be used to assess Shetland fetuses. We have determined fetomaternal beat-to-beat (RR) interval (inversely correlated to HR) and HRV in warmblood (n=6) and Shetland pregnancies (n=7) at days 280 and 300 of gestation by ECG. Maternal RR interval was lower in pony than in warmblood mares (day 280: Shetland: 958±110, warmblood: 1489±126ms, p<0.01) The SDRR (standard deviation of RR interval) and the RMSSD (root mean square of successive RR differences) did not differ between breeds at any time. Also RR interval as well as HRV did not differ between warmblood and pony fetuses (RR interval day 280: Shetland: 606±39, warmblood: 589±38ms). In conclusion, although maternal RR interval is clearly higher in Shetland than in warmblood mares, fetal RR interval in the two breeds is on the same level.

Distribution of ventilation in pregnant Shetland ponies measured by Electrical Impedance Tomography.

The regional distribution of ventilation in conscious standing pregnant Shetland pony mares was investigated by Electrical Impedance Tomography (EIT). Six ponies were repeatedly examined a minimum of four weeks prior to (antepartum, AP) until three weeks after parturition (postpartum, PP). From the cross-sectional ventilation image the ventral to dorsal (V/D), left to right (L/R) ventilation distribution ratio and the relative ventilation in four horizontal regions of interest (ROI) placed symmetrically in the chest was analyzed. Antepartum V/D was 0.74 ± 0.09 on day -28 ± 3 (AP28) and decreased to 0.68 ± 0.10 on day -3 ± 2 (AP3). Postpartum V/D increased significantly (p<0.05) to 0.96 ± 0.08 on day 7 ± 2 (PP7). The L/R ventilation distribution remains unaffected. Ventilation in the most ventral ROI was significantly lower on days AP28 and AP3 compared to PP7. These results suggest that in Shetland ponies late pregnancy compromises the ventilation in ventral (dependent) lung regions. We demonstrated the feasibility of a repeated EIT measurement in standing conscious ponies.

Increasing expression of oxytocin and vasopressin receptors in the equine conceptus between Days 10 and 16 of pregnancy

Oxytocin (OT) and arginine vasopressin (AVP) have been detected in the yolk sac of the pre-attachment equine conceptus. Therefore, we have assessed the presence of OT and AVP receptors in equine conceptuses between Days 10 and 16 of pregnancy by qualitative PCR, quantitative PCR and immunohistochemistry. Expression of OT receptor and of the AVP receptors V1aR and V2R could be verified after sequencing the RT-PCR products of the expected length. The size of conceptuses used for quantitative PCR significantly increased with day of pregnancy (P<0.01) as did their quantitative expression of OTR (P<0.01). Immunohistochemistry of OTR resulted in weak trophectodermal abundance on Day 10, increasing at Day 12. On Day 14, staining intensity increased in individual cells of the trophectoderm while it decreased in other cells; this trend became more apparent on Day 16. The endoderm of the trophoblast and surrounding subtrophoblastic compartments always showed moderate staining for OTR. On Day 10 immunoreactive V2R protein was localised in the trophectodermal apical membrane; on Day 12 it was also present in the basal membrane and weakly in the cytoplasm. On Day 14 only individual trophectodermal cells showed positive supranuclear cytoplasmic areas or V2R, whereas on Day 16 about one-third of the trophectodermal cells were stained entirely and intensely positive. These results suggest an involvement of OT and AVP action in the development and expansion of the early equine conceptus.